ROLE OF CHOLECYSTOKININ RECEPTORS IN THE REGULATION

ROLE OF CHOLECYSTOKININ RECEPTORS IN THE REGULATION

DISSERTATIONES MEDICINAE UNIVERSITATIS TARTUENSIS
3
ROLE OF CHOLECYSTOKININ RECEPTORS IN THE
REGULATION OF BEHAVIOUR AND IN THE ACTION OF
HALOPERIDOL AND DIAZEPAM
bv
Eero Vasar
TARTU 1992
DISSERTATIONES MEDICINAE UNIVERSITATIS TARTUENSIS
3
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ROLE OF CHOLECYSTOKININ RECEPTORS IN THE
REGULATION OF BEHAVIOUR AND IN THE ACTION OF
HALOPERIDOL AND DIAZEPAM
by
E«ro Vasar
TARTU 1992
Department of Physiology, University of Tartu, Tartu, Estonia
Dissertation is accepted for the commencement of the degree of Doctor of Medical
Sciences on October 30th, 1991 by the Council of the Faculty of Medicine of Tartu
University
Official opponents:
Professor Pekka T. Männistö, M.D., Helsinki
Professor Vija Klusha, M.D., Riga
Professor Aleksandr Zarkovsky, M.D., Tartu
Commencement: February 5th, 1992
©
Eero Vaear, 1992
Department of Physiology, University of Tartu, Tartu, Estonia
Dissertation is accepted for the commencement of the degree of Doctor of Medical
Sciences on October 30th, 1991 by the Council of the Faculty of Medicine of Tartu
University
Official opponents:
Professor Pekka T. Männistö, M.D., Helsinki
Professor Vija Klusha, M.D., Riga
Professor Aleksandr Zarkovsky, M.D., Tartu
Commencement: February 5th, 1992
©
Eero Vaear, 1992
CONTENTS
KOKKUVÕTE
5
LIST OF ORIGINAL PUBLICATIONS
8
ABBREVATIONS
10
1. INTRODUCTION
11
2. REVIEW OF LITERATURE
12
2.1. Behavioural effects of CCK agonists
12
2.1.1. Motor depressant and antiamphetamine effect of CCK agonists
12
2.1.2. Interaction of CCK agonists with emotional behaviour in rodents
13
2.1.3. Anticonvulsant action of CCK agonists
14
2.2. Multiple CCK receptors in the brain and the selective antagonists at CCK
receptors
14
2.3. Interaction of neuroleptics and anxiolytic drugs with the CCK-ergic
neurotransmission
15
3. AIMS OF THE PRESENT STUDY
17
4. MATERIALS AND METHODS
18
4.1. Animals
18
4.2. Behavioural experiments
18
4.2.1 Exploratory activity in an elevated plus-maze
18
4.2.2. Locomotor activity in an open-field test
18
4.2.3. Measurement of motor activity in the photocell cages
18
4.2.4. Foot-shock induced aggressive behaviour
19
4.2.5. Interaction of CCK agonists with seizures
19
4.3. Preparation of brain membranes for radioligand binding experiments
19
4.4. Radioligand binding experiments
20
4.5. [^Hj-spiroperidol binding 'in vivo'
21
4.6. Drugs and their administration
22
4.7. Statistical analysis
23
5. RESULTS AND DISCUSSION
24
5.1. Motor depressant and antiamphetamine effect of CCK agonists
24
5.2. Interaction of CCK agonists and antagonists with emotional behaviour
28
5.3. Anticonvulsant effect of CCK agonists
31
5.4. Effect of repeated administration of devazepide and L-365,260 on
motor activity and [3H]pCCK-8 binding in mice
5.5. Comparison of the effects of long-term haloperidol and caerulein
treatment on mice behaviour and I^Hl-radioligand binding in the
3
32
mouse brain
34
5.6. Changes at CCK receptors after long-term treatment with
haloperidol and diazepam
36
6. CONCLUSIONS
41
ACKNOWLEDGMENTS
43
REFERENCES
44
4
KOLETSÜSTOKINIINI RETSEPTORITE TÄHENDUS KÄITUMISE
REGULATSIOONIS NING HALOPERIDOOLI JA DIASEPAAMI TOIMES
Kokkuvõte
Käesoleva töö üheks eesmärgiks oli selgitada, milline on koletsüstokiniini
(CCK)
retseptori
alatüüpide
tähtsus CCK
agonistide
(tseruleiini,
CCK-8,
pentagastriini ja CCK-4) käitumuslikes efektides. Uuriti järgmiseid CCK agonistide
käitumuslikke
toimeid:
sedatiivset,
amfetamiinivastast,
anksiogeenset,
agressiivsusevastast ja krambivastast toimet. Jälgiti, millisel määral üks või teine
CCK agonistide toime on kõrvaldatav valikuliste koletsüstokiniini antagonistide
poolt. Eksperimentides kasutati CCKA ('perifeersete') retseptorite antagonisti
devasepiidi ja CCKB ('tsentraalsete') retseptorite antagonisti L-365,260. Devasepiidi
ja L-365,260 mõju katseloomade käitumisele ja [^H]pCCK-8 sidumisele ajus uuriti
nende ainete ühekordse ja kestva manustamise järgselt. Selgitamaks CCK-ergiliste
mehhanismide
osa
neuroleptilise
ravimi
haloperidooli
efektides,
võrreldi
haloperidooli ja CCK agonisti tseruleiini pikaajalise (14-päevase) manustamise
toimet hiirte käitumisele ja erinevate [^Hj-radioligandite sidumisele ajus. Töö
teiseks eesmärgiks oli selgitada, millisel määral on CCK retseptori alatüübid seotud
neuroleptikumide ja anksiolüütiliste preparaatide toimega. Selleks uuriti muutusi
CCK retseptoritel haloperidooli ja diasepaami kestva manustamise järgselt.
Teostatud farmakoloogilise analüüsi alusel võib väita, et ainult mittevalikulised
CCK agonistid tseruleiin ja CCK-8 pärsivad katseloomade motoorset aktiivsust,
selektiivsetel CCKB agonistidel (pentagastriin ja CCK-4) antud toime puudub. Kuid
valikuliste CCK antagonistide (devasepiid ja L-365,260) vastupidine mõju
tseruleiini ja CCK-8 liikumisaktiivsust pärssivale toimele annab alust arvata, et
CCKA ja CCKB retseptorid omavad vastupidist rolli loomade motoorse aktiivsuse
regulatsioonis. CCK agonistide liikumisaktiivsust vähendav toime realiseerub
eelkõige ССКЛ retseptorite vahendusel. Tseruleiin ja CCK-8, mitte aga
CCKg/gastriini retseptorite agonist pentagastriin, kõrvaldasid amfetamiinist
tingitud hüperlokomotsiooni hiirtel. Devasepiidi manustamine väikestes annustes
(1-100 ng/kg), mis toimivad ainult perifeerset tüüpi CCK retseptoritele, kõrvaldas
täielikult tseruleiini amfetamiinivastase toime. Devasepiidi suur annus (1 mg/kg),
mis avaldab toimet ka CCKB retseptoritele, oli aga ise võimeline kõrvaldama
amfetamiini motoorikat stimuleerivat efekti. Need tulemused viitavad eelkõige
CCKA ja CCKB retseptorite antagonistlikule interaktsioonile dopamiinergiliste
neuronite aktiivsuse regulatsioonis katseloomade ajus.
s
CCK
agonistid
(tseruleiin,
CCK-8,
pentagastriin,
CCK-4)
vähendasid
märkimisväärselt rottide uurimisaktiivsust pluss-puuris. CCK agonistide
'anksiogeenne' toime korrelleerus nende afiinsusega CCKB retseptorite suhtes
ajukoores, kuid mitte CCKA retseptorite suhtes pankreases. Valikuline CCKB
retseptorite blokaator L-365,260 oli tugevam CCK-4 'anksiogeense' toime
antagonist kui devasepiid. Erinevalt tseruleiinist põhjustas CCKB agonisti CCK-4
ajusisene või süsteemne manustamine agressiivse käitumise tunduvat suurenemist
isastel rottidel. Järelikult etendavad CCKB retseptorid väga olulist osa
katseloomade emotsionaalse käitumise kontrollis.
Tseruleiini ja CCK-8, kuid mitte pentagastriini, manustamine antagoniseeris
pikrotoksiini ja pilokarpiini poolt esile kutsutud krampe hiirtel. Erinevate
konvulsiivsete ainete (pikrotoksiin, pilokarpiin ja N metüül-D-aspartaat) krampe
põhjustav
toime
korrelleerus
CCK
retseptorite
tiheduse
vähenemisega
katseloomade ajus. L-365,260 ja devasepiidi ühesugune annus (1 mg/kg) blokeeris
täielikult CCK-8 mõju pilokarpiinist tingitud Timbilistele’ krampidele, mis viitab
CCK retseptori mõlema alatüübi osalusele CCK agonistide krambivastases toimes.
CCK antagonistide L-365,260 ja devasepiidi kestev manustamine põhjustas
erinevaid muutusi loomade käitumises ja I^HJpCCK-8 sidumises hiirte eesajus.
Devasepiidi mõjul vähenes tseruleiini motoorikat pärssiv toime ja tugevnes
amfetamiinist tingitud hüperlokomotsioon. Samal ajal L-365,260 suurendas
märkimisväärselt CCK retseptorite tihedust hiire ajus, avaldamata aga olulist mõju
loomade käitumisele. Järelikult etendavad CCKA retseptorid uuritavates
käitumisavaldustes suuremat tähendust kui CCKB retseptorid.
Haloperidooli ja CCK agonisti tseruleiini pikaajaline kasutamine põhjustas
samaseid nihkeid loomade käitumises ja erinevate (^Hj-radioligandite sidumises
ajus. Tolerantsus kujunes tseruleiini, mustsimooli ja flumaseniili motoorsete
efektide suhtes, kuid amfetamiinist tingitud hüperlokomotsioon on oluliselt
suurenenud 14-päevase haloperidooli ja tseruleiini manustamise järgselt.
Paralleelselt käitumuslike nihetega suurenes hiire ajus opioid ja dopamiin2retseptorite tihedus, kuid vähenes GABAa , bensodiasepiini ja CCK retseptorite arv.
Antud tulemused viitavad CCKA retseptorite olulisele osale haloperidooli
pikaajalise manustamise toimes.
Kestev
haloperidooli ja
diasepaami
manustamine
põhjustas tolerantsust
iseruieiini
käitumist
pärssivate
efektide
(sedatiivne,
amletamnnivastane,
krambivastane ja antiagressiivne toime) suhtes. Haloperidooli ja diasepaami
pikaajalise kasutamise järgselt tuli ilmsiks CCK agonisti tugev proagressiivne
toime. Seejuures on oluline rõhutada, et haloperidooli ja diasepaami kroonilise
süstimise mõjul suurenes CCK retseptorite afiinsus ajukoores. Järelikult tekib
haloperidooli ja diasepaami 14-päevase manustamise vältel alatundlikkus CCKA ja
ülitundlikkus CCKB retseptoritel.
Läbiviidud farmakoloogilise analüüsi alusel võib väita, et CCKA ja CCKB
retseptorite
vahel
eksisteerib
funktsionaalne
antagonism
mitmesuguste
käitumisavalduste regulatsioonis. Haloperidooli ja diasepaami pikaajalisel
manustamisel leiavad aset vastupidised nihked CCKA ja CCKB retseptorite
tundlikkuses, mis on ilmselt seotud organismi adapteerumisega nende
ravimite suhtes.
7
LIST OF ORIGINAL PUBLICATIONS
I. Vasar E., Otter M. and Rägo L. (1982) Intraventricular administration of
cholecystokinin decreases the activity of dopamine- and serotoninergic systems in
the brain. Sechenov Physiol. J. of the USSR, 68: 1218-1222 (in Russian).
II. Vasar E., Maimets M., Nurk A. and Allikmets L. (1984) Caerulein stimulates
[3H]-spiperone binding in vivo after long-term haloperidol administration.
Psychopharmacol. Bulletin, 20: 691-692.
III. Vasar E., Maimets M. and Allikmets L. (1984) The role of serotonin2-receptors
in the regulation of aggressive behaviour. J.Higher Nervous Activity, 34:283-289
(in Russian).
IV. Vasar E., Nurk A., Maimets M. and Allikmets L. (1985) Stimulation with
caerulein, an analog of cholecystokinin octapeptide, of ^H-spiroperidol binding
after prolonged administration of neuroleptics. Bull. Exp. Biol. Med., 99: 72- 74
(in Russian).
V. Vasar E., Rägo L., Soosaar A., Nurk A. and Maimets M. (1985) Modulatory
effect of caerulein on benzodiazepine receptors. Bull. Exp. Biol. Med., 100: 711713 (in Russian).
VI. Vasar E., Maimets M., Nurk A., Soosaar A. and Allikmets L. (1986)
Comparison of motor depressant effects of caerulein and N-propylnorapomorphine
in mice. Pharmacol. Biochem. Behav., 24: 469-478.
VII. Vasar E., Soosaar A., Maimets M. and Allikmets L. (1986) Reduced sensitivity
of the brain cholecystokinin receptors under the effect of haloperidol prolonged
administration. Bull. Exp. Biol. Med., 102: 583-585 (in Russian).
VIII. Vasar £., Allikmets L., Soosaar A. and Lang A. (1987) Change of behavioural
and biochemical effects of caerulein, an analogue of cholecystokinin octapeptide
(CCK-8), following long-term administration of haloperidol. J.Higher Nervous
Activity, 37: 696-702 (in Russian).
IX. Vasar £., Allikmets L. and Soosaar A. (1988) Caerulein, an agonist of CCK-8
receptors, antagonizes the behavioural effects of ketamine in mice and rats. Bull.
Exp. Biol. Med., 105: 43-45 rin Russian).
X. Vasar E. Allikmets L., Ryzhov /., Prakhye /., Soosaar A. and M inaev S. (1988)
Interspecies differences in the behaviojral effects of caerulein, an agonist of CCK8 receptors, in mice and rats. Bull. Exp. Biol. Med., 105: 168- 170 (in Russian).
XI. Vasar E., Soosaar, A. and Lang A. (1988) The involvement of cholecystokinin
receptors iri the realization of behavioral and biochemical effects of long-term
haloperidol administration. Proc. Acad. Sei. of the Estonian SSR. 37: 131- 139 (in
x
Russian).
XII, Allikmets L. and Vasar E. (1990) Adaptation^ changes in GAB A.
benzodiazepine and cholecystokinin receptors elicited by long-term haloperidol
administration. Sov.Med.Rev.G.Neuropharm., 1: 101-126.
ХШ. Vasar E., Allikmets L., Soosaar A. and Lang A. (1990) Similar behavioral and
biochemical effects of long-term haloperidol and caerulein treatment in albino
mice. Pharmacol. Biochem. Behav. 35: 855-859.
XIV. Vasar E., Harro J., Soosaar A. and Lang A. (1990) The changes at
cholecystokinin
receptors
after
long-term
treatment
with
diazepam.
In:
Psychosomatic Disorders. (V.Vasar, ed.). Acta et Commentationes Universitatis
Tartuensis. 891: 215-223.
XV. Vasar E., Harro J., Lang A., Põld A. and Soosaar A. (1991) Differential
involvement of CCK-A and CCK-B receptors in the regulation of locomotor
activity in the mouse. Psychopharmacol, 105: 393-399.
XVI. Vasar E., Stephenson J.D and Meldrum B.S. (1991) Changes in motor
activity and forebrain [^H)pCCK-8 binding in mice after repeated administration of
drugs affecting cholecystokinin receptors. Eur.J.Pharmacol, 202: 385-390.
XVII. Vasar
Harro J., Lang A. and Soosaar A. (1991) Pilocarpine-induced
limbic seizures - an involvement of CCK receptors. In: Molecular Pharmacology of
Receptors IV. (L.Allikmets, ed.). Acta et Commentationes Universitatis Tartuensis.
929: 34-41.
XVIII. Vasar E., Harro J., Pöid A. and Lang A. (1991) CCK-8 receptors and
anxiety in rats. In: Multiple cholecystokinin receptors in the CNS (S.T.Cooper and
C.D.Dourish, eds.), Oxford IJniversity Press (in press).
ABBREVIATIONS
CCK
cholecystokinin
CCK-8
octapeptide of cholecystokinin
С СК д receptor 'peripheral' subtype of cholecystokinin receptors
CCK в receptor 'central' subtype of cholecystokinin receptors
[ЗщрССК-8
[propionyl-^HJ-propionylated-cholecystokinin octapeptide
GABA
y-aminobutyric acid
HEPES
4-(2-hydroxycthyl)-l-piperazineethanesullonic acid
icv
intracerebroventricular administration
ip
intraperitoneal administration
L-365,260
selective antagonist at 'central' cholecystokinin reccptors. 3R{+)N-(2,3-dihydro-l-methyl-?-oxo-5-phenyl-lH- 1,4-benzodiazcpin3yl)-N’-(3-methyl-phenyl)urea
MK-329
devazepide, selective antagonist at 'peripheral' cholecystokinin
receptors, l-methyl-3-(2-indoloyl)amino-5- phenyl-3H-l,4benzodiazepin-2-one
NMDA
N-methyl-D-aspartate
NPA
N-propylnorapomoфhine
sc
subcutaneous administration
Tris
Tris(hydroxymcthyl)aminomethan
10
1. INTRODUCTION
Cholecystokinin (CCK) is an important intestinal hormone with a major role in
regulating the control of digestive processes (pancreatic secretion and gall bladder
contraction) and in inhibiting feeding behaviors (Morley, 1987). Vanderhaeghen et
al. (1975) discovered gastrin-like immunoreactivity in the mammalian brain.
Several years later this immunoreactive substance was identified as the sulfated Cterminal octapeptide of cholecystokinin (CCK-8) (Eng et al., 1982). CCK-8 is
probably the most widely distributed neuropeptide in the mammalian brain, which
fulfills many of the criteria for a neurotransmitter (Beinfeld, 1988). CCK-8 is
localized in high concentrations in the cerebral cortex, hippocampus and other
limbic structures, midbrain and spinal cord neurons (Beinfeld, 1983). CCK is
leleased from the rat cerebral cortex synaptosomes, from the rat striatum tissue
slices, and from the rat nucleus accumbens tissue slices, after calcium-, potassium,
and veratridine-induced depolarization (Meyer, Krauss, 1983; Voight et al., 1986;
Vitkroy, Bianchi, 1989). Specific high-affinity binding sites for ( 125ij_(XK-8
have been identified, with the anatomical localization of terminals containing CCK8 (Innis, Snyder, 1980; Saito et al., 1981; Beinfeld. 1983). Over the past decade
major advances have occurred in our understanding of CCK receptors. There are at
least 2 types of CCK-8 receptor designated CCKA ('peripheral') and CCKB
('central') (Dourish, Hill, 1987). Neurophysiological studies of CCK 8 indicate its
function as an excitatory transmitter throughout the central nervous system
(Skirboll et al., 1981; White, Wang, 1984). Behavioural studies have suggested that
high doses of CCK-8 administered systemically have analgesic, sedative or
neuroleptic-like activity (Zetler, 1980; Kadar et al., 1985), while centrally
administered CCK-8 may have opposite functional effects (Faris et al., 1983;
Crawley et al., 1985). The discovery that CCK coexists with dopamine and GABA
in certain neurons of the rat brain (Hökfelt et al., 1980; Somogyi et al., 1984)
aroused great interest in the role of CCK-8 in biochemical and behavioral processes
which might be relevant to the action of anxiolytic and neuroleptic drugs.
In the present work an attempt to learn more about the role of CCK receptors in
the regulation of behaviour was made. The selective CCK antagonists (devazepide
and L-365,260) were used throughout the study to reveal the relevance of the CCK
receptor subtypes in the action of CCK agonists. In the second part of this work the
involvement of CCK receptors in the action of long-term treatment with a
neuroleptic drug haloperidol and an anxiolytic compound diazepam was studied.
11
2^
2. REVIEW OF LITERATURE
2.1. Behavioural effects of CCK agonists
Caerulein and CCK-8, the unselective agonists at CCK receptors, are shown to
induce many behavioural effects after systemic or intracerebral administration.
However, in the present study main attention is directed to the interaction of CCK
agonists with motor activity, amphetamine-induced hyperlocomotion, seizures
induced by picrotoxin, pilocarpine and quinolinate, and emotional behaviour
('anxiogenic-like' effect in an elevated plus-maze, antiaggressive action). These
behavioural effects are believed to be related to the interaction of CCK with
dopamine and GAB A (Zetler, 1985; Harro, Vasar, 1991b) and may be affected by
benzodiazepine tranquillizers, exerting their effect through the facilitation of
GABA-ergic neurotransmission (Haefely et al., 1985), and neuroleptic drugs, the
potent antagonists at dopamine2- receptors (Seeman, 1980).
2.1.1. Motor depressant and antiamphetamine effect of CCK agonists
The motor depressant effect of caerulein and CCK-8 in mice and rats manifested
itself as a reduction both in motility and in frequency of rearings, and also as a
potentiation of central depressant drugs (barbiturates, ethanol). The systemic and
intracerebroventricular administration of CCK-8 and caerulein, but not of CCK-4,
induced the hypolocomotion and blocked amphetamine-induced hyperlocomotion
in the mouse (Zetler, 1985; Moroji et al., 1987; Hagino, Moroji, 1989). The
pretreaunent of mice with a selective CCKA receptor antagonist devazepide
antagonized the sedative effect of systemically and intracerebroventricularily
administered CCK-8, reflecting the involvement of the CCKA receptor subtype in
the action of CCK agonist (Khosla, Crawley, 1988; O'Neill et al., 1991). On the
other
hand,
motor
depression
occurred
in
the
rat
not
only
after
intracerebroventricular administration, but also after microinjection of a few ng
into the periaquaductal grey and ventromedial thalamus (Juma. Zetler, 1981;
Katsuura, Itoh, 1982; Matsushita, Itoh, 1982). The rearing inhibiting potency of
caerulein in mice was many times greater than that of reference drugs as
clonazepam, diazepam, haloperidol and clonidine (Zetler, 1980; 1983; 1984). It is
thought that the motor depressant effect of CCK-8 and the suppression of
dopaminergic activity by large doses of CCK agonists are of peripheral origin.
12
since they could be abolished by abdominal vagotomy in rats (Crawley, Kiss, 1985;
Hamamura et al., 1989). Nevertheless, not all authors have been able to reproduce
the finding that vagotomy can reverse the behavioural effects of CCK agonists in
rodents.
Moroji
and
Hagino
(1987)
have
demonstrated
that
bilateral
subdiaphragmatic vagotomy does not prevent the behavioural effects of
systematically administered caerulein in mice. The suppression of electrical selfstimulation by caerulein is completely insensitive to vagotomy in rats (De Witte et
al., 1986). Altar and Boyar (1989) have shown that peripherally injected CCK-8
interacts through CCKB receptors with central dopaminergic mechanisms.
2.1.2. Interaction of CCK agonists with emotional behaviour in rodents
The systemic administration of CCK agonists (caerulein, pentagastrin, CCK-4)
at very low doses inhibited the exploratory activity of mice and rodents in an
elevated plus-maze (Harro et al., 1988, 1989, 1990a). The pretreatment of animals
with proglumide, an unselective CCK antagonist, attenuated the anxiogenic-like
effect of CCK agonists (Harro et al., 1989). Moreover, proglumide was able to
antagonize the antiexploratory effect of GABA-negative drugs DMCM and
pentetrazole in the plus-maze test (Harro et al., 1989). There it was possible to
select the rats according to their behaviour in the elevated plus-maze. The animals
with "anxious" behaviour had evidently higher density of CCK receptors in the
cerebral cortex as compared with "non-anxious" animals (Harro et al., 1990).
Evidence exists that the CCKB receptor antagonist CI-988 reversed the anxiogeniclike effect induced by the cessation of long-term diazepam treatment in the mouse
(Hughes et al., 1990). Rataud et al. (1991) have shown that the treatment of mice
with the CCKB receptor antagonist L-365,260, but not with the CCKA receptor
antagonist devazepide, causes the anxiolytic-like effect in the elevated plus-maze.
The intracerebroventricular administration of pentagastrin signifl ;žuitly reduced the
exploratory activity of rats in the elevated plus-maze and this effect was reversed
by pretreatment with CI-988 (Singh et al., 1991).
Several times higher doses of caerulein antagonized foot- shock- and isolatio induced aggressiveness in mice (Zetler, Baumann, 1986; Vasar et al., 1987). The
aniiaggressive effect of caerulein was blocked by pretreatment with proglumide
and naloxone, an antagonist at opioid receptors (Vasar et al., 1987). Probably the
aniiaggressive effect of caerulein at high doses is related to its antinociceptive
action (Zetler. 1985; Barber etal., 1989).
13
2.1.3. Anticonvulsant action of CCK agonists
Caerulein and CCK-8 delayed or prevented convulsions induced by picrotoxin,
harman, thiosemicarbazide and isoniazid, whereas they were only weak antagonists
or inactive against other convulsants such as bicuculline, pentetra/ol and strychninc
(Kadar et al., 1983; 1984; Zetler, 1980, 1981, 1985). The inactivity of caerulein
and CCK-8 against convulsants, bicuculline and pentetrazol, and the resistance of
the antiharraan effect of caerulein against the benzodiazepine antagonist,
flumazenil, separates the anticonvulsant action of caerulein and CCK-8 from that of
diazepam (Zetler, 1985). The tonic-clonic convulsions induccd by maximal
electroshock were not prevented by caerulein and CCK-8, but latency to the onset
of clonic seizures and the duration of postictal motor inactivity were prolonged
(Zetler, 1985). The anticonvulsant effect of caerulein against picrotoxin induced
seizures was reversed by pretreatment with an unselective CCK antagonist
proglumide (Vasar et al„ 1987). The benzodiazepine antagonist CGS 8216, but not
flumazenil, also blocked the anti-picrotoxin effect of caerulein (Vasar et al., 1987).
2.2. Multiple CCK receptors in the brain and the selective antagonists at
CCK receptors
Two CCK receptor subtypes have been differentiated according to their affinity
for CCK fragments and analogues (Innis, Snyder, 1980; Moran et al., 1986;
Dourish, Hill, 1987). 'Peripheral' CCK receptors (CCKA) located in organs such as
the gallbladder and pancreas (Sankaran et al., 1980), but also in several discrete
brain regions such as the area postrema, interpeduncular nucleus, nucleus tractus
solitarius, nucleus accumbens and the dorsal raphe (Moran et al., 1986; Hill et al.,
1987; Barrett et al., 1989; Vickroy, Bianchi, 1989; Pinnock et al., 1990). CCKA
receptors exhibit a high affinity for the sulphated octapeptide fragment and a lower
affinity for the desulphated octapeptide, gastrin and cholecystokinin tetrapeptide
(CCK-4). Conversely, central’ CCK sites (CCKB) display a high affinity for all
these CCK fragments and gastrin (Innis, Snyder, 1980). The vast majority of CCK
receptors in the brain are of the CCKB subtype and these receptors are ubiquitous in
the mammalian brain (van Dijk et al., i 984; Hill et al., 1987). The careful analysis
of dissociation curves also revealed the presence of two subtypes binding sites for
[3Hl-pCCK-8 and | ^ i j . c c K - S in the rodents’ brain (Wennogle et al., 1985;
Sekiguchi, Moroji, 1986). There was only the 2-4-fold difference between the
affinities of these binding sites in the different species (Sekiguchi, Moroji, 1986).
14
The preincubation of brain membranes at 37°С converted all the binding sites for
[3H]pCCK-8 into the low- affinity state (Soosaar et al., 1988). The relation of these
binding sites of CCK to CCKA and CCKB receptors remains to be established.
In recent years very specific and highly potent non-peptide CCK antagonists
have been developed, including some that are highly selective for CCK receptor
subtypes and have good brain penetrability. These include the CCKA receptor
antagonists MK-329 [devazepide) (Chang, Lotti, 1986), A65186 (Kerwin et al.,
1989) and lorglumide (Rovati etal., 1987), and the CCKB receptor antagonists L365,260 (Lotti. Chang, 1989), CI-988 (Hughes et al., 1990) and LY-262684
(Howbert et al., 1991). MK-329 (deva/epide) is shown to antagonize the decreased
feeding induced by systemic injection of CCK-8 (Dourish el al.,
1989).
Behavioural studies showed that both MK-329 (devazepide) and L-365,260
increased food intake and postponed the onset of satiety, however, the CCKB
receptor antagonist was 100 times more potent than MK-329 (Dourish et al., 1989).
In contrast in the rat tail flick test, L-365,260 was only 5 times more potent than
devazepide in enhancing of morphine analgesia (Dourish et al., 1990). L-365,260
and CI-988, but not devazepide, exhibited anxiolytic-like properties in several
behavioural tests in rodents (Hughes et al., 1990; Singh et al., 1991; Rataud et al.,
1991).
2.3. Interaction of neuroleptics and anxiolytic drugs with CCK-ergic
neurotransmission
CCK-8 is shown to colocalize with
dopamine
in the mesencephalic
dopaminergic neurons (Hökfelt et al., 1980) and with the major inhibitory
transmitter GAB A in the cerebral cortex and hippocampus (Somogyi et al., 1984;
Hendry et al., 1984). Therefore it is not surprising that the administration of
dopaminergic
drugs,
but
also
compounds
affecting
the
GABA-ergic
neurotransmission, is changing the CCK-ergic activity in the brain. Repeated
administration, but not acute treatment, of different dopamine antagonists
(clozapine, chlorpromazine and haloperidol) evidently increased the amount of
CCK-8 in the striatum and mesolimbic structures (Frey et al., 1983). Chang et al.
(1983) have shown that long-term treatment with haloperidol increases the density
of CCK receptors in the cortical and limbic structures of mice and guinea pigs. By
contrast, chronic treatment with an indirect dopamine agonist metamphetamine
decreased the number of CCK-8 receptors in the rat cerebral cortex (Suzuki,
15
Moroji, 1989). In addition, the repeated, but not acute, administration of dopamine
antagonists (haloperidol, chlorpromazine, clozapine etc.) induced, through the
CCK-8 sensitive mechanisms, depolarization and subsequent inactivation of
dopaminergic neurons in the rat midbrain (Chiodo, Bunney, 1983; Bunney et al.,
1985). The CCKA receptor subtype is shown to be involved in the mediation of this
effect of neuroleptic drugs (Jiang et al., 1988: Zhang et al., 1991; Minabe et al.,
1991).
Benzodiazepine tranquillizers (lorazepam, diazepam), exerting their action
through the facilitation of GABA-ergic neurotransmission in the brain (Haefely et
al., 1985), selectively depressed the CCK-8-induced excitation of rat hippocampal
pyramidal cells (Bradwejn, De Montigny, 1984). The blockade of CCK-8 receptors
by a selective CCKA receptor antagonist lorglumide is shown to augment the action
of diazepam in the rotarod motor performance test (Panerai et al., 1987). The
withdrawal of long-term treatment with diazepam was demonstrated to increase the
density of CCK receptors in the cerebral cortex and hippocampus of rats (Harro et
al., 1990). The CCKB receptor antagonist CI-988 has been shown to antagonize the
behavioural signs of benzodiazepine withdrawal (Hughes et al., 1990).
16
3. AIMS OF THE PRESENT STUDY
The general purpose of the present work was to study the roie of CCK receptors
in the regulation of behaviour, but also in the action of haloperidol (a 'classical'
neuroleptic drug) and diazepam (a widely used anxiolytic compound). In detail the
aims of the present study were:
1. To examine the role of CCKA and CCKB receptors in the different behavioural
effects of CCK agonists (motor depressant, antiamphetamine, modulation of emo­
tional behaviour, anticonvulsant effect).
2. To analyze the interaction of CCK antagonists (devazepide and L-365,260) with
the behavioural effects of CCK agonists.
3. To investigate the effects of long-term treatment with CCK antagonists
(devazepide and L-365,260) on mice behaviour and pH]pCCK-8 binding in the
mouse brain.
4. To compare the effects of long-term administration of caerulein and haloperidol
on mice behaviour and on the parameters of dopamine2-, opioid, CCK-8 and
GABAA-benzodiazepine receptors in the mouse brain.
5. To examine the changes at CCK receptors and in behavioural effects of caerulein
after long-term treatment with diazepam and haloperidol in rodents.
17
3
4. MATERIALS AND METHODS
4.1. Animals
Mate and female albino mice, weighing 20-25 g, and rats, weighing 150-300 g,
were used throughout the study. Mice and rats were maintained at 20±.VC with
food and water ad lib. Every experimental group consisted of 8-16 animals.
4.2. Behavioural experiments
4.2.1. Exploratory activity in an elevated plus-maze.
The method suggested initially by Handley and Mithani (1984) for measuring
exploratory activity was used in rats with our modifications (Harro et al., 1990).
The apparatus consisted of two opposite open arms (50x10 cm) without side walls
and two enclosed arms (50x10x40 cm) with side walls and an end wall, extending
from a central area (10x10 cm). To determine the exploratory activity in the openpart of the plus-maze, the maze (together with the central open square) was divided
by lines into 7 equal squares. The maze was elevated to the height of 50 cm, and
placed in a room exposed to daylight. During a 4-min test session the following
measures were taken by an observer: ( 1) the latency period of the first open part en­
try, (2) the number of lines crossed in the open part, (3) the total time spent in the
open part of plus-maze, and (4) the total number of closed and open part entries. At
the beginning of the experiment the rat was placed at the centre of the plus-maze,
facing usually the right closed arm. The rats clearly preferred the closed arms. An
entry was counted only when all four limbs of the rat were within a given arm.
4.2.2. Locomotor activity in an open-field test.
After testing in the plus-maze the mice and the rats were placed singly into an
open field (for mice 30x30x18 cm; for rats 100x100x40 cm, divided by lines into
16 equal squares) and observed during 3 min. The number of line crossings and
rearings was counted.
4.2.3. Measurement o f motor activity in the photocell cages.
Locomotor activity and (+)-amphetamine-induced hyperlocomotion in the mice
were also measured in individual photocell cages. The cage for the registration of
motor activity was a cylinder with an inner diameter of 40 cm and two photocells
(located in the walls) for detection of motor activity. The motor depressant effect of
18
caerulein was measured between 0 and 30 min after subcutaneous administration of
CCK agonist (15 Mg/kg)- The antiamphetamine action of caerulein (100 pg/kg, sc)
was determined between 15 and 45 min after intraperitoneal injection (+)-amphetamine (an indirect dopamine agonist, 5 mg/kg). Caerulein was given 5 min af­
ter the administration of amphetamine.
4.2.4 Foot-shock-induced aggressive behaviour.
The interaction of caerulein with aggressive behaviour of the mice was detected
by using the foot-shock-induced aggressive behaviour. A pair of mice was placed
into a special box (15x15x15 cm) with a grid floor where during 2 min they re­
ceived 30 foot-shocks with an intensity 1.5 mA. The number of aggressive contacts
(bitings, boxings etc.) was counted during this period. Caerulein (40 pg/kg) was
given subcutaneously 15 min before the experiment. The animals were used only
once.
4.2.5 Interaction o f CCK agonists with seizures.
The interaction of caerulein with picrotoxin and pilocarpine-induced seizures
was detected in the individual observation boxes. The animals were placed there 15
min before the start of the experiment (20x20x20 cm). After this habituation period
each animal was treated with caerulein (20-250 pg/kg sc) or saline. Picrotoxin (10
mg/kg ip), a potent antagonist at chloride channel, and the muscarinic agonist pilo­
carpine (380 mg/kg ip) were given 10 min later. After that the mice were observed
for 60 min and the latencies to onset of clonic seizures, tonic extension and death
were registered. In one part of the experiments the interaction of caerulein with
quinolinate (5 pg icv) and N-methyl-D-aspartatc (0.1 pg icv) induced seizures was
studied. Caerulein was injected (1-50 ng icv or 100-500 pg/kg sc) 5 min before
intraventricular administration of NMDA agonists. The unselective CCK antagonist
proglumide (25-100 mg/kg ip) was injected 10 min before treatment with convul­
sants. The behaviour of the mice was observed for 10 min and the number of mice
with clonic seizures was registered.
4.3. Preparation o f brain membranes fo r radioligand binding experiments
Following decapitation the whole brain was rapidly removed from the skull.
The different brain regions (cerebral cortex, striata, mesolimbic structures [nucleus
accumbens and tuberculum olfactorium| and brainstem) were dissected on ice.
Freehand method was used for dissection of the brainstem, whereas the other struc­
19
3*
tures were dissected according to the method of Glowinski and Iversen (1966).
Brain regions from 5-8 mice were pooled and homogenized in 10 volumes of icecold 50 niM TrisHCl, pH 7.4 at 20"C, using motor-driven Teflon-glass homogenizer for 12 strokes. The homogenate was centrifuged at 40000 x g for 15 min,
resuspended in the same volume of buffer and again centrifuged for 15 min. The
membrane preparation for all radioligands was the same, except for j^Hj-etorphine
binding. In this case the homogenate of the mesolimbic structures was incubated
for 45 min at 3 7 X between two centrifugations (for elimination of endogenous
opioid peptides). In the case of [^H l-m uscim ol binding the membranes were
washed (centrifuged) 7 times at 40000 x g for 15 min.
4.4. Radioligand binding experiments
Different incubation mixtures were used for the radioligand binding experi­
ments. The binding of f^H]-etorphine (36 Ci/mmole, Amersham International),
l^HI-flunitrazepam (81 Ci/mmole, Amersham International) and [^H]-muscimol
(19 Ci/mmole, Amersham International) were performed in 50 mM TrisHCl (pH
7.4 at 20"C). pH]-spiroperidol (77 Ci/mmole, Amersham International) binding
was determined in an incubation buffer consisting of the following: 50 mM Tr­
isHCl (pH 7.4 at 20X ), 120 mM NaCl, 5 mM KCl, 2mM CaCl2, 1 mM MgCl2, 1
mM EDTANa2, 50 pM pargyline and 0.1 % ascorbic acid. [^H]-pentagastrin (81
Ci/mmole, NEN-Dupont) and |propionyl-^H]propionylated-CCK-8 ([3H]pCCK-8,
60-81 Ci/mmole, Amersham International) binding was studied in the following
incubation medium: 10 mM HEPES-KOH (pH 6.8 at 2 0X ), 130 mM NaCl, 5 mM
KC1, 5 mM MgCl2, 1 mM sodium, 0.2 % bovine serum albumine.
For the binding experiments each polypropylene tube (1.5 ml) received 50 pi of
{^H]-ligand, 50 pi of incubation medium or displacing compound and 400 pi of
brain membrane homogenate (1-4 mg of original tissue wet weight). [^H]-flunitrazepam was added in concentrations from 0.6 to 16 nM. The nonspecific binding
was determined by using 1 pm flunitrazepam. The membranes of the cerebral cor­
tex and brainstem were incubated at 0°C for 60 min. [^H]-muscimol was used in
concentrations from 1 to 80 nM. The nonspecific binding was measured by 100 pM
muscimol. The membranes of the cerebral cortex and brainstem were incubated for
10 min at OX. [^HJ-etorphine was added in concentrations from 0.05 to 3 nM, the
nonspecific binding was detected by adding naloxone (10 pM). The incubation of
the mesolimbic membranes performed at 2 5 X for 45 min. [^HJ-spiroperidol was
20
used in concentrations from 0.1 to 2 nM and the nonspecific binding was measured
by adding 1 цМ spiroperidol. The membranes of murine striata were incubated for
30 min at 37°C. |^H]-pentagastrin was added to the incubation medium in
concentrations from 0.1 to 20 nM, nonspecific binding was detected with 1 jiM
caerulein. Incubation of [^HJ-pentagastrin was performed for 75 min at 25 °C. In
the case of [Зщ рССК-8 binding the brain membranes were preincubated for 25
min at 23"C with or without 200 nM CCK-8, after which the radioligand was added
and the samples were carefully mixed. The membranes were incubated in the pres­
ence of radioligand for 120 min at 23 °C.
In all cases the incubation was terminated by rapid centrifugation in a Beckman
microfuge (11000 x g) for 3 min at room temperature. The supematants were care­
fully aspirated and the pellets washed three times with 250 ц1 of ice-cold incubation
buffer before transfer to scintillation vials. Radioactivity of samples was counted
after stabilization in scintillation cocktail within 24 hours, using a Beckman LS
6800 (counting efficacy 50-54%). The binding experiments were repeated at least
three times. The specific binding of [^H]- radioligand was defined as the difference
between the degree of binding in the absence and presence of excess of unlabelled
ligand. Saturation curves were analyzed using a non-linear, regression program
(ENZFITTER, Leatherbarrow, 1987).
4.5. [3HJ-spiroperidolbinding "in vivo"
pH]-spiroperidol (5 pg/kg, 17 Ci/mmole, Amersham International) was injected
subcutaneously into the dorsal part of the mouse’s neck. N-propylnorapomorphine
(NPA, 5 and 50 pg/kg) and caerulein (20-250 pg/kg) were used to inhibit [^H]spiroperidol binding. Two doses of NPA with different action on rodent behaviour
were selected because two sites with different affinity for dopamine and its agonists
existed on dopamine2-receptors (Creese, Leff, 1982; Grigoriadis, Seeman, 1984).
Five Mg/kg NPA is ED50 for suppression of exploratory activity in mice, whereas
50 pg/kg is ED50 for motor excitation in rodents (Bradbury et al., 1983). NPA and
caerulein were administered 15 min before [^HJ-spiroperidol. The animals (6 mice
per group) were sacrificed 20 min after [3H]-spiroperidol treatment by cervical
dislocation. The brains were rapidly removed and the dorsal cortex and subcortical
forebrain structures (striata and limbic structures) were dissected on ice. The dis­
sected brain areas of each group were pooled and homogenized using a glass-teflon
homogenizer by hand during 1 min. The homogenization procedure was performed
in ice-cold Tris-HCl buffer (50 mM, pH 7.4 at 20°c) in the volume of 40 mg tissue
21
per ml. After homogenization 0.5 ml (20 mg tissue) of suspension was pipetted into
6 polypropylene tubes (1.5 ml) and centrifuged during 10 min at 9000g. The super­
natant was carefully discarded and the remaining pellet was washed and cut into
vials. Radioactivity of the samples was counted after stabilization in the Bray cock­
tail within 12 hours in Beckman LS 6800 with the counting of efficacy 43 %. The
binding experiments were repeated at least three times and the data were analyzed
by using Student's t-test.
4.6. Drugs and their administration
The drugs used in the present investigation are caerulein (Bachem; Farmitalia
C arh Erba), CCK-8 (Bachem; Bristol-Myers & Squibb)), pentagastrin (Sanitas),
CCK-4 (Bachem), proglumide (Rotta Pharmaceutici), devazepide, L-365,260
(Merck Sharp & Dohme), haloperidol (Gedeon Richter), spiroperidol (Janssen
Pharmaceutica), naloxone (Dupont), flunitrazepam, flumazenil (Hoffmann-La
Roche), diazepam (Gedeon Richter), muscimol (Serva), pilocarpine (Sigma), picro­
toxin (Sigma), quinolinic acid (Sigma), N-methyl-D-aspartate (Sigma), apomorphine (Sigma), N-propylnorapomorphine (NPA, Sterling-Winthrop) and (+)-amphetamine (Sigma). CCK agonists, proglumide, dopamine agonists (apomorphine,
NPA and amphetamine), muscimol, naloxone and commercial solutions of
haloperidol, diazepam and pentagastrin were prepared in saline (0.9 % NaCl solu­
tion w/v). Devazepide (MK-329, l-methyl-3-(2-indoloyl)amino-5- phenyl-3H-l,4benzodiazepin-2-one),
L-365,260
(3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-
phenyl-lH-l,4-benzodiazepin-3yl)-N'-(3-methyl-phenyl)urea and flumazenil were
suspended in saline with 1-2 drops of Tween-85. Each injection was done in a
volume 0.1 ml/10 g body weight.
Devazepide (2 mg/kg twice daily) and L-365,260 (2 mg/kg twice daily) were
administered for 10 days. The effects of caerulein and amphetamine on motor
activity were studied 24 h after the last injection of CCK-8 antagonists. [^H]pCCK8 binding experiments were carried out also 24 h after the last injection of drugs.
Haloperidol (0.5 mg/kg) and caerulein (0.1 mg/kg) were injected once daily for 15
days. The doses of haloperidol and caeruelin were chosen according to the previous
behavioural experiments. Acute administration of haloperidol (0.5 mg/kg) and
caerulein (0.1 mg/kg) caused significant neuroleptic effects in the mice (catalepsy,
motor depression and the reversal of the behavioural effects of dopamine agonists).
The behavioural and radioligand experiments were performed 72 hours after the
22
cessation of haloperidol and caerulein treatment.
Haloperidol (1 mg/kg), diazepam (5 mg/kg) and saline were injected once daily
for 14 days. The doses of caerulein, haloperidol and diazepam were chosen
according to our previous studies (Vasar et al., 1990; Harro et al., 1990a). The
behavioural and radioligand experiments were done 72 hours after the last injection
of haloperidol and diazepam treatment. This withdrawal period was chosen in order
to be sure that most haloperidol and diazepam had been washed out from the
mouse's body, because large doses of diazepam and haloperidol may substantially
mask the behavioural actions of caerulein in the mouse (Harro et al., 1990a; our
unpublished data).
4.7. Statistical analysis
The behavioural data were analyzed by using a one-way analysis of variance
(ANOVA). Post hoc comparisons between the groups were made by using the
Newman-Keuls test. The Mann-Whitney U-test was also used to analyze the
behavioural experiments. The results of radioligand binding studies were evaluated
by Student's t-test.
23
5. RESULTS AND DISCUSSION
5.1. Motor depressant and antiamphetamine effect o f CCK agonists
Subcutaneous treatment with caerulein and CCK-8 (1-50 pg/kg) in a dosedependent manner depressed the locomotory activity of mice (Table 1). Five (ig/kg
caerulein caused a significant reduction of motor activity. The administration of 10
pg/kg CCK-8 did induce the same effect on the locomotory activity of the mice.
The CCKB receptor agonists pentagastrin and CCK-4 did not change the locomotor
activity up to the dose of 100 (ig/kg. The pretreatment of the mice with an
unselective CCK antagonist proglumide (1-50 mg/kg) failed to affect the motor
depressant action of caerulein (Table 2). The pretreatment of the animals with a
selective ССКЛ receptor antagonist devazepide (0.1-10 Mg/kg) only partially
antagonized the sedative effect of the CCK agonist. By contrast, a selective CCKB
receptor antagonist L-365,260 (0.1- 1000 (ag/kg) significantly enhanced the motor
depressant effect of caerulein. A dopamine agonist apomorphine in a low dose (0. i
mg/kg) also reduced the motor activity of the mice. Pretreatment with L-365,260
( 1-10 pg/kg) significantly enhanced the sedative effect of apomorphine in the
mouse (Table 2). Small doses of devazepide (1-10 pg/kg) only partially attenuated
the sedative effect of apomorphine, whereas high doses (100 and 1000 pg/kg)
enhanced the action of the dopamine agonist. The pretreatment with caerulein (15
pg/kg) significantly potentiated apomorphine-induced hypolocomotion in the
mouse. The co-administration of apomorphine and caerulein caused nearly
complete loss of motor activity in the mice. Neither devazepide, nor L-365,260
could antagonize the effect of concomitant treatment with apomorphine and
caerulein. According to the existing data the motor depressant effect of
apomorphine and caerulein are thought to be related to the decreased activity of
dopaminergic cells in the mesencephalon (Strömbom, 1977; Zetler, 1985). The
behavioural effects of CCK antagonists probably reflect the distinct role of CCKA
and CCKH receptors in the regulation of presynaptic dopaminergic activity in the
mouse's brain. The blockade of CCKB receptors by L-365,260 decreases the
dopaminergic activity, whereas the interaction of devazepide with CCKA receptors
increases it in the mouse's brain. It seems probable that the CCKA receptors at
which caerulein and CCK-8 act to reduce locomotor activity are in the periphery
and are associated in some way through the vagal afferent pathway with
dopaminergic neurons (Crawley, Schwaber, 1984; Crawley, Kiss, 1985; Hommer
et al., 1985). However, it is important to stress that in our study devazepide, in
contrast to the investigation of Khosla and Crawley (1988), only moderately
24
Table I
THE BEHAVIOURAL EFFECTS OF CCK AGONISTS IN RODENTS
Behavioural effect
Caerulein
CCK-8
Pentagastrin
CCK-4
Motor depression
+
+
0
0
Inhibition of
exploratory activity
+
+
+
+*
Antiaggressive effect
+
Antiamphetamine effect
+
+
0
Anticonvulsant effect
+
+
+/0
0
+ - strong effect; +/Ü - moderate effect; 0 - ineffective
Table 2
THE
INTERACTION
OF
CCK
ANTAGONISTS
WITH
TH1
BEHAVIOURAL EFFECTS OF CCK AND DOPAMINE AGONISTS
Behavioural effect
Proglumide
Devazepide
L-365,260
CCK-8-induced hypolocomotion
ineffective
antagonism
potentiation
Antiamphetamine
effect of caerulein
ineffective
antagonism
moderate
antagonism
Antiexploratory
effect of CCK-4
antagonism
at low dose
antagonism
at high dose
antagonism
at low dose
Anticonvulsant
effect of CCK agonists
antagonism
at high dose
antagonism
at high dose
antagonism
at high dose
Apomorphine-induced
hypolocomotion
ineffective
antagonism
potentiation
Am phctam inc-induced
hyperlocomotion
antagonism
at high dose
antagonism
at high dose
ineffective
25
antagonized the motor depressant effect of caerulein. This seems to support the idea
that not only the CCKA receptor subtype is involved in mediating the effect of
caerulein. The concomitant treatment with a low dose of apomorphine and
caerulein causes nearly complete loss of motor activity in the mice. Devazepide and
L-365,260 were completely ineffective against the motor depression induced
by simultaneous administration of caerulein and apomorphine. According to the
studies of Hommer et al. (1986) and Crawley (1989) the CCK receptors that
mediate the potentiation of dopamine-induced hypolocomotion and suppression of
the electrical activity of dopamine neurons in the rat mesencephalon by CCK
belong to the CCKB subtype. Altar and Boyar (1989) have found that the
antagonistic effect of centrally or peripherally administered CCK agonists on the
amphetamine-evoked dopamine release in the mouse is also related to the CCKB
receptor subtype. Nevertheless, it is not clear whether CCK,, receptors are involved
in the potentiation of apomorphine-induced hypolocomotion induced by caerulein
in the mouse and it remains to be established.
An indirect dopamine agonist (+)-amphetamine (5 mg/kg) caused a three fold
increase in the number of motor activity counts. Pretreatment with caerulein (25125 pg/kg) induced a dose-dependent inhibition of (+)-amphetamine-induced
hyperlocomotion (Table 1). CCK-8 significantly antagonized the behavioural effect
erf (+)-amphetamine at a dose 200 pg/kg, whereas pentagastrin was completely
ineffective up to 1 mg/kg. It is worth noting that intraventricular administration of
CCK-33 (1 Ivy Unit) also blocked the behavioural effects of (+)-amphetamine
(stereotyped behaviour and motor stimulation). Taking into account that the
selective CCKA receptor antagonist devazepide (10 jag/kg) also completely
antagonized the behavioural effects of dopamine agonist, it is possible that CCK-33
interacted with (+)-ampfietamine-induced behavioural effects through the CCKB
receptor subtype. This finding is in accordance with the study of Crawley et al.
(1985) where administration of CCK-8 into the anteriolateral part of the nucleus
accumbens reversed (+)-amphetamine-induced hyperlocomotion by interacting
with the CCKB receptor subtype. L-365,260 had no effect on (+)-amphetamine
induced hyperactivity, whereas devazepide in high doses (above 1 mg/kg)
suppressed spontaneous motor activity and completely antagonized the motor
stimulation induced by (+)-amphetamine in the mice. The unselective CCK
antagonist proglumide induced a similar antiamphetamine effect at doses 5-15
mg/kg. The pretreatment of mice with devazepide over a wide dose range (MOO
pg/kg) completely blocked the antiamphetamine effect of caerulein (Table 2) The
administration of L-365,260 at a low dose (1 pg/kg) also did counteract the
26
antiamphetamine effect of the CCK agonist. Proglumide (1-100 mg/kg) could not
affect the antiamphetamine effect of caerulein. The interaction of CCK antagonists
with amphetamine-induced hyperlocomotion and the antiamphetamine effect of
caerulein is somewhat different from their action on caerulein- and apomorphineelicited hypolocomotion. It is suggested that (he different pharmacology of CCK-8
against dopamine-induced hypolocomotion and hyperlocomotion is related to the
involvement of distinct brain regions in the development of two opposite
behavioural effects of dopamine in the rat (Crawley, 1989). The CCK-B/gastrin
antagonist
L-365,260
did
not
significantly
change
amphetamine-induced
hyperlocomotion, but paradoxically it reversed (at a low dose) the antiamphetamine
effect of caerulein. Devazepide antagonized the antiamphetamine effect of
caerulein at low doses, where it probably interacts selectively with CCKA receptors.
However, at a high dose (1 mg/kg), which also interacts with CCKB receptors
(Dourish et al., 1989), devazepide per se reverses the motor excitation induced by
(+)-amphetamine. It is noteworthy that proglumide, which failed to interact with
the sedative and antiamphetamine effect of caerulin, substantially antagonized the
motor excitation induced by (+)-amphetamine (Table 2). According to the studies
of Moroji and Hagino (1987) the antiamphetamine effect of caerulein in mice is
completely resistant to vagotomy. Accordingly, it seems very probable that the
CCKA receptors involved in the antiamphetamine effect of caerulein are distinct
from the CCKA receptors related to caerulein- and apomorphine-induced
hypolocomotion. The idea that these CCKA receptors are located in the mouse's
brain is supported by the study of Hagino et al. (1989), in which the
intraventricular administration of CCK-8 and caerulein, but not desulfated CCK- 8
and CCK-4, antagonizes amphetamine-induced motor excitation in the mouse. The
possible mediation of the antiamphetamine effect of caerulein through the CCKA
receptors in the mouse's brain may reflect the substantial difference between CCKA
receptors in the mouse and rat brains. Crawley et al. (1985; Crawley, 1989) have
shown that CCK-8 by interacting with CCKA receptors facilitates dopamineinduced hyperlocomotion in the posteriomedial part of the nucleus accumbens of
the rat. The different pharmacology of CCKA receptors in the mouse and the rat
brains seems to account for the interspecies differences in the behavioural effects of
caerulein in the mouse and the rat. Namely, systemic treatment with caerulein
reversed the behavioural effects of amphetamine in the mouse, but not in the rat.
()n the other hand, deva/epide at a moderate dose (10 ng/kg) completely reversed
amphetamine-induced stereotypy and motor excitation in the rat, whereas in the
mouse it potently antagonized the antiamphetamine effect of caerulein.
27
4*
In conclusion, the results of the present study reflect the apparently distinct role
of ССКЛ and CCK,, receptors in the regulation of motor activity. The opposite
effect of devazepide and L-365,260 on caerulein- and apomorphine-induced
hypolocomotion is probably related to the antagonistic role of CCKA and CCK,,
receptor subtypes in the regulation of dopaminergic cells. The ССКЛ receptors, at
which caerulein acts to reduced locomotor activity, are possibly in the periphery
and are associated in some way through the vagal afferent pathways with
dopaminergic neurons in the mesencephalon (Crawley and Schwaber, 1984;
Crawley and Kiss, 1985). The antiamphetamine effect of caerulein seems to be
linked to the stimulation of CCKA receptors in the mouse's brain, whereas probably
the blockade of both subtypes of the CCK-8 receptor is involved in the
antiamphetamine effect of devazepide.
5.2. Interaction o f CCK agonists and antagonists with emotional behavwur
The lowest dose of caerulein to cause the anxiogenic- like effect on the
exploratory behaviour of the rat in the elevated plus-maze was 100 ng/kg (Table 3)
pentagastrin had a similar effect after administration of 500 ng/kg. The
subcutaneous treatment with
10 ng/kg CCK-4 in some experiments also
significantly decreased the exploratory activity of the rats. The maximal reduction
of the animals' behaviour was seen after injection of 25 and 50 (ig/kg of CCK-4.
The anxiogenic-like effect of CCK agonists in the elevated plus-maze was in good
accordance with their potency to inhibit pH]pCCK-8 (0.3 nM) binding in the
cerebral cortex, but not in the pancreas (Table 3). According to these results it is
very likely that CCKB receptors have a significance in the anxiogenic-like action
of CCK-8 agonists on the rat. The interaction of different CCK antagonists
(proglumide, devazepide and L-365,260) with the anxiogenic-like effect of CCK-4
(50 jug/kg) was also studied (Table 2). The pretreatment with 1 pg/kg L-365.260,
the selective antagonist at CCKB receptors, moderately reduced the anti-exploratory
effect of CCK-4. But only 10 pg/kg L-365,260 caused statistically evident
antagonism with the anxiogenic-like action of CCK-4. The CCKB receptor
antagonist L-365,260 was nearly 100-fold more effective than the selective CCKA
receptor antagonist devazepide (1 mg/kg) and the unselective CCK antagonist
proglumide (1 mg/kg). Consequently, the experiments with the selective CCK
antagonists obviously support the idea that tho anxiogenic-like effect of
peripherally administered CCK agonists is related to the CCKB receptor subtype in
the rat.
28
Table 3
THE CORRELATION BETWEEN ANXIOGENIC-LIKE EFFECT OF CCK
AGONISTS AND THEIR AFFINITY AT CCK RECEPTORS IN THE RAT
CEREBRAL CORTEX AND PANCREAS
CCK agonist
IC50 values against
Anti-exploratory
[3H]-pCCK-8
effect in plus-maze
cerebral cortex
(pmol/kg)
(nM)
Caerulein
0.074
1.1
Pentagastrin
0.670
10
CCK-4
411
0.9999
17.3
Pearson's у
p=0.008
pancreas
(nM)
0.6
6200
>10000
0.808
p>0.4
The systemic administration of caerulein (2-100 ng/kg), but not that of
pentagastrin, induced a dose-dependent reduction of foot-shock aggressiveness in
the male mice. The failure of pentagastrin, the agonist at CCKB/gastrin receptors, to
reduce the aggressive behaviour, probably supports the opinion that the CCKA
receptor subtype is mediating the antiaggressive effect of caerulein in the mouse.
The pretreatm^nt of mice with the unselective CCK antagonist proglumide (5-25
mg/kg) antagonized the antiaggressive effect of caerulein (40 Mg/kg). The opioid
antagonist naloxone (0.5- 2.5 mg/kg) also blocked the antiaggressive effect of CCK
agonist. Concomitant repeated administration of caerulein (100 ng/kg twice daily,
for 14 days), but not of pentagastrin (250 ng/kg twice daily), with apomorphine (1
mg/kg twice daily) potently antagonized the development of apomorphine
aggressiveness in the male rat. These data seem to support the above mentioned
idea that the antiaggressive effect of caerulein in rodents is linked to the CCKA
receptor subtype. Moreover, intraventricular injection of 200 ng of CCK-4, but not
of caerulein, induced an appreciable enhancement of the foot-shock-induced
aggressiveness. The animals receiving CCK-4, differently from the control animals,
inflicted injuries on one another. When the dose of CCK-4 was further increased, at
lirsi the aggressive reactions were reduced (1000 ngh but then (5000 ng) they again
24
exceeded the control level. Pirenperone, the selective antagonist at serotonin2receptors, in comparison with the dopamine2-receptor antagonist haloperidol, had a
more pronounced effect on the CCK-4-enhance foot-shock aggressiveness. A low
dose of haloperidol (0.01 mg/kg) potentiated the action of CCK-4, and only the
administration of 0.2 mg/kg haloperidol significantly suppressed aggressive
behaviour. Pirenperone, in contrast to haloperidol, significantly lowered the
intensity of aggressive behaviour in a dose of only 0.01 mg/kg. When the dose was
further increased, the antiaggressive action of pirenperone became more profound.
The increase of aggressive behaviour, although not so significant, was also
established after systemic treatment with a nearly 60-fold higher dose of CCK-4
(50 Mg/kg, 12.5 |ig per rat). Accordingly, there is considerable evidence that CCK-4
potentiates the foot-shock aggressiveness through the CCKB receptor subtype. The
strong antagonism of pirenperone against the proaggressive action of CCK-4 seems
to support the role of serotoninergic mechanisms in the action of the CCKB agonist.
In conclusion, the above described results reflect the apparently distinct action
of CCK agonists on the emotional behavior in rodents. At very low doses caerulein,
CCK-8, pentagastrin and CCK-4 induced anxiogenic-like effect on the exploratory
behaviour in the rats. There is good correlation between the minimal effective
doses of CCK agonists and their affinity at CCKB receptors in the rat's cerebral
cortex, but not at С С К д receptors in the pancreas. L-365,260 was a stronger
antagonist of the anxiogenic-like effect of CCK-4 as compared with devazepide.
Accordingly, the anxiogenic-like effect of CCK agonists is related to the CCKB
receptor subtype in the rat. On the other hand, the unselective CCKB/CCKA agonist
caerulein at high doses inhibited through the naloxone-sensitive mechanisms the
aggressive behaviour in the mice. Probably, the antiaggressive effect of caerulein is
related to CCKA receptors. By contrast, the selective CCKB agonist CCK-4
increased the foot-shock-induced aggressive behaviour in the rat after systemic, as
well as after intracerebroventricular administration. The comparison of doses of
CCK-4 affecting the aggressive behavior after the intracerebral and systemic
administration left little doubt that this action of CCK-4 is related to the CCKB
receptor subtype in the brain. However, it seems possible that CCKA and CCKB
receptors have a distinct role in the regulation of negative emotions and this may
explain why CCK-4 and pentagastrin, but not CCK-8, induce generalized anxiety
and panic attacks in man (De Montigny, 1989; Abelson, Nesse, 1990; Bradwejn et
al., 1990).
30
5.3. Anticonvulsant effect o f CCK agonists
The administration of picrotoxin (10 mg/kg) induced fatal seizures in all the
tested animals. Pretreatment of control animals with caerulein (20-500 ng/kg)
obviously delayed the onset of clonic seizures, tonic seizures, and death (Table 1).
Moreover, caerulein (125 |ig/kg) was able to protect 60 % of mice against the fatal
action of picrotoxin. The concomitant administration of the CCK antagonist
proglumide (50 mg/kg) with caerulein evidently antagonized the anticonvulsant
action of CCK agonist (Table 2).
Systemic treatment with muscarinic agonist pilocarpine (380 mg/kg) evoked
fatal seizures in all injccted male mice. The pretreatment of mice with CCK-8 (25200 ng/kg) significantly antagonized the effect of 380 mg/kg pilocarpine (Table 1).
50 ng/kg CCK-8 obviously reversed the effect of muscarinic agonist, the further
increase of CCK-8 dose did not enhance the effect of neuropeptide. 13 mice from
the 39 tested survived pilocarpine-induced seizures after administration of 200
jug/kg CCK-8. The CCKß/gastrin agonist pentagastrin only moderately reduced the
convulsant action of pilocarpine (Table 1). The CCKA receptor antagonist
devazepide at a high dose (1 mg/kg) evidently antagonized the anticonv з.гл т
effect of CCK- 8 (Table 2). The CCKB receptor antagonist L-365,260 also after the
administration of a high dose (1 mg/kg) reversed the anticonvulsant action of CCK.
Intracerebroventricular administration (1-50 ng), but not systemic injection
(100-500 pg/kg sc), of caerulein completely blocked the seizures induced
by
quinolinc acid (5 ng icv) and N-methyl-D-aspartate (NMDA, 0.2 ng icv) in the
mice. The antagonist at CCK receptors proglumide (50 mg/kg) attenuated the
anticonvulsant effect of caerulein. The coadministration of proglumide (25 mg/kg)
with a subthreshold dose of quinolinic acid (2.5 ng icv) induced fatal seizures in all
the tested animals.
Picrotoxin, pilocarpine and NMDA up to 1 mM did not interact with
f^H|pCCK-8 binding in the radioligand studies in 'in vitro'. The anxiogenic dose of
picrotoxin (0.5 mg/kg) increased the density of CCK receptors in the cerebral
cortex and hippocampus (FIGURE 1). The administration of picrotoxin at the
convulsant doses (1 and 2.5 mg/kg) increased the affinity, but decreased the density
of [^H]pCCK-8 binding sites in the rat's forebrain. The injection of a high dose of
pilocarpine (380 .ng/kg) changed the parameters of l ^HJpCCK-8 binding sites in
several lor brain structures of the rat. Pilocarpine also reduced significantly the
31
number of [^H|pCCK-K binding sites in the striatum, frontal cortex and
hippocampus. Simultaneously, their affinity was increased in the striatum and
hippocampus. The anxiogenic dose of NMDA increased also the density
f^HlpCCK-8 binding sites in the mouse's forebrain. By contrast, the administration
of NMDA at convulsant doses (100-200 mg/kg) increased the affinity of CCK
receptors, but decreased their density in the mouse's brain.
The above presented data reflect a significant role of CCK-8 receptors in the
modulation of the epileptogenic effect of picrotxin, pilocarpine and NMDA
agonists. CCK-8 and caerulein potently antagonized the seizures induced by
chemoconvulsants. The selective CCK antagonists devazepide and L-365,260
reversed the anticonvulsant effect of caerulein and CCK-8 against the pilocarpineinduced seizures. However, this happened after the administration of a very high
dose ( I mg/kg) of CCK antagonists. It is noteworthy that the effect of L-365,260
was somewhat stronger as compared with devazepide. Indeed, L-365,260, in a wide
dose range (10-1000 pg/kg), blocked also the effect of CCK-8 on pilocarpineinduced lethality. Nevertheless, both subtypes of CCK-8 (CCKA and CCKB) seem
to be involved in the anticonvulsant effect of CCK-8. However, the site of the
anticonvulsant action
of CCK
agonists remains
to
be
ambiguous.
The
administration of picrotoxin, NMDA and pilocarpine at convulsant doses
significantly decreased the density of CCK receptors in the different forebrain
structures of the rodents. The administration of picrotoxin and kainic acid was
shown to reduce the concentrations of CCK-8-like immunoreactivity in the rat’s
limbic structures (Kato et al., 1988, Gall, 1988). Therefore, it is possible that the
CCKn receptor subtype is, at least partially, involved in the modulation of seizures
induced by different chemoconvulsants (pilocarpine, picrotoxin, NMD> etc.). Last
not least, the unselective CCK agonists (caerulein and CCK-8) seem to be the
unique anxiogenic-like compounds. They possess, differently from the other
anxiogenic drugs, the anticonvulsant action.
5.4. Effect o f repeated administration o f devazepide and L-365,260 on motor
activity and f3HJpCCK-8 binding in mice
A .single injection ol the ССКЛ antagonist, devazepide (2 mg/kg) increased the
frequencies of rearings and line-crossings, whereas the CCKB antagonist. L365,260 (? mg/kg) only increased the number of rearings. Tolerance developed to
the Joiomotor el feels ol the antagonists al ler their repeated administration (for 10
days, iwicc daily). However, tolerance to devazepide was not seen in all the mice,
about 20 % becoming aggressive with repeated treatment. The mice attempted to
bile the backs of other mice in the cage. Administration of a moderate dose of
caerulein (20 pg/kg) 24 h after the last injection of CCK antagonists reduced motor
activity in control animals pretreated with vehicle and in mice treated with L365,260. The sedative effect of caerulein (20 pg/kg) was significantly reduced in
mice pretreated with devazepide. Administration of (+»-amphetamine produced a
54% increase in the number of line crossings and this effect was not altered by 10
days pretreatment with L-365,260. Injection of (+)-amphetamine in animals
pretreated with devazepide increased motor activity more than in animals
pretreated with vehicle. This increase was more marked when compared with the
saline + vehicle group. Repeated treatment with devazepide and L-365,260 altered
[^HJpCCK-8 binding in the mouse’s forebrain. Treatment with devazepide slightly
increased the affinity of pH jpC CK -8 binding sites whereas after L-365,260 there
was no significant change. Devazepide and L-365,260 increased the number of
pH]pCCK-8 binding sites, but only an 83 % increase after 1-365,260 was
significant.
Repeated treatment with devazepide significantly affected the locomotor
activity of mice and their behavioural responses to caerulein and (+)-amphetamine.
The results are interpreted in the light of recent suggestions that CCKA and CCKB
receptors have opposite effects on dopamine-mediated behaviours (Crawley, 1989;
Koshikawa et al., 1990). Chronic treatment with proglumide increased the activity
of dopaminergic cells in the mesolimbic system (Chiodo et al., 1987). A similar
effect occurring after repeated treatment with devazepide might explain the reduced
sedative effect of caerulein. The increased motor stimulant effect of (+)amphetamine is most likely due to increased sensitivity of striatal and mesolimbic
dopamine2-receptors because long-term administration of CCK antagonists
(proglumide, devazepide) increased the number of dopamine2-receptors in the basal
ganglia of the rodents (Csemansky et al., 1987; our unpublished data). Increased
dopamine2-receptors sensitivity would also account for the increased
aggressiveness seen in some mice during repeated treatment with devazepide.
However, it is difficult to explain the discrepancy between the increase in the
density of CCK-8 receptors and the lack of any changes in behaviour after 10-day
treatment with L-365,260. It may be that the behaviours studied are more
dependent on CCKA receptors and that L-365,260 does not affect these receptors
even at high doses.
33
5
5.5. Comparison o f the effects o f long-term haloperidol a n d caerulein
treatment on mice behaviour and l^HJ-radioligand binding in the mouse
brain
According
to our preliminary experiments the cessation of long-term
administration of haloperidol and caerulein did not cause significant signs of
withdrawal. The basal motor activity of mice was unaltered 72 hours alter the last
injection of repeated treatment with saline and haloperidol as well as caerulein. In
addition, we found that quinolinic-acid- and picrotoxin-induced seizures were
identical after the withdrawal of long-term saline, haloperidol or caerulein
treatment. Moreover, there were no significant differences in the binding values of
t^H]-spiroperidol, [^HJ-flunitrazepam and [^H]-pentagastrin if the tissues were
obtained 2 or 72 hours after the last injection of haloperidol and caerulein.
Therefore the changes in mice behaviour and radioligand binding described below
were not caused by the withdrawal of haloperidol and caerulein, but were rather
induced by the repeated administration of both drugs.
Seventy-two hours after the cessation of 15 days of haloperidol (0.5 mg/kg
daily) and caerulein (0.1 mg/kg) treatment the effects of different drugs on mice
motor activity were changed. The motor excitation induced by amphetamine (3
mg/kg) was evidently increased after haloperidol and caerulein treatment (Table 4).
However, tolerance developed to the action of muscimol (1 mg/kg), caerulein (15
Mg/kg) and flumazenil (10 mg/kg). Muscimol and caerulein were not able to
suppress the motor activity of the mice after haloperidol or caerulein
administration. Flumazenil, which increased the motor activity in saline-treated
animals, failed to affect the activity after 15 days of haloperidol or caerulein
treatment.
The prolonged haloperidol and caerulein treatment also affected the binding of
different radioligands to washed brain membranes in a similar way. The density of
[З щ -spiroperidol binding sites in striatum (mainly dopamine2-receptors) was
significantly increased after the administration of both drugs (Table 4). Similar
increase of [^HJ-etorphine (labelling mu-, delta- and kappa-opioid receptors)
binding sites was detected in the mesolimbic structures. Accordingly, our data
suggest that both compounds increase the number of dopamine2-receptors in the
striatum and opioid receptors in the mesolimbic structures. The increased
sensitivity of the mice to the motor stimulating effect of amphetamine, a compound
that increases the release of dopamine, probably reflects the enhancement of
34
dopaminc2-receptors density aller caerulein or haloperidol treatment. Some authors
have demonstrated that opioid receptors play an important role in the regulation ol'
dopamine receptors' sensitivity (Matsuhara, Matsushita, 14X6; Siinus et al., 1986).
It seems probable that the increased sensitivity of opioid receptors is obligatory for
the development of hypersensitivity at dopamine receptors in the mesolimbic area.
Table 4
THE COMPARISON OF LONG-TERM EFFECTS OF HALOPERIDOL AND
CAERULEIN
Haloperidol
Caerulein
tolerance
tolerance
Muscimol-induced
hypolocomtion
tolerance
tolerance
Flumazenil-induced
motor stimulation
tolerance
tolerance
Amphetamine-induced
motor excitation
increase
increase
increased density
increased density
Opioid receptors
in mesolimbic structures
increased density
increased density
CCK-8 receptors
in cerebral cortex
decreased density*
decreased density*
Behavioural effects
Caerulein-induced
hypolocomotion
Radioligand binding studies
Dopamine2-receptors
in striatum
GABAA-benzodiazepine receptors
in cerebral cortex
decreased density
in brainstem
increased density
decreased densit\
increased density
* - increased affinity (decrease of K j values)
Differently from j^H]-spiroperidol and [-^Hj-etorphine binding the number of
Ij-p e n ta g a s trin (a ligand interacting with CCK„/gastrin receptors) binding sites
was evidently decreased, but their affinity was increased in the mouse cerebral
35
5*
cortex (Table 4). The significant reduction of motor depressant effect of caerulein
after haloperidol or caerulein treatment is probably related to the decrease of the
CCK-8 receptor number in the brain. Many behavioural studies now support the
idea that CCK-8 acts as a functional antagonist of dopamine and endogenous opioid
peptides in the brain (Faris et al., 1983; Zetler, 1985; MaLsubara, Matsushita,
1986). Accordingly, the subsensitivity of CCK-8 receptors seems to be necessary
for the development of hypersensitivity at dopamine and opioid receptors. The
changes in [^H]-flumtrazepam and [^H]-muscimol binding were dependent on the
brain region studied. In the cerebral cortex their number was reduced, whereas in
the brainstem the density of [^HJ-flunitrazepam and PH]-muscimol binding sites
was increased after 15-day treatment of haloperidol and caerulein. The similar
alteration of CCK-8 and benzodiazepine-GABAA receptors may be linked to the
finding that CCK-8 and GABA are comediators in the same neurons of the cerebral
cortex and hippocampus (Somogyi et al., 1984). The molecular changes at
benzodiazepine and GABAa receptors are probably associated with tolerance of
behavioural effects of GABAa agonist muscimol and benzodiazepine antagonist
flumazenil. Muscimol did not suppress and flumazenil did not increase the motor
activity of the mice after long-term treatment of haloperidol and caerulein.
In conclusion, the similar actions of haloperidol and caerulein after long-term
treatment seem to be related to the fact that the effects of haloperidol are mediated
not only through dopaminergic, but also via CCK-8-ergic mechanisms. The effect
of CCK-8 seems to be related to the modulation (probably through CCKA
receptors) of the sensitivity of different neurotransmitter receptors (dopamine,
endogenous opioid peptides and GABA).
5.6. Changes at CCK receptors after long-term treatment with haloperidol and
diazepam
Administration of caerulein (15 |ig/kg) to the saline pretreated mice produced an
evident inhibition of the locomotor activity in the animals. After diazepam
withdrawal the motor depressant effect of caerulein was somewhat reduced, as
compared with the saline + diazepam treated mice. But, it was still statistically
significant in comparison with the saline + saline treated group. By contrast,
caerulein was unable to decrease the motor activity of the mice after the long-term
administration of haloperidol (Table 5). The administration of (+)-amphetamine (5
mg/kg) induced a nearly 4-fold increase in the motor activity of the mice as
36
compared wilh ihe saline-treated control mice. The co-administration of caemlein
(100 Mg/kg) with (+)-amphetamine potently antagonized the action of dopamine
agonist. Long-term treatment with haloperidol induced a complete tolerance to the
antagonistic
excitation.
action of caerulein against
Two-weeks
administration
(+)-amphetamine-
of
diazepam
did
induced
not
motor
change
the
antiamphetamine effect of caerulein. There is considerable evidence that the motor
depressant and antiamphetamine effect of CCK agonists (caerulein, CCK-8) are
related to their interaction wilh CCKA receptors in the mouse (Khosla, Crawley,
19X8; Hagino et al.,
1989; Crawley,
1989; O'Neill et al.,
1991). The
intraventricular administration of caerulein (5-50 ng) also decreased the locomotor
activity in the rat. However, after the long-term treatment with haloperidol
caerulein (50 ng icv) significantly increased the motor activity of the animals in the
open-field test. On the other hand, repeated treatment with haloperidol potentiated
the long-term antiamphetamine effect of caerulein and CCKB/gastrin agonist
pentagastrin in the rat. Thus, it is probable that the long-term antiamphetamine
effect of CCK agonists in the rat is related to their interaction with the CCKB
receptor subtype. Accordingly, it is most likely that long-term treatment with
haloperidol causes the subsensitivity at CCKA receptors modulating the activity of
dopaminergic neurons, whereas the CCKB receptor subtype became more sensitized
to the action of CCK agonists. By contrast, repeated administration of diazepam
seems to have only a weak influence on CCKA receptors affecting the activity of
dopaminergic neurons. There exists evidence that long-term haloperidol
administration induced through the indirect interaction with CCKA receptors the
depolarization inactivation of dopamine neurons in the midbrain (Bunney et al.,
1985; Zhang et al., 1991; Minabe et al., 1991). This inactivation of dopaminergic
neurons may explain to some extent the development of subsensitivity at CCKA
receptors after repeated haloperidol treatment. The injection of caerulein at doses
(20-250 |Jg/kg), decreasing the motor activity and blocking (+)-amphetamineinduced motor excitation, inhibited [^HJ-spiroperidol binding in 'in vivo' studies in
the mouse forebrain. The CCK antagonist proglumide (25-50 mg/kg) counteracted
the effect of caerulein. On the other hand, the CCKB/gastrin agonist pentagastrin
(100-2500 pg/kg) was unable to influence [^H]-spiroperidol binding performed in
"in vivo" conditions in the mouse forebrain. It is noteworthy that after long-term
treatment with haloperidol caerulein caused an opposite effect: it stimulated the
binding of [^Hj-spiroperidol in the mouse forebrain. It seems probable that these
changes in [3H]-spiroperidol binding after repeated treatment with haloperidol also
reflect
the
development
of
subsensitivity
at
CCKA
receptors.
37
COMPARISON
OF
LONG-TERM
EFFECTS
OF
DIAZEPAM
AND
HALOPERIDOL ON THE BEHAVIOURAL EFFECTS OF CAERULEIN
AND ON I^HjpCCK-X BINDING IN THE MOUSE BRAIN
Dia/epam
Haloperidol
Complete tolerance
Behavioural effects o f caerulein
Motor depression
Moderate decrease
Aniiaggressive effect
Increased
Anticonvulsant effect
Complete tolerance
Moderate decrease
Antiamphetamine effect
Unchanged
Complete tolerance
Increased
aggressiveness
[3HIpCCK-H binding in the mouse forehrain
Affinity
Increased
Increased
Density
Decreased
Decreased
The injection of 40 ng/kg caerulein to the saline-treated control animals induced
the statistically significant reduction of aggressive behaviour. On the contrary, after
long-term treatment with haloperidol and diazepam, caerulein markedly increased
ihe intensity of aggressive behaviour, especially the number of biting attacks was
increased (Table 5). The antiaggressive effect of caerulein was reversed after 14
days administration of haloperidol and diazepam. The increased aggressiveness
induced by caerulein is in agreement with the above described studies where the
intraventricular administration of a CCKB agonist CCK-4 (0.2 jig per animal)
induced very dramatic potentiation of foot-shock-induced aggressiveness in the
male rat. Moreover, the subcutaneous administration of CCK-4 (25-50 pg/kg)
induced the anxiogenic-like interaction with the exploratory behaviour in the rat,
and this effect had clearly the CCKB receptor subtype pharmacology (Harro, Vasar,
1991). Some evidence exists that CCKH receptors are involved in the mediation of
anxiety-!ike states in the mouse (Hughes et al., 1990; Rataud et al., 1991).
Therefore, it is likely that the increased aggressiveness induced by caerulein, after
two-weeks administration of haloperidol and dia/epasn. could be explained by the
increased sensitivity of the CCK,, receptor subtype.
The systemic treatment with caerulein is shown to antagonize ihe seizures
induced by picrotoxin, an antagonist at chloride channel, in the mouse (Zeller,
19X5).
Two-weeks
treatment
with
haloperidol
moderately
reduced
the
anticonvulsant effect of caerulein, especially the latency to death was shorter, as
compared with the saline-treated control mice (Table 5). After two-weeks
administration of diazepam caerulein was unable to affect the development of
picrotoxin- induced seizures in the mice. The site of caerulein's interaction with
picrotoxin-induced seizures is still unclear. However, the development of tolerance
to the antipicrotoxin effect of caerulein alter repeated administration of diazepam
may support the involvement of GABA-ergic mechanisms in the action of CCK
agonist. Harro et al. (1990a) have established that two-weeks administration of
diazepam induced tolerance to the anxiogenic-like effect of caerulein. These
behavioural data are in accordance with the electrophysiological study of
Bouthillier and De Montigny (1988), showing that long-term treatment with
diazepam significantly reduced the responsiveness of hippocampal pyramidal
neurons to the application of CCK. There is a possibility that the anti- picrotoxin
effect of CCK agonists is related to the CCKB receptor subtype. Indeed, the nearly
similar doses of sulfated CCK-8 and unsulfated CCK-8, the selective agonist at
CCKB receptors, antagonized picrotoxin-induced seizures in the mouse (Kadar et
a l, 1985). It is possible that CCK receptors, functionally linked to the GABA-ergic
system, are involved in the anti-pierotoxin action of caerulein. Probably, long-term
treatment with diazepam induces the subsensitivity at these CCK receptors.
Haloperidol and diazepam (up to 1 mM) failed to inhibit [^H]pCCK-8 binding
in "in vitro" studies. Acute administration of haloperidol (1 mg/kg) and diazepam
(5 mg/kg) did not influence [Зн]рССК-8 binding in the mouse forebrain.
However, alter the cessation of long-term treatment with diazepam and haloperidol
the parameters of [^H]pCCK-8 binding sites were significantly changed (Table 5).
Namely, the affinity of [^H]pCCK-8 binding sites was increased (Kd values were
decreased), but their number was evidently decreased in the mouse forebrain.
Long-term treatment with haloperidol as well as with diazepam is shown to
increase the density of CCK receptors in the forebrain structures of rodents (Chang
et al., 1983; Harro et al., 1990b). In the light of these findings the data of the
present study are really unexpected. Indeed, two-weeks haloperidol and diazepam
treatment significantly decreased the density of [^HlpCCK-8 binding sites, but
simultaneously increased their affinity. The careful analysis of dissociation curves
for pH]pCCK-8 and [ '^IIC C K -K revealed the existence of two distinct binding
sites for CCK in the different brain structures of the rodents (Wennogle et al.,
39
1985; Sekiguchi, Moroji, 1986). It might he possible that long-term diazepam and
haloperidol treatment differently alters these subtypes of the CCK receptor. Thus,
one could speculate that the high-affinity binding sites for |^H|pCCK-8 started to
prevail over the low-affinity sites after long-term treatment with haloperidol and
diazepam.
Long-term treatment with diazepam and haloperidol reduced or induced
tolerance to the inhibiting effects of caerulein on the mouse behaviour.
Simultaneously, a clear proaggressive action of CCK agonist became evident al ter
the cessation of haloperidol and diazepam administration. These behavioural
changes seem to be in favour of the opinion that the subsensitivity developes at one
subtype of CCK receptors, whereas the other subtype of CCK receptors becomes
more sensitized to the action of caerulein. There is evidence (the development of
tolerance to the sedative and antiamphetamine effect, but also to the inhibiting
effect of caerulein on [^H]-spiroperidol binding in 'in vivo') that the sensitivity of
CCKA receptors is decreased after two-weeks haloperidol treatment. By contrast,
the changes occurring at the CCKB receptor subtype during long-term haloperidol
and diazepam treatment are still less clear. Nonetheless, the possibility remains that
the hypersensitivity develops at one part of CCKB receptors (behaviourally related
to the regulation of aggressive behaviour). This idea is supported by the increased
affinity of CCKB receptors in the mouse forebrain after long-term administration of
haloperidol and diazepam.
40
6. CONCLUSIONS
1. The unselective ССКЛ/ССК„ agonists caerulein and CCK-8. bul nol the CCKH
agonists pentagastrin and CCK-4, dose-dc|>endcnlly inhibited the locomotor
activity of rodents. A selective ССКЛ receptor antagonist devazepide attenuated the
motor depressant effect of CCK agonists, whereas a selective CCK,, receptor
antagonist L-365,260 potentiated their action. Therefore. ССКЛ and CCKB
receptors have the opposite role in the regulation of motor activity in the rodents.
2.
Caerulein,
but
not
pentagastrin,
antagonized
(+)-amphetamine-induced
hyperlocomotion in the mice. Pretreatment with devazepide at low doses (1-100
pg/kg), interacting with CCKA receptors, blocked the antiampheiamine effect of
caerulein. Accordingly, the antiamphetamine effect of caerulein is related to the
stimulation the CCKA receptor subtype in the brain. The blockade of both subtypes
of the CCK receptor by a high dose (1 mg/kg) of devazepide completely blocked
the locomotor stimulation induced by (+)-amphetaminc.
3. CCK agonists (caerulein, CCK-8, pentagastrin, CCK-4) reduced the exploratory
activity of the rodents in the elevated plus-maze. The anxiogenic-like effect of
CCK agonists correlated with their affinity at C’CKB receptors in the cerebral
cortex, but not at CCKA receptors in the pancreas. L-365,260 was a stronger
antagonist of anti-exploratory action of CCK-4 as compared with devazepide.
Thus, the CCKB receptor subtype is involved in the anti-exploratory effect of CCK
agonists.
4. Systemic treatment with the unselective CCKA/CCKB agonist caerulein, but not
with the CCKB/gastrin agonist pentagastrin, blocked the foot-shock-elicited
aggressiveness in the mouse and antagonized the development of apomorphine
aggressiveness in the ra t Intraventricular and subcutaneous administration of the
CCKB agonist CCK-4, differently from caerulein, increased the intensity of footshock-induced aggressiveness in male rats. Consequently, the CCKA and CCKB
receptor subtypes are play an opposite role in the regulation of aggressive
behaviour in the rodents.
5. Systemic treatment with caerulcin and CCK-8, but not with pentagastrin,
significantly antagonized picrotoxin- and pilocarpine-induced seizures in the
mouse. Intraventricular, but not subcutaneous, administration of caerulein potently
blocked quinolinate- and NMDA-induced seizures. Proglumide attenuated the
41
6
anticonvulsant effect of caerulein against picrotoxin- and quinolinate-induced
convulsions. The selective CCK antagonists L-365,260 and devazepide at a high
dose (1 mg/kg) blocked the anticonvulsant effect of CCK-8 against pilocarpine
seizures. Accordingly, both subtypes of CCK receptors are related to the
anticonvulsant action of CCK agonists.
6. Long-term treatment with devazepide and L365.260 induced different changes in
the behaviour and in the l^H]pCCK-8 binding in the mouse forebrain. Repeated
administration of devazepide reduced the motor depressant effect of caerulein and
increased (+)-amphetamine-induced hyperlocomotion in the mice. L-365,260 failed
to change the behavioural effects of caerulein and (+)-amphetamine, but
significantly increased the density of CCK receptors. These data obviously support
the involvement of the CCKA receptor subtype in the sedative and antiamphetamine
action of caerulein.
7. Long-term treatment with haloperidol and caerulein caused very similar changes
in the mice's behaviour and [^H]-radioligand binding in the mouse forebrain.
Tolerance developed to the locomotor effects of caerulein, muscimol and
flumazenil, whereas amphetamine-induced hyperlocomotion was increased after
15-days haloperidol and caerulein treatment. Simultaneously, the number of opioid
and dopamine2-receptors was increased, however, the density of GABAabenzodiazepine and CCK-8 receptors was reduced in the mouse forebrain. It is
most likely that CCKAreceptors play a role in the long-term effects of haloperidol.
8. Long-term diazepam and haloperidol treatment decreased or induced tolerance to
the inhibiting effects (motor depressant, antiamphetamine, anticonvulsant and
antiaggressive) of caerulein on the mouse behaviour. Simultaneously, the
proaggressive action of caerulein became evident. The number of [Зщ рССК-8
binding sites was decreased, whereas their affinity was increased after the
withdrawal of long-term haloperidol and diazepam treatment. Therefore, long-term
administration of diazepam and haloperidol induced the subsensitivity at one
subtype of the CCK receptor (mainly the CCKA subtype), whereas the others
(mainly the CCKB subtype) became more sensitized to the action of CCK agonists.
42
ACKNOWLEDGEMENTS
This study was performed at the Psychopharmacology Laboratory, Institute of
General & Molecular Pathology o f Tartu University.
I wish to express my deepest gratitude and respect to all colleagues and friends who
made this study possible:
My teacher, Professor Lembit Allikmets for his constant guidance and support
throughout this work.
My very close co-workers, Doctors Jaanus Harro, Aavo Lang, Andres Soosaar,
Anu Pflld and Tiina Ööpik for a very inspiring and enthusiastic collaboration,
and for valuable help in every way.
Professor Brian S. Meldrum and John D. Stephenson, for a stimulating
collaboration and for providing possibility to work at the Department of
Neurology, Institute of Psychiatry, University of London.
Doctor Susan D. Iversen and Doctor Colin T. Dourish (Merck Sharp & Dohme),
for the generous gift of CCK antagonists.
The financial support from the British Council is gratefully acknowledged.
Finally, my warmest and most sincere thanks go to my family and my parents, for
interest, encouragement and support.
4'
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somatostatin or cholecystokinin-im m unoreactive material. N euroscience, 14: 2590-2603.
82 Soosaar, A., Vasar, E. and Lang E (1988) The parameters o f cholecystokinin receptors in
50
dif ferent brain structures o f the rat A cla et Commentationes Universitatis Tartuensis, 839: 95-108.
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different neuroleptics elicits behavioural supersensitivity
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infusion
into nucleus
accumbens. Biol.Psychiatry 21;34-48.
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doses o f apomorphine. J.Neural Transm. 40: 191-194.
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52
PUBLICATIONS
Ф И З И О Л О Г И Ч Е С К И Й Ж У Р Н А Л СССР им. //. М. СЕЧЕ НО Ih
S E C H E N O V P H Y S I O LOGICAL J O U H N a J T ü F
L X V I I I ■ № 9 ■ 1082
1
THE U s s i l
y;u( crj.sji.t
И Н ТРА Ц ЕРЕВРО ВЕ И Т РИ К У Л Я РП О Е В В Е Д Е Н И Е
ХОЛЕЦИСТОКИНИИЛ УГНЕТАЕТ АКТИВНОСТЬ
ДОФАМИН- И СЕРОТОИИНЕРГИЧЕСКОЙ СИСТЕМ МОЗГА
Э. Э. Васар, М. Я. Оттер и Л. Ii. Ряго
Кафедры физпологпн (зав. Э. Ф. Васар) и фармакологии (зав. Л . X . Аллпкметс)
Государственного университета, Тарту
В опытах па крысах-самцах лпнпи Впстар псследопали поведенческие п биохимическиеаффекты интрацеребровентрикулярного введения холецистокинина. Холецистокиннн вызывал
специфические, зависящие от дозы, изменения в поведении животных. При малых его дозах
превалировало угнетающее влияние на поведение, при больших дозах паблюдалпсь стерео­
типное поведение, встряхивания головой и повышенная реактивность иа болевые раздраж и­
тели. Холецистокиннн заметно угнетал по сравнению с физиологическим раствором круго­
оборот серотонина о дофамина в мозговых структурах. Введение холецистокинина на фоне
фенамина и 5-окситриптофана на короткое время полностью устраняло вызываемые этими
веществами поведенческие эффекты. На основе полученных данных можно предполагать,
что холецистокиннн является эндогенным модулятором активности моноаминергическнх
систем мозга.
К лю чевые слова: холецистокиннн, стереотипное поведение, кругооборот моноаминов,
фенамин, 5-окситриптофан.
В последние годы установлено [5> 10, 12], что холецистокиннн, кроме ж елу­
дочно-кишечного тракта, в значительных количествах содержится в разных
структурах головного мозга. Особенно высокие концентрации этого пептида и
его фрагментов выявлены в коре больших полушарий, лимбических структу­
рах, гипоталамусе и стриатуме [13], т. е. в структурах, получающих обильную
афферентную иннервацию от моноаминергическнх систем мозга. Fuxe и соавт. [91
вашли, что холецистокиннн в малых дозах угнетает кругооборот дофамина
в передних ядрах стриатума и прилегающем ядре. О функциональной роли
холецистокинина в центральной нервной системе пока известно относительна
мало. Некоторые авторы связывают его функцию с регуляцией пищепого по­
ведения [4], причем, по их мнению, его действие опосредуется через серотонинергические механизмы.
Основываясь на вышеизложенных фактах, в настоящей работе поставлена
задача исследовать поведенческие эффекты интрацеребровентрикулярного вве­
дения холецистокинина, а также его влияние на центральные серотонин- и
дофаминергические процессы.
МЕТОДИКА
Опыты проводились на крысах самцах линии Вистар (массой 250—300 г). Под эфирным
наркозом по координатам атласа Фвфковой и Маршала [1] в черепе просверливались отвер­
стия и спустя 4—5 ч после этого животвые брались в опыт. Холецвстокинин (фирма «Boots»,
Англия) рааводили в физиологическом растворе. Через билатеральные отверстия пептид
в общем объеме 10 мкл вводили в течевие 60 с в боковые желудочки мозга. Контролем слу­
ж или инъекции физиологического раствора в том ж е объеме. Холецвстокинин вводился
в равных дозах — от 0.1 до 8 Ед по Ivy. Сразу после ввутрвмозговых инъекций животных
пом етали в клетку размерами 6 0 X 6 0 x 3 5 см и наблюдали за их спонтанным поведением
и реакцией на внешние раздражители. Спустя 10 мин после введения холецистокинина
в дозе 0.1, 1, 4 Ед п ов од и л и сь также биохимические исследования. По методике Earley
и Leonaid [6] определяли и Е м е н е н г я в содержании дефамива и его главных метаболитов —
1218
гомованвлиновой кислоты и 3,4-диокспфенилуксусной кислоты в стриатуме и лимбических
структурах. Содержание серотонипа и его метаболита 5-оксииидолуксусной кислоты опре­
делялись в черном веществе и стволе мозга по методике Curzon, Green | SJ. Все яти определе­
ния проводились с помощью флуоресцентного спектрофотометра МПФ-2А фирмы «Хитачи».
В отдельной серии опытов исследовалось влияние средней дозы холпцистокипппа (1 Ед)
на поведенческие аффекты фенамина (2.5 мг/кг) и 5-окситринтофана (150 мг/кг), влияющих
на активность дофамин- и серотопинергнческой системы через пресипаитические м еха­
низмы I14, и ). Внутрижелудочковыо введения физиологического раствора пли холецистокпнина проводили через 30 мин после подкожной инъекции фенамина. Стереотипию оцени­
вали по методике, предложенной C ostall, N aylor |*Jt перед внутримозговым введением и
спустя 7 мин после введения. В опытах с 5-оксптринтофаиом холецистокинин и физиологи­
ческий раствор вводились через 45 мин поело предшественника серотонина. Число встряхи­
ваний головой подсчитывалось и период от 3 до 10 мин после внутр»желудочковых инъекций.
Все экспериментальные дапныо подвергались статистической обработке с использованием
t -теста Стьюдента.
РЕЗУЛ ЬТЛТЫ
ИССЛ ЕДОВЛ НИ Я
Интрацсребровентрикулярное введение холецистокинина вызывало опре­
деленные изменения в поведении животных, находившихся в тесной корреля­
ции с вводимой дозой. Доза 0.1 Е д it о т л и ч и е от физиологического раствора
вызывала полное прекращение д в и г а т е л ь н о й а к т и в н о с т и и заметную сонли­
вость у некоторых животных. С п о в ы ш ен и ем д о зы через 3 — 4 мин после вве­
дения появлялись повороты т ел а в с т о р о н у п е р в о й инъекции, а затем стерео­
типные движения в виде п р и н ю х и в а н и я и ж е в а н и я в течение 3 — 4 м и н . После
Вшишю (1нграцэребровенгр'.1КУЛЯ|)1|.1п> н > дем ш \ >
ги'с.пнша на содержание дофамина,
гомовшилиповой кислоты, 3 ,4 -диокс11фен11.|ук суг| 1.>|| кислоты ii стриатуме и лимбических
структурах; серотонина и 5-океииндолукч-уснои k m c . i o i u и черном вещестпе и стполе мозга
крысы (приведены средние данные и мкг на I г тк ан и мин а и достоверные границы к ним)
Л с ш е с п и , л ица
Д оф ам ин
Интактные жи­
вотные
Контроль
Холецистоки­
нин в дозе:
0.1 Ед
1 Ед
4 Ед
Г о м п в а н и .н ш о в а н к и с л о т а
отриатум
лим бические
структур«
етрнатум
6.64 ± 0.38
2.9« M US
0.40 ± 0 .0 6
0.55 ± 0.06
9.13 ± 0.42
2.27 ± 0.26
1.44 ±0.12
1.08 ± 0 .1 2
7.36 ±0.36*
6.77 ±0.31*
6.03 +0.32*
2.30 + 0 .3 0
2.72 + 0.18
2.89 ± 0.31
1.5 3 + 0 .1 2
0.99 + 0.11 *
1.00 + 0.08 *
<1.85 J 0.09
0 .9 0 + 0 .1 2
0.54 +0.04*
лим бические
структуры
П родиллсение
В ещ ество , д о за
3 ,4 -л и о к с 1 1 ф с н и л у к с у с н а и
ки слота
стриатум
лим бические
структуры
С еротони н
черное
вещ ество
ств о л м овга
5 -о к с и и н л о л у к с у с н а я
кислота
черное
вещ ество
Интактные жи­
вотные
0.78 ± 0 .0 5
0.56 ± 0 .0 5
1.81 ± 0 .1 8
0.70 ± 0 .0 4
Контроль
Холецистокинин в дозе:
0.1 Ед
1 Ед
4 Ед
1.10 ± 0.11
1.09 ± 0 .0 9
5.12 ± 0.45
0.77 ± 0 .0 7
1.70 + 0.16
1.28 ± 0 .1 0
0.79 ±0.09*
0.67 + 0.07
0.66 +0.07*
0.6!) ±0.08*
0.47 +0.06*
2.24 +0.22*
2.09 +0.25*
2.44 + 0.22*
0.77 + 0.04
0 .8 8 + 0 .0 8
0.81 + 0.07
1.51 +0.19
1.39 fO.15
0.78 +0.04*
/1 0
м и л ),
” 0 |,Т Р '>ЛЬ - “ " ’'р а ч о р е б р о в в и т р и и у л н р ч и е
— д остоверное р азл и ч и е с к о н тр о л ем п р и р < 0 .0 5 .
введении
0.86 ± 0 .0 7
ство л м овга
0 .5 4 + 0 .0 4
0 .7 4 + 0 .0 8
0.61 + 0.06
0.54 + 0.04*
0.52 +0.03*
ф и зи ологического
а1
раствора
1219
э т о г о д в и г а т е л ь н ы е р е а к ц и и п о л н о с т ь ю п р е к р а щ а л и с ь и у ч а ст и кры с р а з в и л а л а с ь с о н л и в о с т ь . В д о з а х 4 и 8 К д х о л е ц и с т о к и н и н в ы зы в а л п о р о д п о я в л е н и е м
у к а з а н н ы х с т е р е о т и п н ы х р е а к ц и й к р а т к о в р ем ен н ы е в с т р я х и в а н и я г о л о в о й .
Р е а к ц и я ж и в о т н ы х на б о л е в ы е р а з д р а ж е н и я н а х о д и л а с ь в п р я м о й к о р р е л я ц и и
с в в о д и м о й д о з о й п е п т и д а . В м ал ы х д о з а х он а н е о т л и ч а л а с ь от т а к о в о й у ж и ­
в о т н ы х к о н т р о л ь н о й г р у п п ы , о д н а к о бы л а н и ж е п о с р а в н е н и ю с н н т а к т н ы м и
к р ы са м и . П о с л е в н у т р и м о з г о в о г о в в е д е н и я х о л е ц и с т о к и н и н а в д о з е 4 и л и 8 Е д
в т о ч ен и е 1 ч н а б л ю д а л а с ь б у р н а я р е а к ц и я н а б о л е в ы е р а з д р а ж е н и я , п р и э т о м
м еж д у ж ивотны м и л егк о п р ов оц и р ов а­
лись драк и.
В в еден и е ф и зи ол оги ч еск ого р аств ора
(см . т а б л и ц у ) в зн а ч и т е л ь н о й с т е п е п и
повы ш ало п о ср а в п ен и ю с интактны м и ж ивотны м и си н тез и к р уго о бо р от
Р ис. 1. Интенсивность реакции на введе­
ние фенамина (2.5 мг/кг) у крыс после
интрацеребровентрикулярного
введения
физиологического раствора и холецистокинина (1 Бд).
Рис. 2. Число встряхиваний головой, вызван­
ных 5-окситрнптофаном (150 мг/кг) у интактных животных и после интрацеребровентрикулярного введения физиологического рас­
твора и холецистокинина (1 Ед).
П о оси о р д и н а т — и н т ен си вн о ст ь стер ео т и п н о го
поведен ия, в
баллах.
С т о л б и к и : б е лы е — д о ,
»^ш т рихованны е — п о сл е
введен и я
ф и зи ологи ­
ческого
раствора
( р < 0 .0 5 ),
черны е — п о сл е
в в е д е н и я х о л е ц и с т о к и н и н а ( р < 0 .0 1 ).
П о оси о р д и н а т — ч и с л о в с т р я х и в а н и й го л о в о й .
С т о л б и к и : б елы е — и н т а к т н ы е ж и в о т н ы е , » a iu m p u ^
хованны е — п о сл е вв ед ен и я ф и зи ологи ческого р ас­
т в о р а , черны е — п о с л е в в е д е н и я х о л е ц и с т о к и н и н а
(РС0.01).
моноаминов во всех нами исследованных мозговых структурах. Холецисто­
кинин в зависимости от дозы угнетал синтез и оборот моноаминов, причем
в дозе 4 Ед отмечалось снижение содержания моноаминов и их метаболитов
до уровня, наблюдаемого у интактных животных. Особенно заметными были
эффекты холецистокинина в лимбических структурах (кругооборот дофамина)
и черном веществе (кругооборот серотонина), причем в этих структурах д ей ­
ствие пептида проявлялось уж е в дозе 0.1 Ед.
При интрацеребровентрикулярном введении физиологического раствора
(рис. 1) через 30 мин после инъекции фенамина (2.5 мг/кг) вначале стерео­
типные двигательные реакции прекращались, а затем в течение 8 — 10 мин
наблюдалось достоверное усиление их интенсивности. Холецистокинин (1 Ед)
на 10—12 мин полностью устранял все признаки действия фенамина у всех
подопытных животных. Введение физиологического раствора (рис. 2) через
45 мин после инъекции 5-окситриптофана (150 мг/кг) ВЕ1чале также несколько
уменьшало число встряхиваний головой и интенсивность тремора* однако по-
1220
том эти проявления становились более интенсивными по сравпению с интактными животными. Холецистокиннн в дозе 1 Ед на 10—12 мин полпостью бло­
кировал встряхивания головой у всех животных.
ОБСУЖ ДЕНИЕ
РЕЗУЛЬТАТО В
Полученные результаты показывают, что холецистокинип вызывает специ­
фические сдвиги в поведении животных, и эти сдвиги находятся в тесной кор
реляции с вводимой дозой. После введения малых доз наблюдается седативное
действие, а более высокие дозы вызывают усилепне двигательных реакций
(повороты тела в сторону первой инъекции и стереотипные движения). При
д о за з 4 и 8 Ед перед стереотипными реакциями возникают встряхивания го­
ловой. Zetler [16] установил, что холецистокипин обладает болеутоляющим
действием, причем данный эффект устраняется введением налоксона — опиат
ного антагониста в очень малых дозах. Однако наши результаты, напротив,
показывают, что после введения холецистокипин» в больших дозах болевые
раздражения могут вызвать бурпые реакции и даж е агрессивность — эффекты,
во многом напоминающие действие дофаминомиметиков. Холецистокиннн
угнетает по сравнению с физиологическим раствором метаболизм дофамина
и серотонина, что особенно выражено в лимбических структурах и черном
веществе. В тесной корреляции с биохимическими данными находится влия­
ние холецистокинина на поведенческие эффекты фенамина и 5-окситриптофана, повышающих активность дофамин- и серотонинергической систем через
пресинаптические механизмы [14, 16]. Холецистокиннн в отличие от физиоло­
гического раствора на 10— 12 мин устраняет действие обоих веществ. Все эти
данные свидетельствуют о том, что под влиянием холецистокинина резко по­
нижается активность пресинаптических механизмов дофамин- и серотонинер­
гической систем. Однако наряду со снижением активности пресинаптических
механизмов наблюдается повышение чувствительности постсинаптических ре
цепторов этих нейромедиаторных систем, о чем свидетельствуют поведенче­
ские эффекты после интрацеребровентрикулярного введения холецистокинина
(встряхивания головой, повороты тела, стереотипные движения и повышенная
реактивность на болевые раздражители). В пользу этого мнения указывают и
наши предыдущие исследования, где холецистокиннн заметно потенцировал
1интенсивность апоморфиновой стереотипии у крыс, т. е. повышал чувствитель­
ность постсинаптических дофаминовых рецепторов. Аналогичные резуль­
таты получили K ovacs и соавт. Iй ], которые нашли, что холецистокиннн зна­
чительно усиливает синдром «лазанья», вызываемого апоморфином у мышей.
Исследованиями последних лет установлено Iе’ 7], что в передних мозго­
вых структурах холецистокиннн содержится в форме СООН-концевого окта­
пептида. Вполне возможно, что наблюдаемые нами модулирующие влияния
на дофамин- и серотонинергические процессы реализуются именно через ре­
цепторные механизмы, связанные с холецистокинином.
Л И ТЕРАТУРА
(1J B u r e s J
Pe t r a n М ., Z a ch a r I . Electrophysiological m ethods in biological research.
Prague, 1967.
[2] C o sta ll B . , N a y lo r R . Stereotyped and circling behavior induced by dopam inergic
agonists after lesions of the midbrain raphe nuclei. Eur. J. Pharm acol., 1974, 2 9 , 206—222.
[3] C v rzo n G ., Green A . R . Rapid m ethod for the determ ination of 5-hydroxy-tryptam ine
and 5-hydroxyindolacetic acid in sm all regions of rat brain. B rit. J. Pharm acol., 1970, 3 9 ,
653—655.
[4] D a jn y N . , J a cobson E . D . C holecystokinin and central nervous regulation о / appetite.
In: G astrointestinal Hormones. A Sym posium . A ustin — London, U niv. T exas Press, 1975.
643—649.
[5] D o c k ra y G. J , I ii m unochem ical evidence of cholecystokinin-like peptides in brain.
Nature, 1976, 2 6 4 , 5 6 8 — 570.
[6J D o c k ra y G. J . , Gregory R . A . , H u tc h is o n J . B . , H a r r is J . / . , R u n s w ic k M . J . Isolation,
structure and biological a ctiv ity of two cholecystokinin octapeptides from sheep brain. Nature.
1978, 2 7 4 , 711— 713.
(7)
D o c k ra y G. J . C holecystokinins in rat cerebral cortex: id en tifica tio n , purification and
characterization by im m unochem ical methods. Brain R es., 1980, 1 8 8 , 155— 165.
1221
8
[8] E a r le y С . J . , L e o n a r d В . E . Isolation and assay of noradrenaline, dopi.ra.ne, 5-bydroxytryptam ine and several m etabolites from brain tissue using disposable Bio-Rad colum ns packod
witn Sephadex G-10. J. Pharmacol. Methods, 1978, 1 , 67—79.
[9] F u x e K ., A n d e rsso n K . , L o c a te lli V ., A g n a t i L . F ., I lö k f e l t Т ., S k ir b o ll L . , M a tt V'.
Cholecystokinin peptides produce marked reduction of dopam ine turnover in discrete areas
in rat brain follow ing in Ira ventricular injection. Eur. J. Pharm acol., 1080. 6 7 , 329—331.
[10] I n n i s R . B . , Correa F . M ., U h l G. R . , S c h n e id e r B . , S n y d e r S . C holrcystokiniu octapeptide-like im m unoreactivity histochem ical localization in rat brain. Proc. N at. Acad.
Sei. USA, 1979, 7 6 , 5 2 1 - 5 2 5 .
[11] K o v a c i G. L . , S za b o G ., Р е п ке В ., T e leg d y G. E ffects of cholecystokinin octap ep tid e
on striatal dopamine m etabolism and on apomorphino-indueed stereotyped cage-clim b in g
in m ice. Eur. I. Pharm acol., 1981, 6 9 , 313—320.
[12] R e h fe ld J . F . C holecystokinins and gastrins in brain and gut. Acta Pharra. T o x ico l.,
1977, 41, 24.
[13] S a ito A . , S a n k a r a n H . , G o ld fin e / . , W illia m s J . A . C holecystokinin receptors in the
brain: characterization and distribution. Science, 1980, 20 8 , 1155—1156.
[14] S lo v ite r R . S . , D r u s t E . G ., C o n n o r J . D . Serotonin agonist actions of p-chlorophenylalanine. Neuropharmacol., 1978, 1 7 , 1029—1033.
[15] T a y lo r R . , S n y d e r S . D ifferential effects of d- and 1-amphetamine on behavior and
on catecholam ine disposition in dopamine and norepinephrine containing neurons of rat brain.
Brain R es., 1971, 2 8 , 2 5 9 - 3 0 9 .
[16] Z e tle r G. A nalgesia and ptosis caused by caerulein and cholecystokinin octapeptide
(CCK-8). Neuropharmacol., 1980, 19, 415—422.
Поступило 13 VII 1981
INTRA VENTRICULAR ADM IN ISTR A TIO N OF CHOLECYSTOKININ
DECREASES TH E ACTIVITY OF DOPAM INE- AND S E R O rO N IN E R G IC
SYSTEMS IN THE BR AIN
E . E . Vasar, M . Y a . O tter and L . K .
Rägo
State U niversity, Tartu
In male W istar rats, intraventricular adm inistration of cholecystokinin caused specific
dose-dependent behavioral changes: low doses (0.1—0.25 U) depressed the exploratory a c tiv ity
whereas higher doses (0.5—8.0 U) caused head tw itch es, stereotyped gnawing and hyperreacti­
v ity . C holecystokinin suppressing markedly dopamine and serotonin turnover in various brain
structures, com pletely blocked the behavioral effects of am phetam ine (2.5 m g/kg) and 5-hydroxytryptophan (150 m g/kg). The data obtained suggest that cholecystokinin suppresses presynaptic
dopamine- and serotoninergic mechanisms but ehances the sen sitiv ity of postsynaptic receptors
of these system s.
V O L. 20. N O . 4. К А П . 1У84
Caerulein Stimulates [3H]-Spiperone
Binding in vivo After Long-Term
Haloperidol Administration
Eero Vasar, M .D .,' Matti Maimets,
M .D .,1 Ants Nurk, M .D .,1 and Lembit
Allikmets, D.Sc.
R ecently th e in v itro m o d u la tio n o f affin ity
a n d d e n s ity o f n e u ro le p tic b in d in g sites in
str ia tu m by c h o le c y sto k in in o c ta p e p tid e (CCK 8) was d e s c rib e d (A g n a ti et al., 1983). C C K -8
in c re a se d th e a ffin ity o f n e u ro le p tic s to its
b in d in g sites b u t r e d u c e d th e ir d e n sity . C o n ­
c o m ita n t u se o f d iff e re n t C C K -re la te d p e p ­
tid es w ith n e u ro le p tic d r u g s in n e u ro le p tic re s is ta n t s c h iz o p h re n ic p a tie n ts was r e p o r te d
to r e d u c e th e in te n s ity o f d ise a se s y m p to m a ­
tology (M oroji e t al., 1982; N a ir et al., 1982).
A c c o rd in g to th e p ro p o s e d h y p o th esis, CC K 8 a n d re la te d c o m p o u n d s m ay b e a c tin g by
p o te n tia tin g th e e ffe c t o f n e u ro le p tic d r u g s in
n e u ro le p tic -r e s is ta n t p a tie n ts (B lo o m e t al.,
1983). In th e p r e s e n t w o rk an a tte m p t to c la r­
ify th e p o ssible m e c h a n ism o f in te ra c tio n b e ­
tw e e n c a e ru le in , an a n a lo g u e o f C C K -8, a n d
n e u ro le p tic d ru g s, with th e he lp o f in vivo [:'H ]s p ip e r o n e (:,H -S P I) b in d in g a fte r lo n g -te rm
h a lo p e rid o l tre a tm e n t was d o n e .
A c c o rd in g to th e stu d ie s o f B a tta g lia a n d
T ite le r (1982), th e r e a re tw o e x istin g b in d in g
sites (w ith low a n d h ig h a ffinity ) fo r N -p ro p y ln o r a p o m o r p h in e (N PA ) o n d o p a m in e -j-re c e p to rs, w h e re a s n e u ro le p tic d r u g s possess h ig h
affin ity fo r b o th . T o s tu d y th e p ossible c h a n g e s
in do p a m in e - 2 - a n d s e ro to n in -j-re c e p to r s u b ­
p o p u la tio n s, N P A as a d isp la c e r in d iff e re n t
d oses was used.
M ale m ice w e ig h in g 25-27 g w ere u se d . In t r a p e r i t o n e a l h a lo p e r id o l ( G e d e o n R ic h te r,
H u n g a ry ) 0.25 m g /k g tw ice daily o r salin e in ­
je c tio n s w e re g iven fo r 14 days. S ev en ty -tw o
h o u rs a fte r w ith d ra w a l, in vivo 'H -S P I (A in ers h a m I n te r n a tio n a l, U .K ., 17 C i/m m o le) b in d ­
11nstitute ol General and Molecular Pathology. Tartu State University, Tartu, Estonia, U.S.S.R.
8*
691
ing studies were performed. NPA (5 and 50
ц-g/kg, Sterling-Winthrop, USA) and caerulein
(200 ц-g/kg, gift from Prof. R. De Gastiglione,
Farmitalia, Italy) were injected subcutaneously. Five ц-g/kg NPA caused suppression o f
motor activity whereas 50 ц-g/kg induced ster­
eotyped behavior in control animals. *H-SPI
(5 ц-g/kg SC) followed 15 min after NPA and
caerulein. T he animals (6 per group) were sac­
rificed 20 min after 3H-SPI. T he brains were
rapidly removed, and frontal cortex and forebrain subcortical structures (limbic structures
and striata) dissected on ice. The dissected brain
areas o f each group were pooled and hom og­
enized in glass-teflon hom ogenizer by hand in
ice-cold Tris HC1 (50 mM, pH 7.4, 20°C) within
1 min in the volume o f 40 mg tissue per ml.
After homogenization 0.5 ml o f suspension was
pipetted into six polypropylene tubes (1.5 ml)
and centrifuged at 9000 r.p.m. during 10 min.
T he supernatant was discarded and pellet
washed four times with 1 ml ice-cold buffer
and cut into vials. Radioactivity was counted
after stabilization in Bray scintillation cocktail
12 hours in Beckman LS 6800 with counting
efficacy 43%. The experim ent was repeated
three times and the data analyzed using Stu­
dent’s (-test.
In the figure, inhibition o f SH-SPI binding
by NPA 5(j.g/kg was expressed as 100%, show­
ing the amount o f high affinity sites for NPA.
As the higher dose o f NPA could inhibit 3HSPI binding to both high and low affinity sites,
the difference between 50 and 5 ц-g/kg NPA
shows the number o f low affinity sites. After
2 weeks’ saline treatment the ’H-SPI displac­
ing potency o f 5 ц-g/kg was lower than the ac­
tion o f 50 ц-g/kg NPA Caerulein inhibited 5HSPI binding in subcortex and in frontal cortex.
After chronic haloperidol 50 (Ag/kg NPA more
readily inhibited SH-SPI binding, whereas the
displacing potency o f 5 ц-g/kg NPA was sig­
nificantly reduced. Caerulein, in contrast to
the inhibition o f ^H-SPI binding in chronic
saline group, stimulated it after chronic halo­
peridol.
It was concluded that long-term haloperidol
treatment increased the affinity o f 3H-SPI to
692
P S Y C H O P H A K M A C Ü l.O G Y ü U L l . t I IN
number o f CCK binding sites h a s b e e n shown
to increase twice after chronic neuroleptic
treatment (Chang et al.. 1983). T h e present
work supports the stu d y o f Agnati et al. (1983)
showing the ability o f CCK-8 to increase the
affinity o f neuroleptics to its binding sites. It
seems that the same mechanism m ay be in ­
volved in the beneficial action o f CCK-related
p ep tid es in n eu ro le p tic-resista n t sch izo ­
phrenic patients.
F IG U R E . T h e e ffe c t o f N -p ro p y ln o r a p o m o r p h in e and
c a e r u le in o n sH -s p ip e r o n e b in d in g a fte r c h r o n ic sa lin e
a n d h a lo p e r id o l. E ach c o lu m n is th e m ean o f th ree in ­
d e p e n d e n t ex p e r im e n ts, th e vertical bars represen t SEM.
T h e N P A 5 fig /k g d isp la c e a b le p art o f total b in d in g is
100% (1 8 1 4 0 ± 1 2 3 0 D P M /g tis s u e in su b co rte x and
15 3 5 0 ± 1 1 0 0 D P M /g t is s u e in fr o n ta l c o r te x ) in the
c h r o n ic s a lin e g r o u p . S ig n ifica n t d iffe r e n c e s b etw een
sa lin e a n d h a lo p e r id o l p r e tre a ted a n im a ls: * ~ <r<0.05,
** * /> < 0 .0 1 . L ig h t c o lu m n s sh o w th e e f fe c t o f N P A 5
Hg/kg, str ip e d c o lu m n s th e d iffe r e n c e b etw e en th e e f ­
fe c t o f N r A 5 0 м-g/kg a n d 5 ^g/k g; th e dark is fo r cae m le tn .
h ig h -a ffin ity b in d in g site s for N PA on
dopamine!- and serotonin*-receptors but d e­
creased it towards low-affinity sites bound by
high concentrations o f NPA. Considering the
stim u latin g action o f caerulein on 3H-SPI
binding, it seems possible that endogenous
CCK-8 increased the interaction o f ’H-SPI with
h ig h -a ffin ity b in d in g sites fo r N PA on
dopam ine 2- and serotonina-receptors. T he
References
A g n ati, L .F , F u x e , K ., B e n fe n a ti, F. e t al. D iffe re n tia '
m o d u la tio n by C C K -8 a n d C C K -4 o f 5H -s p ip e r o n e
b in d in g sites lin k e d to d o p a m in e a n d 5 -h y d ro x y try p ta m in e r e c e p to rs in th e b r a in o f th e ra t. N m ra sa . Leu.,
3 5 :1 7 9 -1 8 3 , 1983.
B a ttag lia, G ., a n d T ite lle r, M. * H -N -p r o p y ln o r a p o m o rp h in e a n d JH - jp ip e r o n e b in d in g in b r a in in d ic a te tw o
sta le s o f D s -d o p a m in e re c e p to r. E ur. J . Pharmacol.,
8 1 :4 9 3 -4 9 8 , 1982.
B lo o m , D .M ., N a ir, N .P .V ., a n d S c h w artz, C . C C K -8 in
th e tr e a tm e n t o f c h r o n ic s c h iz o p h re n ia . Psychopharmacot. b u ll., 19(3 ):3 6 1 -3 6 3 , 1983.
C h a n g , R .S.L ., L o tti, V .l., M a rtin . G .E ., a n d C h e n , T .B
In c re a se of b ra in '" [ -c h o le c y s to k in in (C C K ) r e c e p to r
b in d in g fo llo w in g c h ro n ic h a lo p e r id o l tr e a tm e n t, intra c is te rn a l 6 -h y d ro x y - d o p a m in e . o r v e n tra l te g m e n ta l
lesions. L ife Sei., 3 2 :8 7 1 -8 7 8 . 1983.
ЖУРНАЛ ВЫСШЕЙ НЕРВНОЙ ДЕЯТЕЛЬНОСТИ
ТОМ XXXIV
1984
ВЫ П . 2
У ДК 612.82/.83 + 615.78 + 612.821
РОЛЬ СЕРОТОНИНгРЕЦЕПТОРОВ В РЕГУЛЯЦИИ
АГРЕССИВНОГО ПОВЕДЕНИЯ
В А С А Р Э. Э., М АИ М ЕТС Л1. О., А Л Л И К М Е Т С Л . X.
Л аб орат ория п си х о ф а р м а к о л о ги и и ка ф ед р а ф а р м а к о л о ги и
Тарт уского го суд а р ст вен но го университета
С. Пероутка и С. Снайдер [1 1 ], основываясь на экспериментах со свя­
зыванием меченых лигандов, разделяли серотонииовые рецепторы на два
подтипа. По их классификации существуют с е р о т о н и н , -рецепторы, свя­
занные с действием серотонина, и серто1ШН2-рецеиторы , опосредующ ие
эффекты галлюциногенных сер отони н ом и м ети к ов. В поведенческих ис­
следованиях установлено, что крысы очень быстро обучаются по методи­
ке самовведения отличать введение галлю ц и н оген ны х с ер от он и н ом и м е­
тиков от введения других веществ [5 ], причем только пнренперон — из­
бирательный антагонист серотониновых рецепторов, блокировал в низ­
ких д озах данный эффект. С другой стороны, из работы [13] видно, что
мескалин — стимулятор сер отон и н г-рец сп торов. вызывает патологиче­
скую агрессивность у крыс-самцов. Авторы [ 1 4 1 нашли, что введение вы­
соких доз апоморфина вызывает у кошек п о в е д е т е , не отличающееся от
поведения при действии другого стимулятора сер отоп и н 2-р еп еп торои
диэтиламида лизергиновой кислоты. Имеются данные, что высокие дозы
опиатного антагониста налоксона усиливают поведенческие и электрофнзнологические эффекты галлюциногенных серотониномиметиков (6 ].
В настоящем исследовании приводятся данные, показывающие, что в
ЦНС существуют два подтипа се р о т о т 1Н2-рецепторов, противоположно
влияющие на агрессивное поведение. Вероятным модулятором активно­
сти серотониновых рецепторов, связанных с агрессивным поведением, яв­
ляется тетрапептид холецистокинина.
М ЕТОДИКА
Опыты проведены на 300 крысах-самцах линии Вистар, разделенных
на 29 групп, по 10— 12 животных в каждой. Влияние квииазниа (фирма
«Майлз Л абе», Англия) и пиренперона (фирма «Янссен», Бельгия) на
связывание 5Н-спироперндола (фирма «Амершам Интерпейзоиал». Анг­
лия) изучалось в стриатуме и фронтальной коре, где места связывания
нейролептиков соответственно относятся к дофамин2- и серотоншь реиепторам [9 ]. Связывание изучали по методике [4 ]. Действие спироперпдола и пиренперона на связывание 3Н-спироперидола (0,5 нмоль, удельная
активность 21 Ки/ммоль) определялось в пределах концентрации
1 нмоль — 10 мкмоль, влияние стимулятора серотониновых рецепторов
квип4зина— 10 нмоль — 50 мкмоль. Содержание белка в суспензии оп­
ределяли по методике [1 0 ]. Результаты опытов связывания обрабаты ва­
ли с помощью анализа Скетчарда.
Во второй части исследования сравнивали влияние системною ьнедения галоперидола (0,01—0,2 мг/кг внутрибрюшинно) и пиреннерона
(0,07—0,3 мг/кг внутрибрюшинно) на поведенческие эффекты апомор­
фина (0,5 мг/кг подкож но). Галоперидол и пиренперон всегда вводили
за 1 ч до введения апоморфина. Все исследуемые вещества вводили в пе­
чение 10 дней, 2 раза в день. Поведенческие тесты проводили на 1. 3, 7-й
и 10-й день длительного введения. Интенсивность стереотипии опреде­
ляли по методике Б. Косталл и соавт. [7 ], агрессивного поведения ;ю ме­
тодике, разработанной Л. X. Алликметсом и соавт. [3]
В третьей части исследования изучали влияние разных доз налоксо­
на (0,5— 15,0 мг/кг подкожно, фирма «Эндо Л абе», США) на развитие
апоморфиновой (0,5 мг/кг подкожно) агрессивности. Развитие спонтан­
ной агрессивности оценивали у сгруппированных крыс (по 10— 12 ж ивот­
ных в группе). Апоморфин и налоксон вводили в течение 10 дней 2 раза
в день. Через 48 ч после их отмены определяли поведенческие эффекты
квипазнна (2,5 мг/кг) — число встряхиваний головой в течение 40 мин
и спонтанную агрессивность.
В четвертой части исследования изучали взаимодействие тетрапепти­
да холецистокинина (ХЦК-4, И. К. И., Англия), который, по нашим пред­
варительным данным, из всех пептидов холецистокининового ряда обл а­
дает самым выраженным усиливающим влиянием на агрессивное пове­
дение с серотонин2-рен,епторамн. ХЦК-4 вводили в боковые желудочки
мозга по методике [1 ]. ХЦК-4 применяли в разных дозах (200— 5000 нг)
через 10 мни после впутрибрюшинного введения квипазнна (2,5 мг/кг).
Число встряхивании головой подсчитывали с 5-й по 10-ю минуту после
введения ХЦК-4. Параллельно исследовали влияние этих ж е доз ХЦК-4
на агрессивность, вызванную электроболевым раздражением. С пустя
5 мин после введения ХЦК-4, двух крыс подвергали электроболевым р аз­
дражениям (96 включений тока в течение 2 мин, напряжением 35 В ).
Интенсивность агрессивного поведения оценивали по числу агрессивных
контактов между животными. Определяли также влияние разных доз
пиренпероиа (0,01—0.1 мг/кг) и галоперидола (0,01—0,2 мг/кг) на уси­
ленную ХЦК-4 (200 нг) электроболевую агрессивность. Оба вещества
вводили за 60 мин до введения ХЦК-4. В отдельной серии опытов иссле­
довали влияние ХЦК-4 (200— 5000 нг) на электроболевую агрессивность
после 10-дневного предварительного введения апоморфипа (0,5 мг/кг.
2 раза в ден ь). ХЦК-4 вводили спустя 48 ч после заключительной инъ­
екции апоморфпна. Все результаты поведенческих исследований подвер­
гали статистической обработке с использованием t -теста Стьюдента.
РЕ ЗУ Л Ь ТА ТЫ И С С Л Е Д О В А Н И Й
ГТиренперон и квипазин в разной степени вытесняли 3Н-сппроперидол
из мест связывания в стриатуме и фронтальной коре (табл. 1). В стриа­
туме вытеснение меченого еппроперндола достигало только 30%, в то
время как во фронтальной коре этот показатель был около 90%. Однако
сродство квипазнна к местам связывания спироперидола было значитель­
но ниже по сравнению с пнренпероном. Этот факт объясняется сущ ест­
вованием неодинаковых мест связывания для агонистов и антагонистов
серотонин2-рецепторов.
Галоперидол и ппренперон по-разному влияли на поведенческие эф ­
фекты апоморфпна (табл. 2). Малая доза галоперидола (0,01 мг/кг), не
блокирующая стереотипного поведения, потенцировала развитие апо­
морфиновой агрессивности. Только 0,2 мг/кг галоперидола, которое пол­
ностью подавляло все признаки стереотипного поведения, устраняло разТ аблица 1
Влияние спироперидола (I нмоль— 10 мкмоль), пиренперона
(1 нмоль — 10 мкмоль) и квипазина (10 нмоль — 50 мкмоль)
на связывание 3Н-спироперидола с мембранами фронтальной коры
и стриатума
Стриатум
Фронт альная корэ
Вещ ество
Спиропепидол
Пиренперон
Квипазин
2 ,1
4 ,8
7 8 ,0
® вмлкс
КД
Свмакс
285±21
8 0 + 10*
88 ± 7 *
1 5
4 ,0
8 3 ,0
370125
3 3 0 -1 2 3
315+28
П рим ечание. Приведены средние гетичины трех независимых нгслед'м.лний. К д __константа днссоцигщии, нмилг . С.иМ(,к с — максимальное число м«-от
связывания, из которых и сследуем ы е п еш ее т а нытеспнлн меченый сиироперидол (фм оль/мг белк а).
* /< 0 ,0 5 .
281
в иTi ic апоморфиновой агрессивности. 0,07 мг/кг пиренперона такж е з а ­
метно ускоряло развитие апоморфиновой агрессивности, в то время как
более высокие дозы оказывали доза-завнсим ое снижение агрессивности.
При этом пиренперон существенно не изменял параметров апоморфино­
вой стереотипии, т. е. оказывал в
отличие от галоперидола избира­
тельное влияние на апоморфиновую агрессивность.
Одновременное введение 15 мг/
/кг палоксона с апоморфином р ез­
ко ускоряло развитие апоморфихювон агрессивности. После отм е­
ны этой комбинации отмечалось
достоверное уменьшение числа
встряхиваний головой, вызванных
квипазином, по сравнению с груп­
пой, получавшей только апоморВлияние длительного предварительного вве­
фнн (табл. 3). Следует отме­
дения апоморфина (0,5 мг/кг в течение
10 дней, 2 раза в день) на усиленную тет­
тить, что квипазин мог вызывать
(ХЦК-4)
у таких крыс спонтанную агрес­ рапептидом холецистокинина
электроболевую агрессивность. ХЦК-4 вво­
сивность, которая не отличалась дили внутрижелудочково через 48 ч после
от апоморфиновой. Малая доза
отмены апоморфина. По оси ординат—чис­
иалоксопа (0,5 мг/кг) оказывала ло агрессивных контактов в течение 2 мшг.
по оси абсцисс — доза ХЦК-4, нг.
Кон­
аналогичное с высокой дозой
троль — внутрижелудочковое введение фи­
(15 мг/кг) действие, хотя и более зиологического раствора. 1 — после дли­
слабое. Средняя доза налоксона
тельного введения физиологического
рас­
(5 мг/кг) при одновременном вве­ твора и 2 — апоморфина. * — р < 0,05; « • —
р<0,02
дении с апоморфином несколько
тормозила развитие апоморфино­
вой агрессивности. После отмены этой комбинации наблю далось значи­
тельное увеличение числа встряхиваний головой, вызванных квипазином, однако не отмечались агрессивные реакции.
Таблица 2
Влияние длительного (в течение 10 дней, 2 раза в день) одновременного введения
пиренперона ( 0 ,0 7 — 0,3 м г/кг) либо галоперидола ( 0 ,0 1 —0 ,2 м г/к г) с апоморфином
( 0 ,5 м г/к г) на интенсивность апоморфиновой стереотипии и агрессивности.
Приведены средние величины (баллы) для всех групп и стандартные ошибки
А гресси в ­
ность
Стереотипия
Стереотипия
А гр ес си вн о сть
В ещ ество, доза (м г/кг)
1 -й день
Апоморфин
раствор
Апоморфин
Апоморфин
Апоморфин
Апоморфин
Апоморфин
Апоморфин
3-й день
0,5 -f- физиологический
3, 1 ± 0 ,18
0
2 ,7 ± 0 ,1 9
0 ,3 + 0 ,2 2
0,5 + пиренперон 0,07
0,5 + пиренперон 0,15
0,5 + пиренперон 0,30
0,5 -j- галоперидол 0,01
0 , 5 + галоперидол 0,10
0 , 5 + галоперидол 0,20
2 ,8 ± 0 ,2 1
2 ,7 ± 0 ,2 6
2 ,4 + 0 ,3 4
3 ,0 + 0 ,2 2
0*
0*
0
0
0
0
0
0
2 ,6 + 0 ,2 3
2 ,5 ± 0 ,1 8
2 ,4 ± 0 ,2 2
3 ,2 ± 0 ,2 0
0*
0*
2 ,9 + 0 ,2 5 *
0
0
2 ,2 ± 0 ,3 2 *
0
0
Стереотипия
А г ресси вн ость
Стереотипия
А грессионость
В ещ ество, до за (м г/к г)
7-П д е н ь
Апоморфин
раствор
Апоморфин
Апоморфин
Апоморфин
Апоморфин
0,5 + физиологический
0,5 +
0,5 +
0,5 +
0,5 +
Апоморфин 0 , 5 +
Апоморфин 0,5 +
пиренперон 0,07
пиренперон 0,15
пиренперон 0,30
галоперидол 0,01
галоперидол 0 ,1 0
галоперидол 0,20
10 й д е н ь
2 ,2 ± 0 ,1 9
2 ,3 + 0 ,1 8
2 ,0 + 0 ,1 7
3 ,2 ± 0 ,2 2
2 ,7 + 0 ,2 2
2 ,3 + 0 ,2 2
2 ,2 + 0 ,1 7
3 ,0 + 0 ,1 9
1 ,4 + 0 ,1 6
0*
3 ,6 + 0 ,1 5
0 ,8 ± 0 ,4 2 *
0 ,3 + 0 ,2 2 *
3 .5 .t 0 , 23*
0 ,6 + 0 ,3 2 *
0*
2 ,2 ± 0 ,2 4
1 ,7 + 0 ,1 9
3 ,0 + 0 ,2 4 *
1 ,0 ± 0 ,4 5 *
0 ,3 ± 0 ,2 2 *
3 ,9 + 0 ,1 0
2 ,3 + 0 .2 5
0*
2 , 4 - 1: 0 , П
0*
Т аблица 3
Корреляция м еж ду развитием апоморфиновой агрессивности и поведенческими эффектами
квипазина ( 2 ,5 м г/кг) после отмены длительного (в течение 10 дней, 2 раза в день)
одновременного введения апоморфина ( 0 ,5 м г/к г) и налоксона ( 0 ,5 — 15 ,0 м г/кг)
Квилазин
В ещ е с т в о , д о за (м г/к г)
число в стр я хи ­
агрессивность
ваний головой
Физиологический раствор
Апоморфин 0,5 + физиологический раствор
Апоморфин 0,5 + налоксон 0,5
Апоморфив 0,5 + налоксон 5,0
Апоморфин 0,5 -f- налоксон 15,0
3 2 ± 3 ,4
5 5 + 4 ,2 *
31 ± 5 ,5
8 8 ± 5 ,8 * *
1 9 + 2 ,7 *
+
+ +
Апоморфиновпя
агрессивность
(-)
(8-й день) *
1 (5-й день)
1 (10-й день)
Т | (2-й день)
Примечание. « + » , «—» — нал ичи е или о т сутст в и е спонтанной гг^ессивностн п осле введения квипазина.
Т* 4 — уси лен и е или осла б л ен и е апоморфиновой агрессивности п од влиянием налоксона. * — в скобка* 1р н в ед ен ден ь д ли тел ьн ого в ведени я , когда в се ж ивотны е в гр уп п е становились агрессивными.
* /)<о,ог>.
** ,1- 0,01.
Т аблица 4
Влияние внутрижелудочкового введения тетрапептида холецистокинина (Х Ц К -4)
на электроболевую агрессивность и встряхивания головой, вызванные квипазином
( 2 ,5 м г/к г). Действие пиренперона (0 ,0 1 — 0,1 м г/к г) и галоперидола (0 ,0 1 — 0 ,2 м г/к г)
на усиленную ХЦК-4 электроболевую агрессивность
Ч и сло встряхиваний голо­ Электроболевая агрессивность,
вой, с б-й по 10-ю минуту число агрессивных контактов
в течение 2 мин
п осле введения ХЦК-4
В е щ е с т в о , доза
Физиологический раствор
ХЦК-4
200 нг
1000 »
5000 »
ХЦК-4 200 нг+галоперидол 0,01 мг/кг
0 ,0 5 >
0 ,2
»
ХЦК-4 200 нг - f пиренперон 0,01 мг/кг
0 ,0 5
»
0 ,1
»
8 ± 1 ,5
2 2 ± 2 ,5
5 ± 0 ,9
8 ± 1 ,7
4 ± 1 ,2
5 0 ± 4 ,2 * *
1 9 ± 2 ,6
4 0 ± 3 ,9 *
—
—
—
6 2 ± 4 ,5 *
3 3 ± 4 ,0
1 5 + 2 ,6
—
3 1 ± 2 ,8
2 ± 0 ,8 * *
0**
_
—
* р < 0 ,0 5 .
* • р < 0,02.
Внутрижелудочковое введение 200 нг ХЦК-4 вызывало заметное уси­
ление электроболевой агрессивности, причем животные наносили по­
вреждения друг другу. При дальнейшем повышении дозы ХЦ К-4 внача­
ле агрессивные реакции ослаблялись (1000 нг), а потом (5000 нг) опять
превышали контрольный уровень (табл. 4 ). Следует отметить, что эти
дозы ХЦК-4 оказывали такж е разное влияние на число встряхиваний го­
ловой, вызванных квипазином (2,5 мг/кг). Введение 200 и 5000 нг ХЦК-4
угнетало, в то время как 1000 нг не изменяло поведенческого эффекта
квипазина (табл. 4 ). Пиренперон обладал по сравнению с галоперидолом
более выраженным влияние на усиленную ХЦК-4 электроболевую агрес­
сивность. М алая д оза галоперидола (0,01 мг/кг) потенцировала дейст­
вие ХЦК-4 и лишь введение 0,2 мг/кг галоперидола значительно подав­
ляло агрессивное поведение. Пиренперон в отличие от галоперидола уж е
в доле 0,01 мг/кг достоверно понижал интенсивность агрессивного пове­
дения (табл. 4 ). При дальнейшем повышении дозы антиагрессивное дей ­
ствие пиренперона углублялось. П осле отмены длительного введения апоморфииа n p o a iресспвное действие ХЦК-4 усиливалось (рисунок), при­
чем такое изменение было особенно очевидно после введения 1000 нг
ХЦК-4.
/
О Б С У Ж Д Е Н И Е РЕ ЗУ Л Ь Т А Т О В
Анализ связывания меченого спнроперидола свидетельствует, что оп­
ределенная часть мест связывания нейролептиков имеет отношение к серотонинергическим механизмам переднего мозга. Наши данные согласу­
ются с мнением автора [1 2 ], что в стриатуме места связывания нейро­
лептиков в первую очередь относятся к дофамииергической, в то время
как во фронтальной коре — к серотонинергической системе. Существо­
вание неоднородных мест связывания для нейролептиков согласуется и
с тем, что пиренперон и галоперидол по-разному изменяют поведенче­
ские эффекты апоморфина. Антиагрессивное действие галоперидола кор­
релирует с подавлением апоморфиновой стреотипии, в то время как пи­
ренперон в дозах, подавляющих апоморфино'вую агрессивность, не изме­
няет интенсивности стереотипного поведения. Известно, что снижение
стереотипного поведения под влиянием нейролептиков в первую очередь
реализуется через блокаду дофаминовых рецепторов в полосатом теле
[4 ]. Пиренперон не взаимодействует в использованных нами д озах с эти­
ми рецепторами и поэтому не влияет на интенсивность стереотипного
поведения. За антиагрессивное действие пиренперона ответственны др у­
гие структуры переднего мозга, где места связывания нейролептиков
имеют тесную связь с серотониновыми рецепторами — в лимбической си­
стеме и фронтальной коре. Полученные нами данные свидетельствуют
также о том, что серотонинергическим механизмам принадлежит более
существенная роль в развитии апоморфиновой агрессивности по срав­
нению с дофаминергическими. Это мнение подтверждается и фактом, что
после длительного совместного введения апоморфина с определенными
дозами налоксона квипазин, стимулятор серотонин2-рецепторов, вызы­
вает спонтанную агрессивность, причем агрессивное поведение не отли­
чается от апоморфиновой агрессивности. С другой стороны, эти резуль­
таты дают нам возможность полагать, что среди серотонин2-рецепторов
существуют функционально неоднородные подтипы рецепторов. О дно­
временное введение определенных доз налоксона и апоморфина повы­
шает чувствительность серотониновых рецепторов, включающих агрес­
сивное поведение, однако параллельно развивается состояние понижен­
ной чувствительности к рецепторам с противоположным влиянием на
агрессивное поведение.
В предыдущих наших исследованиях установлено, что пептиды холецистокининового ряда обладаю т апоморфин-подобным действием [2 ].
Описанные выше данные показывают, что ХЦК-4 участвует в регуляции
чувствительности серотониновых рецепторов. ХЦК-4, как и одновремен­
ное введение определенных доз налоксона и апоморфина, неодинаково
изменяет чувствительность разных подтипов серотонин2-рецепторов и в
связи с этим ведет к усилению оборонительных реакций организма.
Таким образом, в результате проведенных исследований можно сд е­
лать заключение, что в мозге имеется два подтипа серотонин,-рецепторов,
опосредующих противоположные влияния на агрессивное поведение. О д­
новременное введение определенных доз налоксона с апоморфнном вы­
зывает сдвиг в сторону активации рецепторов, включающих агрессивные
реакции. Результаты настоящего исследования свидетельствуют, что
аналогичным действием обладает также ХЦК-4. Известно, что ХЦК-4 со­
держится в структурах переднего мозга в значительных концентрациях
[8 ]. В связи с этим можно полагать, что ХЦК-4 является эндогенным м о­
дулятором чувствительности серотониновых рецепторов, связанных с ре­
гуляцией агрессивного поведения.
ВЫ В О Д Ы
1.
Пиренперон, антагонист серотонин2-рецепторов в отличие от га­
лоперидола является избирательным антагонистом апоморфиновой агрес­
сивности. Длительное совместное введение определенных доз налоксона
с апоморфнном выявляет разные подтипы серотонин2-рецепторов, что вы­
■'
9
287
ражается в угнетении встряхиваний головой и появлении спонтанной
агрессивности после введения квипазина, стимулятора серотонинг-рецепторов.
2. Тетрапептид холецистокинина значительно усиливает электробо­
левую агрессивность и в тех ж е дозах уменьшает число встряхиваний го­
ловой, вызванных квипазином. Пиренперон является избирательным блокатором действия тетрапептида холецистокинина.
3. Тетрапептид холецистокинина, возмож но, является эндогенным мо­
дулятором чувствительности серотониновых рецепторов. Высказывается
предположение, что его функциональная роль заключается в активации
серотонинг-рецепторов, включающих оборонительные агрессивные реак­
ции.
Л итература
1. А ллик м ет с Л . X . Влияние адрен- и серотонинсргических веществ на поведение амигдалэктомироваиных крыс и на агрессивность, вызванную интраамргдалярным вве­
дением ацетилхолина.— Ж . высш. нервн. деят., 1975, т. 25, № 1, с. 164.
2. В а с а р Э. Э., Оттер М . Я ., Р я г о Л . К. Интрацеребровентрикулярное введение холе­
цистокинина угнетает активность дофамин- и серотонинергической систем мозга.—
Физиол. ж. СССР, 1982, т. 68, № 9, с. 1218.
3. A llik m e ts L. H ., S t a n le y М ., G e rsh o n S . The effect of lithium on chronic haloperidol
enhanced apomorphine aggression in rats.— Life Sei., 1979, v. 25, p. 165.
4. B iir k i H. R. Correlation between 3H-haloperidol binding in the striatum and brain
amine metabolism in the rat after treatment with neuroleptics.— Life Sei., 1978, v. 23,
p. 437.
5. C o lp a e rt F. C , N ie m e g e e rs C. J. E., J a n s se n P. A . J. A drug discrimination analysis
of lysergic acid diethylamide: in vivo agonist and antagonist effects of purported
5-hydroxytryptamine antagonists and of pirenperone, an LSD antagonist.— J. Pharma­
col. and Exptl Therap., 1982, v. 221, p. 206.
6. C o n tre ra s C. M ., G u z m a n -F lo r e z C., D o r a n te s M . E., E r v in F. R ., P a lm o u r R . Naloxone
and phencyclidine: interacting effects of the limbic system and behavior.— Physiol.
Behav., 1981, v. 27, p. 1019.
7. C o s ta ll B„ N a y lo r R. J. Stereotyped and circling behaviour induced by dopaminergic
agonists after lesions of the midbrain raphe nuclei.— Europ. J. Pharmacol., 1974,
v. 29, p. 206.
8. G o lte r m a n n N . R., S te n g a a r d - P e d e r s e n K-, R e h je ld J. F., C h r is te n s e n N . J. N ew ly syn­
thesized cholecystokinin in subcellular fractions of the rat brain.— J. Neurochem., 1981,
v. 36, p. 959.
9. L e y s e n J. E., G o m m e r e n W., L a d u r o n P. M . Spiperone: a ligand of choice for neurolep­
tic receptors. I. Kinetics and characteristics of in vitro binding.— Biochem. Pharma­
col., 1978, v. 27, p. 307.
10. L o w ry O. H ., R o s e n b r o u g h N . J.. F a rr A . L., R a n d a ll R . J. Protein measurement with
the Folin reagent — J. Biol. Chem., 1951, v. 193, p. 265.
11. P e r o u tk a S . J., S n y d e r S . H . Multiple serotonin receptors: differential binding of
3H-5-hydroxytryptamine, 3H-lysergic acid diethylamide and *H-spiroperidol.— Mol.
Pharmacol., 1979, v. 16. p. 687.
12. S e e m a n P. Brain dopamine receptors.— Pharmacol. Rev., 1981, v. 32, p. 229.
13. S b o r d o n e R . J., G orelick D. A.. E llio tt M. L. An cthological analysis of drug-induced
pathological aggression.— In: Multidisciplinary approaches to aggression research/
I Eds. P. F. Brain, D. Benton. Elsevier North-Holland Biomedical Press, 1981, p. 369.
14. T r u lso n M . E., C risp T. Behavioral and neurochemical effects of apomorphine in the
cat.— Europ. J. Pharmacol., 1982, v. 80, p. 295.
Поступила в редакцию
28.VI.1983
TH E ROLE OF SE R O T O N IN 2-R E C E P T O R S
IN TH E RE G U LA TIO N O F A G G R E S S IV E BE H A V IO U R
K /IS 4R E. E . i M M M E T S M. 0 .t A L L I K M E T S L - H .
C h a ir o f P h a r m a c o lo g y , T a r tu U n iv e rs ity , T a r tu
Q uipazine and pirenperone, the drugs in teractin g with serotonin2-receptors, more readily displaced 3H -spiroperidol from its binding sites in the
iruiih'l cortex than in the striatum. Pirenperone (0,07— 0,3 m g /k g ), an ta­
gonist of serotonin2-receptors, selectively decreased the intensity of apomorphine a g g resiv en ess. The an tiaggressive action of haloperidol (0,01 —
0.2 m g/kg) w as in correlation with its nntistereotypic activity. Long-term
adm inistration of naloxone (0,5; 15.0 m g /k g ), together with apomorphine
(0.5 m g/kg) reduced 1lie number of iiead-tw itehes caused by quipazine
(2,5 m g /k g ). TJie adm inistration of quipazine 48 hours after (he last inje­
ction of naloxone and apomorphine caused spontaneous aggressiven ess
that did not differ from apomorphine a g gressiven ess. Intracerebroventricular injection of cholecystokinin tetrapeptide (CCK-4) m arkedly enhanced
the foot-shock a g gression . The sam e dose of CCK-4 also decreased the in­
ten sity of quipazine (2,5 m g/kg) head-tvvitches. Compared to haloperidol,
pirenperone w as a more selective an tagon ist of CCK-4. After long-term
apomorphine treatment (0,5 m g/kg during 10 days, tw ice d aily), the effect
o f CCK-4 on a g g ressiv e behaviour w as m arkedly enhanced. It is possible
that two subtypes of serotonim -receptors exist in the brain and have oppo­
site action on the ag g ressiv e behaviour. CCK-4 may play the role of an
en dogenous m odulator of sen sitivity of serotonin2-receptors involved in
the control of ag g ressiven ess.
ISS N 0365-9615
БЮЛЛЕТЕНЬ
ЭКСПЕРИМЕНТАЛЬНОЙ
БИОЛОГИИ И МЕДИЦИНЫ
УД К в 15.355:577.! 75.734 -.IH5 1:612 -.42?. J
К л ю ч е в ы е с л « и а: церр.н'пн: II-прогш лнорапо
морфин', ъН -спироперш Ьл. дофаминовы е и се ротон иное ые
рецепторы.
Э. Э. Васар, Л.
Л . X. Алликметс
М.
Н урк.
М. О
Мийметс,
СТИМУЛЯЦИЯ Ц Е Р У Л Е И Н О М -А Н А Л О Г О М
ОКТАПЕПТИДА
ХОЛЕЦИСТОКИНИНА —
СВЯЗЫВАНИЯ 3Н-СПИ РО ПЕРИДОЛА
ПО­
СЛЕ д л и т е л ь н о г о
Вв е д е н и я
ней ро­
лептиков
Л аб о р ато р ия психофармакологии Н И И общей и молеку­
лярной патологии, кафедра фармакологии Тартуского уни­
верситета
П редставлена акад. АМН СССР А. В. В ад им ам ™
Имеются данные о том. что пептиды холецистокининового ряда в низких концентрациях м оду­
лируют взаимодействие еннронеридола с дофамин2- и серотоиин2-р е ц е т о р а м и [2]. В исследо­
ваниях поведенческнх реакций выявлено, что холецистокинин и его аналоги при внутрнмозговом
и периферическом введении оказы ваю т действие,
подобное влиянию как нейролептиков [4, 15],
так и апоморфина [1]. П араллельн о поведенче­
ским сдвигам холецистокинип и его аналоги вы ­
зы ваю т подавление м етаболизма дофам ина и се­
ротонина в структурах переднего мозга [1. 7].
В настоящ ее время появились данные, свиде­
тельствующ ие о том. что холецистокиннн и его
ан алог церулепн оказы ваю т на больных ш изо­
френией, резистентных к нейролептикам, зн ачи ­
тельное антипсихотическое действие [10. I I] .
В связи с этим представляло практический ин­
терес изучение влияния церулеина — вы сокоаф ­
финного аналога октапептида холецистокинина
[15] на связы вание 3Н-сппроперидола в опытах
in vivo. Учитывая апоморфиноподобное действие
церулеина, данный биохимический ан ализ прово­
дили в сравнении с Н-пропилнорапоморфнпом
(Н И Л ) - иыеокоаффннным ан ало ю м апоморф и­
на.
М е т о д и к а и с с л е д о в а н и я . Опыты про­
водили на белых беспородных м ы ш ах-самцах
массой 2 0 - 25 г. В течение 2 нед 2 раза в день
животным вводили галоперидол (0.25 мг/кг, ф и р ­
ма «Gedeon R ichter», В енгрия), пиренперон
(0.25 мг/кг, фирма « Jan ssen P h arm aceu tica» ,
Бельгия) или физиологический раствор. Спустя
72 ч после отмены длительного введения ставили
опыты по связы ванию in vivo: 6 животным из
каж дой группы (физиологический раствор, галоиерндол и пиренперон) вводили только 3Н-спироперидол в д озе 5 мкг/кг подкож но (уд. р ад и о ак­
тивность 17 Ки/ммоль, фирма «Am ersliam », Анг­
ли я) и спустя 20 мин их декапитировали. Ос­
тальным мышам из тех ж е групп (по 6 ж и вот­
ных) перед меченым спироперидолом вводили
вытесняющ ие вещества. Г алоперидол в дозе
2,5 мг/кг был введен внутрибрюш инно за 40 мин
до введения меченого лиганда, а церулеин в д о ­
зе 0.4 мг/кг (подкож но, фирма « F a rm ita lia » , И т а ­
лия) н (Н П А в д озах 5 и 50 м кг/кг (ф ирма «R e­
search Biochem icals Inc.», СШ А) вводили за
15 мин перед 3Н-спироперидолом. После декапитацин животны х на льду быстро извлекали мозг
н препарировали подкорковые образован и я пе­
реднего мозга (лим бическая система и стриатум)
и ф ронтальную кору. Выделенные структуры го­
могенизировали в 25 объемах трис-НС1-буфера
(50 м.М pH 7,4 при 2 0 °С ). Затем пробы центри­
фугировали при 9000 об. в течение 10 мин. Супернатант вы ливали и осадок осторож но промы ­
вали несколько раз с помощью холодного трисHC I-буфера. Радиоактивность проб (5 п а р а л л е ­
лей) определяли в сцинтилляторе 13рен на счет­
чике ß -частии «У льтро-Бста 1210» (ф ирма L.KR,
Ш веция). Опыты повторяли 3 р аза.
Р е з ультаты
иссл сд о в а н и я .
Вытес­
няющее действие высокой дозы галоперидола
(2.5 мг/кг) сущ ественно не изменялось после
длительного введения галоперидола (0,25 мг/кг)
и пиренперона (0,25 м г/к I) по сравнению с по­
казателем и I руппе животных, получавш их фи-
Влнянне галоперидола (2 ,5 м г'к г), НПА (5 и 5» м кг'кг) и церулеина (0,4 мг/кг) на связы вание ^Н-спироиеридола (5 мкг/кг)
в опытах in vivo после длительного введения галоперидола (0,25 мг кг) и пиренперона (0,25 м г'кг)
сЬпнолопиич кип P.ICTIIOP
йещестк,
Галоперидол, 2,5 мг кг
НИЛ:
50 м кг/кг
5 м кг/кг
Церулеин, 0,4 мг кг
ППЧКОККИНМС
"S S W
9 050 jr840
11 100 .±800
8«Ю0+7()0
7 3001:670
1 1Ю±:Ю0
и 40 0
12 2 5 0± 1 OJO
7 3 0 0 -И» 10
I III poi иерон
Г-Л..П РИД..,,
9 700 ±7 8 0
12 400 1-800*
1 100-h400*‘
i -.чиоо i-40^>*
пммя
11 200 L740
,
1I 800 M 020*
v
7Г>0
100 : 250’
■1иЛЦ|>|
VTpyi туры
фронтальнач
•м оо. д а )
5 Г,14) *890*
12 ЯК) *-7‘К)
2 700 >Ji001 100 1 1*0'
10 7Гч) »1 010
.{;$."*0 < <>50" *
2 450 . 1ОО* -
Примечание
Приведены средние ра (лнчии (число импхльсии нл 1 г з канн) че v i y i рун м.utti. п.. к ч лишим и mi. п.ко
3Н спнроперидол или '‘Н спироиерндол на фоне вытесняющих веществ. Знак • плюсстнмулнр'.гочич’ влияние па <им.im
ванне *Н-спироперндола Одна звездочка — Р < 0 ,0 .г). две - /-*<0,02 но сравнению 4. Kwirpo.wv (фн ш ологм чк'к ий раствор).
72
зиологнческий раствор (см. табли цу). Только
после многократного введения серотонпн2-антагониста инрснперона ум еньш алось его вы тесняю ­
щее влияние во фронтальной коре. В отличие от
действия галоперидола влияние НПА заметно
изменялось после длительного введения галоперидола и ниренперона. В дозе 50 мкг/кг НПА
значительно сильнее вытеснял 3Н-спнроперндол
в подкорковых структурах после длительного
введения как галоперидола, так и пиренперона.
Во фронтальной коре вытесняющий эф ф ект НПА
в этой дозе д аж е несколько о слаб лялся после
длительного введения нейролептиков. Такое р а з­
личие в действии НГ1А в двух регионах м оз­
га, по-видимому, объясняется его более с л а ­
бым
агонистическим действием
на серото­
н ин-рецепторы
по сравнению с доф ам ино­
выми. У становлено, что только высокие д о ­
зы апоморфпна вы зываю т подобные галлю ­
циногенным
серотониномиметикам
поведенче­
ские эфф екты [14] и значительны е концент­
рации апоморфина вытесняют 3Н-кетансерин —
антагоннст серотонин2-рецепторов — из мест свя­
зы вания в префронтальной коре [8 ]. К вытес­
няющему действию малой дозы НП А (5 м кг/кг)
разви валась, однако, толерантность после д л и ­
тельного введения галоперидола и пиренперона
(см. табли цу). Этот ф акт свидетельствует о том,
что на определенных местах связы вания 3Н-спироперидол сильнее взаимодействует с доф ам ино­
выми и серотониновыми рецепторами после д л и ­
тельного введения
нейролептиков. Р езу л ьта­
ты
настоящ его
исследования
во
многом
согласую тся с данными литературы [5 ], соглас
но которым нейролептики и апоморфин неодина­
ково взаимодействуют с доф ам ин2-рецепторами.
П оказано, что взаимодействие апоморфина с 3Нспироперидолом иа дофам ин2-рецепторах осу­
щ ествляется через низко- и высокоаффинные м е­
ста связы вания, в то время как нейролептики име­
ют на этих рецепторах только высокоаффинные
места связы вания [5]. Выявлено, что константы
диссоциации этих двух мест связы вания для апо­
м орфина отличаю тся приблизительно в 10 раз.
Учитывая различное действие разны х доз НПА
после длительного введения галоперидола и пнренперона, можно полагать, что аффинность ней­
ролептиков к этим двум местам связы вания для
апоморфина изменяется неодинаково. По-вндимому, понижается аффинность 3Н-спироперндола
к низкоаффинным местам связы вания для апо­
м орфина, в то время как на высокоаффннных м е­
стах. связы вание 3Н-спироперндо. 1а существен
ним образом увеличивается. Вероятно, что изме­
нение чувствительности ннзкоаффннных мест
связы вания отраж ает развитие гнперчувствитрльностч к дофамино- и серотониномиметикам и
ослабление разных эффектом нейролептиков, в то
время как повышение аффинности 3Н-епиропериаола к высокоаффнниым местам связы вания для
апоморфина, по всей вероятности, связано с р а з ­
витием антипсихотического действия и процессе
длительного введения нейролептиков.
После длительного введения нейролептиков не
изменяется только действие НПА, а такж е церулеина — высокоаффинного аналога октапепти­
да холецистокинина [15]. Сущ ествует мнение,
что ряд эфф ектов холецистокинина и его а н ало ­
гов реализуется при периферическом введении
через афферентны е механизмы блуж даю щ его
нерва [9, 13]. О днако наши исследования свиде­
тельствую т о том. что церулеин проникал в мозг
и вытеснял у контрольных животных 3Н-спироперидол из мест связы вания (см. таб ли цу). П ос­
ле 2-недельного введения галоперидола и пирен­
перона действие церуленна, однако, стало проти­
воположным. Ц ерулеин не вытеснял, а стимулиро­
вал связы вание спироперидола в обоих нами ис­
следованны х регионах переднего мозга. Следует
отметить, что после длительного введения а н та­
гониста серотонин2-рецепторов пиренперона сти­
мулирую щее действие церуленна было более в ы ­
раж енным во фронтальной коре (см. таб ли цу),
а после галоперидола, взаимодействую щ его преи­
мущественно с дофаминовы ми рецепторам и, это
действие церулеина было более значимы м в под­
корковых структурах. Эти данные согласую тся с
исследованиями [12], показавш им и, что во ф рон­
тальной коре места связы вания для нейролепти­
ков в основном относятся к серотонин2-рецепторам, в то время как в подкорковых структурах
превалирует взаим одействие с дофаминовыми ре­
цепторами. Если учитывать, что длительное вве­
дение галоперидола (2 —3 мг/кг) повышает плот­
ность холецистокининовых рецепторов почти в
2 раза [3 ]. можно полагать, что увеличение св я ­
зы вания 3Н-спироперидола на высокоаффинных
местах связы вания для апоморфина обусловлено
именно усиленным действием эндогенного октапептида холецистокинина после длительного вве­
дения нейролептиков.
Таким образом , полученные данные свиде­
тельствуют о том, что после длительного введе­
ния нейролептиков усиливается их взаим одейст­
вие с высокоаффинными местами связы вания для
апоморфина на дофам ин2- и серотонин_>-рецепто
рах. Этот механизм, по-видимому, леж и т в осно­
ве антипсихотического действии нейролептиков.
О днако, как показывают эксперим ентальны е [6]
и клинические исследования [1 0 |, это' действие
нейролептиков реализуется только на фоне дос­
таточных концентраций окгапептида холецисто­
кинина. Установлено, что у больных ш изофрени­
ей, резистентных к нейролептикам, после смерти
вы является низкое содерж ание холецистокинина
в лимбических структурах [6 |.
ЛИТЕРАТУРА
1. Пасар Э. Э . Оттср М. H., Ряго Л. К — Физиол. ж.
СССР. 1482. № 9, с. 1218— 1222.
2 A g n a il /.. F.. Fuxe A.. Heafotuiti F. et ;il
N rurnsci
Lett., 1983. v. 35, г- '7 9 183.
73
,i. C hang R. S L . L o tti V. / , M o rtin G. E. et a l . - Life
Sei., 1983, v. 32. р. 8 7 1 - 878.
4. Cohen S. L., K night /Vf.. Ta n vn in g a C. A et al. — E urop.
J. P harm acol., 1982, v. 83, p. 213—222.
5. Creese /.. L e ff S. A . — J. clin. P sy chopharm aeol., 1982,
v. 2, p. 329—335.
*». F erner N., R o b erts G. W., Crow T. J. et al. — Life Sei.,
1983, v. 33, p. 4 7 5 -4 8 2 .
7. F uxe K-, A n d ersson K., Locatelli V. et al. — Europ. J.
P harm acol.. 1980, v. 67, p. 329—331.
8. L eysen J. E., N iem egeers C. J. E., Van N ueten J. M.
et al. — M olec. P harm acol., 1982, v. 21, p. 301—304.
9. L o ren z D. N.. G oldm an S . A. — Physiol. Behav., 1982,
v 29 p 599__604
10. Nair N. P. V., B loom D. M ., N estoros J. N. — P rogr.
N eu ro-Psychopharm acol. Biol. P sy chiat., 1982, v. G,
p. 808—812.
11. M oroji Т., W atanabe M., A o k i N. et al. — Arch. gen.
P sychiat.. 1982, v. 39, p. 485—486.
12. S e e m a n P. — P harm aco l. Rev., 1981, v. 32, p. 229—313.
13. S m ith G. P., Jerom e C., C ushin B. /. e t al. — Science,
1981, v. 231, p. 1036— 1037.
l t. Trulson M. E., Crisp T. — Europ. J. P harm acol., 1982,
v. 80. p. 295—309.
15. Z etler G. — N europharm acology. 1981, v. 20, p. 277—
283.
Поступила !2tfl|.S1
STIM U LA TIO N W IT H C E R U L E IN , AN ANALOG O F C H O ­
L E C Y STO K IN IN O C T A PE PT ID E , O F 3H -S P IR O P E R ID O L
B IN D IN G AFTER PR O L O N G E D A D M IN ISTRA TIO N OF
N E U R O L E PT IC S
E. E. Vasar, A . M. N urk, M. O. M a im ets, L. Kh. A llik m ets
R esearch In s titu te
T a rIu U niversity
of
G en eral
and
M olecular
Pathology.
It h as been estab lish ed in experim ents on w hite m ale
ra ls th a t p rolonged ad m in istra tio n (tw ice a day for 14 days)
of haloperidol (0.25 m g /k g ) an d pyreneperone (0.25 m g /k g )
resu lted in the reduced in teractio n betw een 3Il-spiroperidol
an d low a ffin ity b in d in g site s for ap om orphine in subcorlical stru c tu re s, w h ereas 5H -spiroperidol b in d in g w ith high
a ffin ity b in d in g site s for apom orphine increased both in
the fro n tal cortex an d sub co rtical stru c tu re s of the forebrain.
A fter p rolonged ad m in istra tio n of neuroleptics the d isp lac ­
in g effect of cerulein, an a n a lo g of cholecystokinin octapepticlr, w as replaced by the stim u la n t action on 3H-spiroperidol b inding. It is assum ed th a t in creased in tera ctio n b et­
w een ’H -spiroperidol an d high affin ity b in d in g site s for
apom orphine o n dopam ine2- and serotonin*-receptors u n d er­
lies the an tip sy ch o tic actio n of n eu ro lep tics afte r their p ro ­
longed ad m in istratio n . C holecystokinin octapeptide is a n e ­
c e ssary factor for re a liz a tio n of th is action of neuroleptics.
74
УДК 618.281-07:616-008.949.4 :« I S .2 8 t.8 - 615.281.8033:618.2
К л ю ч е в ы е
м ант адин
слова:
берем енност ь,
р е­
Н. Ф. Проедина, В. М . Шобухов, И Г. Петрова.
С. Г. Тулъкес, Г. А . Галегов
КИНЕТИКА НАКОПЛЕНИЯ И ВЫВЕДЕНИЯ
•Н-РЕМАНТАДИНА В ТКАНЯХ БЕРЕМЕННЫХ
МЫШЕЙ И ПЛОДАХ
И нсти тут ви русологи и им. Д . И . И ван овского АМН
СССР, .Москва Н И И по биологическим испы таниям х и ­
мических соединений
П редстпвленл
акад. АМН СССР В. В. Закусопым
Аминопроизводные адамантана, в частности реман­
тадин (а-метил-Ьадамантанметнламин),
применя­
ются в качестве средств профилактики и лечения
гриппа [2, 4, 71. Одна из важных фармакокинети­
ческих характеристик химиопрепаратов, исполь­
зуемых для широких контингентов населения, а к
ним, безусловно, относится ремантадин, является
проницаемость через плаценту и динамика накоп ­
ления и особенно выведения их из плодов. В ли те­
ратуре подобные данные относительно ремантадина
отсутствуют.
В связи с этим задачей настоящего исследования
явилось изучение кинетики накоплении и элими­
нации 3Н-ремантадина в плодах и тк ан hx беремен­
ных мышей.
М е т о д и к а и с с л е д о в а н и я . В работе
использован 3Н-ремантадин, полученный нами по
ранее описанной методике [5]. Удельная радио­
активность препарата 30 мКи/ммоль. Опыты с та­
вили на беспородных белых мышах-самках массой
30 г на 15— 16-е дни беременности, а такж е на
беспородных белых мышах массой 10— 12 г. *Н-ремантаднн вводили в 0,3 мл физиологического раст­
вора перорально (2,8 мг/кг). В установленные сро­
ки (15 и 30 мин, 1, 2, 6 и 12 ч после введения пре­
парата) мышеи декапитировали и извлеченные
органы помещали в 5 н . NaOH (7 мл — на плоды,
5 мл — на печень и по 3 мл — на почки и селезен­
ку). Ткани гомогенизировали и к гомогенату до­
бавляли равный объем бензола для экстракции ме­
ченого ремантадина. После интенсивного встря­
хивания в течение 10 мин суспензию центрифуги­
ровали 15 мин при 4000 об/мин для отделения бен­
зола от водной фазы. От каждой пробы отбирали
аликвоты (0,2 мл — для печени и по 1 мл — для
плодов, селезенки и почек) и определяли радио­
активность в 10 мл толуолового сцинтиллятора
на жидкостном сцинти.пляцнонном спектрометре
SL-30 фирмы «Intertecknique» (Ф ранция).
Результаты
и с с л е д о в а н и я . Д ан­
ные, представленные в табл. 1, отраж ают распре­
деление :‘Н-ремантадина в плодах, печени, почках
и селезенке беременных мышей в течение 12 ч
ISSN 0365-9615
БЮЛЛЕТЕНЬ
ЭКСПЕРИМЕНТАЛЬНОЙ
БИОЛОГИИ И МЕДИЦИНЫ
Бюл. эксперим, биологии и медицины 1985,
№ 12, 641—768
Ü LJE
A
M
ной иерархии низшие ранги. Подобные особи
обычно характеризую тся высокой чувствитель­
ностью к стрессориым ф акторам и вы раж енной
склонностью к развитию эксперим ентального а л ­
коголизма, основанной на нормализую щ ем в л и я ­
нии, которое этанол оказы вает на эмоциональную
сферу и поведение этих особей [ I, в]. Следова
тельно, полученные нами данны е свидетельству­
ют об общности причин, вызывающ их влечение
как к никотину, так и к алкоголю .
Рис. 2. В лияние никотина в до зах 0,05 мг/кг (/) и 0,1 м г/к г
( // ) на длительность иммобилизации крыс.
ЛИТЕРАТУРА
По оси ординат — время (в с). / — ниэкоактнвные крысы. 2 — вы ­
сокоактивные крысы. Светлые столбики — фон, заш трихованные —
1. Борисенко С А , К ам пов-Н олевой A h — Ж урн. «нсш.
нервн. деят., 19Я0, Л» 3. с 513.
2. Б у р о в Ю. В — Вестн. АМН СССР. 1982. № 5. с 72—
77.
3. Б у р о в Ю В., К ампов-П олевой А. Б.. Каминка Л . И. —
Ж ури высш. нервн. деят., 1983, JV» 5, с. 941.
4. К ам пов-П олевой А. Б — Бюл. экепер. биол., (978, N i 9,
с. ЗОВ.
5. К ам пов-П олевой А. Б . — Д епонировано
в
ВИН ИТИ
1979, № 825—79.
6. К ампов-П олевой А. Б. — В кн.: Ф армакология экеперн
ментального алкоголизма. М., 1982, г. 130— 135
7. П лохин ский Н А. Биометрия М., 1970
8 M eddis R. - Brit. J P s y c h o l, 1975, vol. 66, p. 225.
(контроль) вводили 0,15 мл физиологического р а ­
створа. При этом введение 0,05 мг/кг никотина
в ы зы вало значительное повышение ЭПС «подчи­
ненной» крысы н в больш инстве случаев (в 6 из
8) происходила смена доминирующ ей особи
(рис. 3 ). Введение 0,1 мг/кг никотина «подчинен­
ной» особи та к ж е повы ш ало ее Э П С , однако см е­
на дом инирования произош ла только в 4 из 8
случаев.
При изучении влияния никотина в широком
ди ап азон е доз на двигательную активность мы
шей какого-либо значим ого эфф екта не н аб л ю д а­
лось.
Таким образом , можно с д е л ан , вывод, что ни­
котин оказы вает специфически активирую щ ее
действие па животны х с низким адаптивны м по­
тенциалом , отличное от действия психостим уля­
торов, чем, по-видимому, и обусловлено их в ы р а­
женное влечение к никотину.
Проведенные эксперименты свидетельствую т о
наличии у части особей в популяции самцов бес­
породных белых крыс выраж енной предрасполо­
женности к развитию никотиновой токсикомании.
П редставляет интерес тот ф акт, что данна,; п а­
тология разви вается у животных с низким а д а п ­
тивным потенциалом , заним аю щ их в зоосоциаль-
Поступил» 28.05 85
TH E FO LE O F N IC O TIN E PSY C H O TR O PIC E FFE C T S IN
IN D U C IN G IN C LINATION TO 1NCOT1NE IN RATS
T.
K hodzhageldyev
In stitu te of P harm acology, Academ y of M edical Sciences of
the U SSR , Moscow
The experim ents have proved som e m ale noninbred w hile
ra ts to of inclined to toxicom ania developm ent. It is of in­
terest th at such pathology develops in anim als w ith low
ad ap tiv e potential occupying the low est rank in zoosocial
h ierarchy These a nim als a re usually characterized by high
sen sitiv ity to stre ss factors and pronounced inclination to
the developm ent oi experim ental alcoholism . Thus, it s u g ­
g e sts the existence of common rea so n s c au sin g the in clina­
tion to both nicotine and alcohol consum ption.
УДК 615.357.34:577.175.7341.017:615.213.015.2:61>.21J .22:547.B91.2
Ключевые
с л о в а : церулеин.
проглум ид.
пикротоксиновые судороги, оенэодиазепиновы е ре­
цепторы, uohoifwp хлора
Э. Э. Васар, JI. К. Ряго, A. X. Сиосаар,
А. М. Н урк, М. О. Майметс
М ОДУЛИРУЮ Щ ЕЕ ВЛИЯНИЕ ЦЕРУЛЕННА
НА БЕН ЗО ДИ АЗЕП И Н О ВЫ Е РЕЦЕПТОРЫ
Л аб оратори я психофармакологии Н И И обшей н м олекуляр­
ной патологии Тартуского университета
П редставлена акад. АМН СССР А. В. Вальдчамом
Риг. 3 Влияние никотина в разных до зах на двигательною
10
У становлено, что октапептид холецнетокшнша
(Х Ц К-8) и его ан алог более сильного действия
церулеин вы зы ваю т при системном введении эф ­
фекты , подобные эфф ектам транквилизаторов
животные не погибали, то реакция мышей по всем
исследованным
парам етрам
соответствовала
30 мин. Антагонист ХЦ К-8 проглумид (ф ирма
«R olta Farm aceu tici» , И тали я) [3] вводили внут­
рибрюш инно в д озах 5 и 25 мг/кг за 5 мин до вве­
дения церулеина. П ар ал л ел ьн о с изучением пове­
денческих реакций исследовали влияние церулеи­
на на связы ван и е 3Н -ф лун и тразеп ам а в опы тах
in v itro и in vivo. П роглум ид (5 и 25 мг/кг) и церу­
леин (20—500 м кг/кг) вводили за 5 мин до под­
кожной инъекции меченого ф лун и тразеп ам а в
д озе
0,3 м кг/кг
(уд ельн ая радиоактивность
84 Ки/мм оль, фирма cA m ersham », А н гли я). Ж и ­
вотных (по 6 мышей из каж дой серии) декапитировали через 30 мин после введения изотопа. П е­
редний мозг ж ивотны х одной группы объединили
в один пул и гом огенизировали с помощью гомо­
генизатора П оттера в 40 объем ах трис-НС1-буферного раствора (50 мМ pH 7,4) при 20 "С. С пе­
цифическое связы вание ф лун и тразеп ам а опреде­
ляли при добавлении Ю мкМ немеченого ф лун и ­
тр азеп ам а к гом огенатам м озга. Разн и ц а меж ду
п оказателям и радиоактивности проб без лиганда
и с немеченым лигандом х а р актер и зо в ал а специ­
фическое связы вание ф л у н и тразеп ам а. И н к у б а­
цию проводили при 0 °С в течение 60 мин. После
инкубации пробы ф ильтровали через фильтры
Г Ф /Б (ф ирм а «W hatm an», А н гли я), которые з а ­
тем промы вали д важ д ы 5 мл б уф ера. Р а д и о а к ­
тивность фильтров оп ределяли в сцинтилляторе
Брея на счетчике ß-частиц Л С-7500 (ф ирмы «Be­
ckm an», С Ш А ). Опыты по связы ванию 3Н-флунитраэепам а in v itro в переднем мозге мышей
проводили по описанной ранее методике [1 ].
Р е з у л ь т а т ы и с с л е д о в а н и я . П р ед вар и ­
тельное подкожное введение относительно вы со­
ких доз церулеина (более 100 м кг/кг) зам ед л ял о
развитие пикротоксиновы х судорог (см. таб л и ц у ),
при этом удлинялись латентны е периоды клоннческих и тонических судорог, а та к ж е увеличива­
л ась продолж ительность ж изни мышей. Ц ерулеин
в д озе 250 м кг/кг о казы в ал наиболее вы раж енное
бензодиазепинового ряда [ 6 ,8 1 . Относительно
высокие дозы церулеина и Х Ц К -8 удлиняю т л а ­
тентные периоды проявления судорог, вы званны х
ти осем икарбазидом и гарм аном , и повыш ают т а к ­
ж е пороговую д озу пикротоксина, необходимую
дл я вы зы вания судорог у мышей [6, 8 ], причем
церулеин по своему противосудорож ном у д ейст­
вию превосходит ди азеп ам . Аналогичное противосудорожное действие н аблю дается при внутриж елудочковом введении низких доз Х Ц К -8 (1 и
100 нг) [4]. В то же время Х Ц К-8 и церулеин о к а ­
зы ваю т действие, сущ ественно отличаю щ ееся от
влияния тран кви ли заторов бензодиазепинового
ряда. Ц ерулеин в отличие от д и азеп ам а потенци­
рует судороги, вы званны е антагонистом ГАМКрецепторов бикукуллином [6]. Введение Ro 151788 — ан тагониста бензодиазепинов — не уст­
ран яет седативное и противосудорож ное действие
церулеина и Х Ц К-8 [8 ].
В настоящ ей работе исследовали участие бензодиазепиновы х
рецепторов в противосудорожном действии церулеина.
М е т о д и к а и с с л е д о в а н и я . Опыты про­
водили на м ы ш ах-сам цах массой 25— 30 г. При
исследовании пикротоксиновы х судорог церулеин
(ф ирм а « F a r m ita lia — C a rlo E rba», И тали я) вво­
дили в разны х дозах (20—500 м кг/кг) подкож но
за 10 мин до внутрибрю ш инного введения пикро­
токсина в дозе 8 м г/кг (ф ирма « Serva», Ф Р Г ).
В I серии экспериментов оп ределяли влияние це­
рулеина (5— 1000 нМ) на связы ван и е 3Н-флунитр азеп ам а (1 нМ) в среде 50 мМ трис-НС1-буферного раствора. Во II серии опы тов к этой среде
добавл ял и 120мМ К С1. В каж дой серии было по
16— 20 животных. О пределяли 3 различны х п а­
рам етра пикротоксиновых судорог: латентны е пе­
риоды клонических судорог, латентны е периоды
тонических судорог и п родолж ительность ж изни
мышей после введения 8 м г/кг пикротоксина. Р е ­
акцию мышей на введение пикротоксина н аб л ю ­
д а л и в течение 30 мин. Если в течение этого вр е­
мени у ж ивотны х не разви вал и сь судороги или
В л а т к е церулеина
(A f if f li л * 3)
н
Препараты
Фнзиологичесхип раствор
Ц ерулеин
П роглумид
П роглумид
церулеин
П роглум ид
церулеин
П роглумид • церулеин
проглумида
на
пякротоксиноаые
судороги н саязыаанне ’ Н-флунитразепама а опытах ln vivo
Доз»
Специфическое связывание
*Н -ф лунитразепама в п е­
реднем м озге, имп. на
20
50
100
250
500
5
25
54100
2 5 ! 100
25 ■ 250
14 9 7 0 ± 8 2 9
14 8 4 0 ± 8 5 0
14 020 ± 7 9 0
12 200 ± 6 8 0
6 145±420***
5 720±380” '
150Э 0± 790
15840±760
10 820 ± 8 6 0 * ‘
15 6 2 0 + 6 7 0
11 640± 870*
Латентные периоды пикротоксиновы х судорог
клон нчсскнх, с
тонических, мни
прод олж и тел ьн ость
ж нэнн мышей. мин
417±23
428±36
486±42
593 ± 4 1 *
674±58‘ *
5 7 3 ± 62*
432±32
406 ± 2 5
644 ± 4 8**
504 ± 3 6
13, 4 ± 1,4
1 3 ,5 ± 1 .8
1 5 .6 ± 1 ,5
1 9 ,3 ± 2 ,3 *
1 9 , 8 ± |, 5 ‘ *
2 0 ,4 ± 2 ,7 *
1 3 ,6 ± 1 ,5
1 2 .8 ± 1,7
2 0 , 8 ± 1 ,5**
1 7 ,4 ± 1,9
1 3 ,8 ± 1 ,4
1 4 ,0 ± 1 ,7
16,2 ± 1,8
2 1 , 3 ± 2 ,5 *
2 1 ,6 ± 2 ,0 *
2 1 ,0 ± 2 .8 *
1 4 ,2 ± 1 .5
13.1 ± 1 , 8
2 Ч .4 ± 1 .9**
18,Г>±2,0
П р и м е ч а н и е . Одна звездочка — Р < 0 ,0 5 , две — Р < 0 ,0 1 . три — Р < 0 .0 0 1 .
m
30
y r
60
40
20 -
I
y
i
5
25
----------- A /
50 WO 250
ЮОО
В лияние церуленна (5— 1000 нМ) на связывание •Н-флунвтразепама (1 нМ) в опытах in vitro.
По оси абсцисс — концентрация церуленна (в нМ), по оси орди­
нат — процент угнетения церулеином связывания *Н-флуннтразепама. За 108 % принято ингибирующее действие 1 мкМ немеченого
ф лунитразепама. / — действие церуленна в среде 60 мМ трис-ИС),
2 - в среде Б иМ КС1 и 120 мМ NaCI ■ 50 иМ трис-НС!.
Одна звездочка — Ж 0 .0 6 ,
две — /*<0,01, три /*<0,001, четыре —
/*<0,0001 (по критерию t Стью дента). Представлены данны е 3 не­
зависимых опытов.
в эти эфф екты церулеина свидетельствует усиле­
ние последних под влиянием низкой лозы
(5 мг/кг) проглумида, а такж е их ослабление
после введения более высокой дозы (25 м кг/кг)
проглумида. И звестно, что низкие дозы церулеи­
на (27 м кг/кг) отчетливо удлиняю т гексобарбиталовый сон [8]. По сущ ествующ им представлени­
ям, действие пикротоксина и барбитуратов на по­
ведение животны х реализуется через их непосред­
ственное влияние на хлорный кан ал [2, 5]. В опы ­
тах in v itro церулеин ингибировал связы вание
3Н -ф лун и тразеп ам а только в присутствии сущ ест­
венных концентраций аниона хлора, что свиде­
тельствует та к ж е в пользу взаим одействия церу­
леина с ионофором хлора. Н а основании этого
м ож но полагать, что именно через ионофор хлора
реализуется противосудорожное действие ХЦ К-8
и церулеина, а т а к ж е модулирую щ ее влияние це­
рулеина на связы вание 3Н -ф лунитразепам а.
ЛИТЕРАТУРА
влияние на пикротоксиновые судороги, дальн ей ­
шее увеличение дозы церуленна не приводило к
усилению его
противосудорожного действия.
В д озах, угнетающ их пикротоксиновые судороги,
церулеин достоверно ингибировал связы вание
3Н -ф лунитразепам а в опы тах in vivo. В д о зах 250
и 500 мкг/кг церулеин вы зы вал более чем 50 %
уменьшение специфического связы вания флунитразеп ам а в переднем мозге. Антагонист ХЦ К-8
проглумид в исследованных дозах сущ ественно
не изменял пикротоксиновых судорог и лишь в
дозе 25 мкг/кг незначительно повышал специф и­
ческое связы вание 3Н -ф лун и тразеп ам а (см. т а б ­
л и ц у). В дозе 5 мг/кг проглумид потенцировал
угнетающ ее влияние церуленна как на пикроток­
синовые судороги, та к и на специфическое св язы ­
вание флунитразепам а. В дозе 25 мг/кг проглу­
мид ока за л противоположное действие: уменьш ал
антипикротоксиновый эфф ект 100 м кг/кг церуле­
нна и устранял ингибирующе влияние церуленна
(250 м кг/кг) на специфическое связы вание ф л у ­
н итразепам а. В опы тах in vitro церулеин (5—
1000 нМ)
противодействовал связы ванию 3Нфлунитразепам а только в присутствии 5 мМ К.С1
и 120 мМ NaCI (см. рисунок). В этих условиях
п араллельн о повышению концентрации церулеина н аблю далось его ингибирующее влияние на
связы вание меченого ф лунитразепам а.
Р езультаты настоящ его исследования сви де­
тельствую т о модулирую щем влиянии церуленна
на бензодиазепиновые рецепторы. Н ачиная с д о ­
зы 100 мкг/кг церулеин угнетал как пикротокси­
новые судороги, та к и специфическое связы вание
3Н -ф лунитразепам а в переднем мозге мышей в
опытах in vivo. Эти данные показы ваю т, что ин­
гибирующее влияние церулеина на пикротокси« и н е судороги и на связы вание 3Н -флунитразем м реализуются через одни и те ж е механизмы.
О м м м е я и о с т н холецнстокининовых рецепторов
1. В асар Э. Э , Ряго Л. К., Алликметс JI. X. — Ж урн.
высш. нервн. д е я т , 1983, т. 33, с. 864.
2. B raesirup С., N ielsen М. — In: H andbook of P sychophar­
m acology. New York, 1983, vol. 17, p. 285.
3. H ahne W. F., J anssn R Т., Lem p G. F. Gardner J. D. —
Proc. n at. Acad. Sei. USA, 1981, vol. 78. p. 6304.
4. K adar Т., P esti A., Р епке В . T elegdy G. — N e uropharm a­
cology, 1984, vol. 23, p. 955.
5. Pole P., B o n etti E. P., S ch a ffn er R., H aefely
07. —
N aunyn-S chm iedeberg's Arch. P harm akol., 1982, Bd 321,
S. 260.
6. Z eller G. — N europharm acology. 1981, vol. 20, p. 277.
7. Z eller G. — Neurosci. Lett., 1982, vol. 25, p. 287.
8. Z etler G. — P sychopharm acol. Bull.,
1983, vo! 19,
p. 347.
П оступила 21.03.85
MODULATORY E FFE C T O F CA ER U LEIN ON BEN ZO D IA ­
Z E P IN E REC EPT O R S
E. E. Vasar, L. K.
M. O. M aim ets
Rägo,
A. Kh. Soosaar,
A. .V.
Nurk,
In stitu te of G eneral and M olecular P a thology, T artu S ta te
U niversity, T artu
S ubcutaneous
ad m inistration
of
caerulein
(100—
500 n g /k g ) sig n ific an tly reduced the developm ent of picroto­
xin (8 m g /k g ) seizures in m ale mice. The sam e doses of
caerulein inhibited
3H -flunitrazepam b inding in in v iv o
experim ents. P roglum ide, an a n ta g o n ist of cholecystokinin
receptors, in low dose (5 m g /k g ) p o tentiated the effects
of caerulein (100 n g /k g ), w hereas the ad m in istratio n of
proglum ide in high dose (25 m g/kg) reduced the action of
caerulein on 3H -flunitrazepam b in d in g and picrotoxin sei­
zures. C aerulein (5-1000 nM) decreased 3lf-flu n itraz ep a m
b in d in g in in vitro experim ents o n ly after supplem entation of
the b inding m edium w ith 120 mM NaCI and 5mM KCI. The
resu lts su g g e st the possible interaction of caerulein with
chloride ionophor. It seem s probable that the direct in te r­
action of caerulein w ith chloride ionophor in involved in the
inhibitory effect of caerulein on picrotoxin seizures and
3H -flunitrazepam binding.
Comparison of Motor Depressant Effects
of Caerulein and N-Propylnorapomorphine
in Mice
E E R O V A S A R , M A T T ! M A IM E T S , A N T S N U R K ,
A N D R E S S O O S A A R A N D L E M B IT A L L IK M E T S
Laboratory o f Psychopharniacolo^y , Institute of General and M olecular Pathology. Tartu State University,
34 Burdenko Street. 202 400 Tartu, Estonia. U .S .S .R .
R e c e iv e d 18 A p ril 1985
VASAR, E .. M. MAIMETS, A. NURK, A. SOOSAAR AND L. ALLIKM ETS. Comparison o f m otor depressant effects
oj caerulein and N-prupyhwraponunphine in mice. PHARMACOL BIOCHEM BEHAV 24(3)469-478, 1986.—The motor
depressant effects o f caerulein and N-propylnorapomorphine (NPA) were compared in male mice. Caerulein (l-50/ig/kg
SC) in a dose dependent manner depressed the exploratory activity, whereas NPA in lower doses (0.5-10 ^g/kg SC)
decreased the motor activity, but in higher doses (over 50/xg/kg) had stimulating effect on the exploratory behavior. In mice
selected according to their motor response after administration of 100 Mg/kg NPA to weak and strong responders, the low
dose of NPA (1 Mg/kg) similarly suppressed motor activity in both selected groups, while the effect of caerulein (2 jtg/kg)
was apparently higher in weak responders. Destruction of catecholaminergic terminals by 6-hydroxydopamine (60/Ag ICV)
reversed completely ihc motor depressant effect of N PA, whereas degeneration of serotoninergic terminals (5.7-dihydroxyiryptamine 60
ICV or p-chloroampheiainine 2x15 mg/kg IP) enhanced the sedative effect of NPA. The motor depressant
effect of caerulein remained unchanged after lesions of monoaminergic terminals in forebrain. Subchronic haloperidol (0.25
mg/kg IP. twice daily during 14 days) treatment, reducing significantly the density of high-afftnity dopamine]- and
serotonin.-receptors, decreased the motor depressant action of caerulein. It is possible that motor depressant effect of
caerulein. differently from the action of NPA, is mediated through the high-affinity dopamine.-receptors and in lesser
extent through the high-affinity serotonin.-receptors.
Exploratory activity
Caerulein
N-propylnorapomorphine
T H E s u p p re s s io n o f s p o n ta n e o u s lo c o m o to r activ ity by low
d o se s o f a p o m o rp h in e in ro d e n ts is a w idely stu d ie d b e h a v ­
ioral p h e n o m e n o n . It is g e n e ra lly a c c e p te d th at th e sed a tiv e
ac tio n o f ap o m o rp h in e a n d its m ore p o w erful ana lo g
N -p ro p y ln o ra p o m o rp h in e (N P A ) is m ed iated th ro u g h the
stim u latio n o f d o p a m in e “ a u to re c e p to r s ," inhibiting the
d o p am in erg ic n e u ro n s ac tiv ity [9, 10. 38. 45]. T h is o p in io n is
su p p o rte d by v a rio u s in v e stig a tio n s. T h e su b c u ta n e o u s a d ­
m in istra tio n o f a p o m o rp h in e in low d o se s inhibited th e firing
rate o f d o p a m in e rg ic n e u ro n s in m e se n c ep h a lo n (2), d e ­
c re a se d d o p a m in e re le a se an d s u p p re sse d d o p am in e tu rn ­
o v e r in fo reb rain stru c tu re s (32,44]. L esio n o f dop am in e rg ic
te rm in a ls by 6 -h y d ro x y d o p a m in e an d a d m in istratio n o f dif­
fe re n t n e u ro le p tic d ru g s in low d o se s re v e rse d th e inhibiting
a c tio n o f ap o m o rp h in e o n b e h a v io r and dop am in e rg ic
n e u ro n s a c tiv ity [3. 42, 46]. H o w e v e r, som e re c e n t in v e sti­
g a tio n s d e m o n s tra te d a m o re c o m p lic ated n a tu re o f a p o m o rp h in e ‘s a c tio n in low a n d m o d e ra te d o se s. It w as found (16)
th a t h alo p erid o l a n d su lp irid e re v e rse d the se d a tiv e effe ct o f
m o d e ra te d o s e (150 ^ g /k g ) o f a p o m o rp h in e , w h e re a s Ihe a c ­
tio n o f low d o se (25 M£/kg) o f ap o m o rp h in e w as re sista n t to
th e an ta g o n iz in g a c tio n o f n e u ro lep tic d ru g s. T h e co m p li­
c a te d n a tu re o f a p o m o rp h in e 's a c tio n in low d o se s w as d e ­
s c rib e d a lso in c h ro n ic sc h iz o p h re n ic p a tie n ts, ev id en tly re ­
s is ta n t to n e u ro le p tic m e d ic a tio n . T h e red u ctio n o f s c h iz o ­
Dopamine..-rcceplors
Serotonin..-receptors
ph re n ic sy m p to m a to lo g y w as d e m o n stra te d in a p p ro x ­
im ately 50% o f p a tie n ts, su ffering m ainly fro m para n o id
sc h iz o p h re n ia (47,48]. It w as q u ite su rp risin g th a t a p o m o r­
p h ine p o sse sse d its b eneficial ac tiv ity w h en c o a d m in iste re d
w ith n e u ro le p tic d ru g s, b u t n o t alo n e (1, 21, 37].
O b v io u sly sim ila r s u p p re ssio n o f a n im a ls’ s p o n ta n e o u s
b e h a v io r w as fo u n d a fte r sy ste m ic ad m in istra tio n o f
ch o le c y sto k in in o c ta p e p tid e (C C K -8) an d ca e ru le in in m ice
(56, 57, 58]. C C K -8 and c ae ru le in sig n ificantly p o te n tia te d
ap o m o rp h in e-in d u c ed inhibition o f d o p a m in erg ic n e u ro n s in
m e se n ce p h alo n (30]. T h e re is s tr ic t e v id e n c e th a t C C K an d
d o p a m in e co e x ist in so m e m e se n c ep h a lic c e lls in n e rv atin g
fore b rain lim bic and co rtic a l re g io n s [31]. In a d d itio n , C C K
h a s b e en re p o rte d to d e c re a se d o p am in e tu rn o v e r in th e d is­
c re te regions o f c a u d a te -p u ta m e n (24|. H o w e v e r. C C K also
d e c re a se d se ro to n in tu rn o v e r (51], w h e re a s ap o m o rp h in e
had th e o p p o site e ffect o n se ro to n in m etab o lism (26). R e­
ce n tly the rapid an d lo ng-iasting re d u c tio n o f p sy ch o tic
s y m p to m s, m ainly n e g ativ e , in sc h iz o p h re n ic p a tie n ts a fte r
a d m in istratio n o f diffe ren t C C K -relate d p e p tid e s w as
d e m o n stra te d (7, 39, 40].
T h e m ain task o f the p rese n t investigation w as to co m pare
the m echanism s o f inhibiting ac tio n o f apom o rp h in e and C C K
on the an im als' b ehavior. T h e atte n tio n w as dra w n to th e study
o f interaction o f cae ru le in and N PA w ith dopam ine- and
VASAK. M AI Ml
s e m lo n in e rg ic m e c h a n ism s. C a e ru le in ;uui NPA w ore
se le c te d lu r Ilk* p re se n t in v e stig a tio n a s th e m ost c lfe c h v e
c o m p o u n d s a m o n g , re s p e c tiv e ly , C C K 8 an d a p o m o ip h m c
IS , N t J K K . S O O S A A K A N D A I I I K M I I S
------
■... i 4
analogs 18. 55. 561.
(ilv N L R A L M IT M O I)
sZ i 4 .
U,
+
A n im a ls
..
—
M ale a lb in o m ice w e ig h in g 2 5 ± 3 g w ere u se d . M ice w ere
m a in ta in e d at 2 0 ± 2 °C a n d o n 12 h r light, b etw ee n 8 a.m . and
8 p .m .. w ith fo o d an d w a te r allo w ed a d lib.
+
M e a s u re m e n t o f S p o n ta n e o u s /,<д -om otor A c tiv ity
S p o n ta n e o u s lo c o m o to r a c tiv ity w a s m e a su re d in gro u p e d
a lb in o m ic e , 10 an im a ls in e a c h g ro u p , b e tw e e n 10 a.m . a n d 4
p.m . Im m e d ia te ly a fte r sy ste m ic a d m in istra tio n o f d ru g s a
g ro u p o f m ice w as p laced in th e m iddle o f an o p e n-field cage.
T h e o p e n -fie ld c o n siste d o f a 1 x 1 m a re a su rro u n d e d by a 40
cm high w all. T h e lo c o m o to r ac tiv ity o f anim a ls w as c o u n te d
b y 5 in d e p e n d e n t p h o to c e lls lo c a te d in w alls. In te rru p tio n s
o f th e light b e a m s w ere re c o rd e d e le c tro m e c h a n ic a lly and
th e lev el o f lo c o m o to r a c tiv ity w as e x p re sse d in c o u n ts p e r
15 o r 30 m in p e rio d . T h e e x p e rim e n t w as rep e a te d w ith e a c h
d ru g c o m b in a tio n at le a st th re e tim e s on differe n t d a y s an d
th e d a ta a n a ly z e d u sin g S tu d e n t’s /-te s t.
S e le c tio n o f M ic e A c c o r d in g to T h eir M o to r R e sp o n se lo
A d m in is tr a tio n o f N P A
T h e re e x is ts th e p o ssib ility o f se le c tin g ra ts a cco rd in g to
ih e ir m o to r re s p o n s e a f te r 50 Mfc^kg N PA tre a tm e n t 115). A
sim ilar a tte m p t w as m a d e fo r s e le c tio n o f m ice. In the p re s ­
e n t s tu d y th e se le c tio n w a s p e rfo rm e d w ith su b c u ta n e o u s
a d m in is tra tio n o f 100 /ug/kg N PA in 400 m ale m ice . T he e x ­
p e rim e n t w a s c a rrie d out in indiv id u al c a g es. T h e ca g e for
m e a su rin g in d iv id u a l lo c o m o to r a c tiv ity w as a c y lin d e r w ith
an in n e r d ia m e te r 40 cm an d 2 p h o to c e lls for d e te c tio n o f
lo c o m o to r a c tiv ity . L o c o m o to r a c tiv ity w as c o u n te d b e ­
tw e e n 15 an d 30 m in a f te r s u b c u ta n e o u s N P A (1(H) /xg/kg)
tre a tm e n t.
FIG. 1. The effect of different doses of N-propylnorapomorphine
and caerulein on exploratory behavior in mice. Each point in the
figure represents mean value of three independent studies in
grouped mice (10 animals in group). Abscissa—the dose of NPA or
caerulein in /xg/kg. Caerulein— A.. NPA— B, The mean value for
saline treated group was I182±170 counts during 30 min. Statisti­
cally evident differences from saline treated mice: */><0.05;
**/»<0.01 (Student's/-test).
TA BLE 1
THE ACTION OF CONCOMITANT ADMINISTRATION OF
CAERULF.IN AND NPA ON MICH SPONTANEOUS
LOCOMOTOR ACTIVITY
Spontaneous locomotor activity of r
L e s u m s o f B rain M in u /a n u n e r y ic T e rm inals
Drug do.se
M o n o a m in e rg ie n e u ro to x in s 6 -h y d ro x y d o p a m in e (6O H D A ) an d 5 ,7 -d ih y d ro x y try p ta m in e (5.7 -D H T ) w ere dis­
so lv e d in 0.1% so lu tio n o f a s c o rb ic a cid . 6 -O H D A (60 /xg in 5
^il) a n d 5 ,7 -D H T (60 ^ g in 5 \xI) w e re in je c te d in to th e right
la te ra l v en tric le o f m ice u n d e r th e e th e r a n e sth e sia . T h e b e ­
hav io ra l a n d b in d in g e x p e rim e n ts w ere c a rrie d o u t 8 d a y s
a f te r th e in je c tio n o f n e u ro to x in s . F in a lly , th e inje ctio n site s
w e re c o n firm e d h isto lo g ically to be lo ca te d w ithin th e right
la te ra l v e n tric le . p -C h lo ro a m p h e ta m in e in n e u ro to x ic d o se
(2 x 1 5 m g/kg 8 a n d 7 d a y s b e fo re th e e x p e rim e n t) w a s a lso
u se d fo r lesioning ol sc ro to n in crg ic te rm in a ls J5J. T he e ffec t o f
n e u ro to x in s o n th e c o n te n t o f in o ita in m es a n d th e ir m a jo r
m e ta b o lite s in b rain s tr u c tu r e s w as a s s e s s e d bioc h em ic ally
u sin g fliio rim ctric a s s a y J20J.
In I Mil 4hSptp eri> n c H indin^
‘Jl-s p ip c ro n c (5 /ig /k g . 17 C i.m m o le . A m e rsh a m I n te r n a ­
tio n a l. U .K .) w as in je c te d s u b c u la n e o u s l\ into th e d o rsal
p art o f m o u se n eck N PA (5 a n d 50 /tg 'k g ) a n d v a em lem
120-250 p g /k g ) w e re u se d to in hibit 'H -sp ip c ro n e binding.
T w ti4 lo s c s o f N'PA w ith d iffe re n t a c tu m o n ro d e n i b e h a v io r
w e re se le c te d b e c a u s e tw o site^ w ith differe n t alfinilN for
Saline
NPA 0.5 /ig/kg
Caerulein 2 Mg/kg
NPA f Caerulein
NPA 10 /tg/kg
Caerulein ! м^/kg
NPA + Caerulein
15 min
608
380
352
170
158
560
33
.t
±
*
±
.♦
T
r
58
42*
38*
I6t
121
57
4$
Counts during
30 min
<7t
100
63
58
28
26
92
5
1230
780
746
276
240
1080
61
±
t
±
±
2
±
i
162
68*
55*
24t
J2t
182
8}
%
100
63
61
22
20
88
5
The mean values o f four independent experiments on grouped
mice (10 animals in group) are presented. */»<0.05; t/><0.0l:
t/»* -0.001 (Student s paired / test, in relation to saline treated
animals).
do p a m in e a n d its ag o n ists e x iste d on d o p a m in e..-rece p to rs
118 .2 7 1. F iv e /*g/kg N P A is ED-*, for su p p re ssio n o f e x p lo ra ­
to ry a c tiv ity in m ice , w h e rea s 50 /*g/kg N PA is ED *, fo r
m o to r e x c ita tio n in ro d e n ts 18}. N P A a n d c a e ru le in w ere
a d m in iste re d I* n u n b efo re 'H -sp tp ero n e- T h e a n im a ls (6
m ice p e r g ro u p ) w e re sacrific ed 20 m m a fte r ‘H -sp ip e ro n e
tie a lin e n t by c e rv ic a l d islo c a tio n . T h e b ra in s w e re rap id ly
471
C A E R U L E IN A ND DOPAM INERGIC MECHANISMS
TABLE 2
THE EFFECT O F C AERULEIN AND NPA ON EXPLORATORY ACTIVITY AND 'H-SPIPERONH IN VIVO
BINDING IN MICE SELECTED WITH 100м */к| NPA
Inhibition of locomotor activity
to 100 /Ag/kg NPA
Weak Responders
Strong Responders
Motor activity counts during 30 min
Drug/dose
Saline
NPA 1 ftg/kg
Caerulein 2 ^g/kg
%
1168 ± 98
550 2: 58
292 ± 34*
100
47
25
%
1224 ± 115
630 ± 52
690 ± 68
100
52
56
Inhibition of ‘H-spiperone binding
to 100 Mg/kg NPA
Weak Responders
Strong Responders
cpm per gram tissue
NPA 5 Mg/kg
NPA (50-5) Mg/kg
C aerulein 100 jxg/kg
Subcortex
Dorsal cortex
Subcortex
Dorsal cortex
+ 1600 ± 280t
5180 ± 380*
+ 1800 - 360+
+ 750 ± 200t
3750 ± 280*
*■1200 ± 300+
9900 ± 1020
10200 ± 980
11840 г 930
10950 ± 1200
6900 ± 520
11150 ± 1060
The experim ents were carried out 10-12 days after mice selection.
The mean values of three independent experiments are advanced in table. + —Stimulation of ;,Hspiperone binding. */><0.05; +/><0.01 (Student's paired /-test, compared to strong responding mice).
r e m o v e d a n d d o rs a l c o rte x a n d s u b c o rtic a l fo re b ra in s tru c ­
tu re s (stria ta a n d lim b ic str u c tu re s ) w ere d isse c te d on ice.
T h e d iss e c te d b ra in a r e a s o f e a c h g ro u p w e re p o o le d and
h o m o g e n iz e d u sin g a g la ss-te flo n h o m o g e n iz e r by han d d u r ­
ing 1 m in. T h e h o m o g e n iz a tio n p ro c e d u re w as p erfo rm e d in
ice-co ld T ris-H C I b u ffe r (50 m M . pH 7.4, 20°C) in the volum e
o f 40 mg tis s u e p e r m l. A fte r h o m o g en iza tio n 0.5 ml (20 mg
tissu e ) o f su s p e n s io n w a s p ip p e te d into 6 p o ly p ro p y le n e
tu b e s (1 .5 m l) a n d c e n trifu g e d d u rin g 10 min a t 9 0 0 0 x g . T h e
su p e rn a ta n t w a s c a re fu lly d is c a rd e d an d rem a in in g p ellet
w as w a sh e d a n d c u t in to vials. R a d io a c tiv ity o f sa m p le s w as
c o u n te d a f te r s ta b iliz a tio n in B ra y scin tilla tio n co c k ta il
w ithin 12 h o u rs in B e c k m a n L S 6800 w ith co u n tin g effic ac y
43%. T h e b in d in g e x p e rim e n ts w e re r e p e a te d a t le a st th re e
tim e s a n d th e d a ta a n a ly z e d u sin g S tu d e n t's z -te s l.
D ru g s
D ru g s u se d in th e p re s e n t in v e stig a tio n w ere c a eru lein
(C e ru le tid e , F a rm ita lia C a rlo E rb a , Italy ), h alo p erid o l (G ed eo n R ich ter, H u n g a ry ), N -p ro p y ln o ru p o m o rp h in c (S terlingW in th ro p , U SA ), p -c h lo ro a m p h e la m in e, 6-h y d ro x y d o p am in e,
5 .7 -d ih y d ro x y try p ta m in e (S igm a, U SA ). C ae ru le in , c o m m e r­
cial solu tio n o f halo p erid o l and p-ch lo ro am p h etam in e w ere
d isso lv ed in saline. T h e in jectio n solution o f N PA w as prep a red
in 0.001 N H CI. E a c h injection w as do n e in a volum e o f 0.1
m l/10 g b o d y w eight.
E X P E R IM E N T I: T H E I N V O L V E M E N T O F
D O P A M IN E R G IC M E C H A N IS M S IN T H E M O T O R
D E P R E S S A N T A C T IO N O F C A E R U L E IN A N D
N -P R O P Y L N O R A P O M O R P H IN E
T h e aim o f e x p e rim e n t I w as to s tu d y th e role o f
d o p a m in e rg ic m e c h a n is m s in th e se d a tiv e e ffe c ts o f c aeru -
F1G. 2. The changes in motor depressant effect of caerulein and
N-propylnorapomorphine after intraventricuJar administration of
6-hydroxydopamine. White signs— the action of NPA; black
signs—caerulein. Triangles—after administration of 6-OHDA;
squares—after mlraventricular injection o f 0. \% ascorbic acid.
Abscissa— the dose of NPA or caerulein in мв/kg. The mean value
for saline medicated mouse was 1098± 156 counts during 30 min in
the case of 0.1% ascorbic acid and 780±78 in case of 6-OHDA.
Statistically evident differences from ascorbic acid pretreated group:
*/?<0.05; **//<0.01 (Student's /-test).
Icin an d N P A . T he p ro b le m s u n d e r ex a m in a tio n w e re: ( I) th e
a c tio n o f diffe ren t d o se s o f c aeru le in a n d N P A on e x p lo ra ­
to ry ac tiv ity in m ice; (2) the e ffec t o f c o n c o m ita n t u se o f
c a e ru le in an d N P A on lo c o m o to r a c tiv ity in m ice; (3) the
a c tio n o f ca e ru le in and N P A on ex p lo ra to ry a c tiv ity and :,H-
VA SAR. M AIM ET S, N U R K , SO O SA A R A N D A L L I K M E T S
472
TABLE 3
THE CHANOES IN •H-SPIPERONE BINDING AFTER INTRACEREBROVENTRICULAR ADMINISTRATION OF ^HVÜROXYDOPAM INF AND
LONG-TERM ADMINISTRATION OF HALOPERIDOL AND P-CHLOROAMPHETAMINE
_____________________
Inhibition of 3H-spiperone binding
с pm per gram tissue
Drug/dose
Saline
6-OHDA 60 >xg
PC A 2X15 mg/kg
Haloperidol 0.25 mg/kg
Subcortex
7800
14400
4240
N00
±
2
±
±
580
930t
560*
120+
Dorsal cortex
6950
11800
3320
2150
Caerulein 50 fj.g/kg
NPA (50-5)
NPA 5 /ig/kg
±
±
±
±
620
1060*
3l0t
380*
Subcortex
5200
1020
5800
10400
±
±
±
±
640
200t
670
980t
Dorsal cortex
4000
2040
3900
6700
±
±
±
£
480
240*
350
530*
Subcortex
5250
3000
3500
+ 3600
±
±
±
±
420
470*
430
320}
Dorsal cortex
4750
2600
3600
+ 400
±
±
±
±
390
320
410
I20t
The binding of ’H-spiperone after intraventricular administration of 0 .1% ascorbic acid did not differ from the binding after long-term
saline treatment. + —Stimulation o f ’H-spiperone binding. *p<0.05; t/> < 0.0l: tp < 0 .001, compared to saline treated mice (Student’s r-test).
s p ip e ro n e bin d in g p a ra m e te rs in p h arm ac o lo g ica lly selec ted
m ice. T h e an im als w ere se le c te d a c c o rd in g to th e ir m o to r
re s p o n se a fte r a d m in istra tio n o f 100 M g/kg N P A into tw o
g ro u p s— w e a k an d s tro n g re sp o n d e rs. T he u n ev e n m o to r r e ­
a c tio n a fte r N P A a d m in istra tio n re fle c ted the differen t d e n ­
sity o f p o s ts y n a p tic d o p a m in e .-re c e p to rs in ro d e n ts (14, 15,
29] ; (4) th e e ffe c ts o f c a e ru le in a n d N P A on lo c o m o to r a c tiv ­
ity in m ice a n d -'H -sp ip ero n e bin d in g p a ra m e te rs a fte r d e ­
s tru c tio n o f p re s y n a p tic d o p a m in e rg ic term in als by
6 -h y d ro x y d o p a m in e .
METHOD
T h e g ro u p o f m ice w as p la c e d in to the c e n te r o f a n openfield c a g e im m ed iately a fte r s u b c u ta n e o u s injectio n o f caeru lein (1 -5 0 M g/kg) o r N P A (0 .2 -5 0 д а /kg). A fter se le ctio n o f
a p p ro p ria te d o s e s , giv in g m a rk e d su p p re ssio n o f s p o n ta n e ­
o u s lo c o m o to r a c tiv ity , th e e ffe c t o f co n c o m ita n t u se o f
caeru lein an d N PA w as stu d ied . T h e a c tion o f N PA (I д а /kg)
an d c a eru lein (2 y.g/kg) w as also exam in ed in m ice se lec te d a c ­
c o rd in g to th e ir m o to r re s p o n se to th e a d m in istra tio n o f N P A
in a hig h d o s e (100 д а /kg). T h e g ro u p s o f w eak an d stro n g
re s p o n d e rs to 100 p.g/kg N P A w e re s e le c te d am o n g 400 m ice.
T h e m otor a c tiv ity w a s a s s e s se d in in d ividual c a g e s fro m 15
to 30 m in a f te r 100 д о /kg N P A ii\jectio n. T h e m e an v alu e o f
m otor a c tiv ity f o r th e first g ro u p (w eak re sp o n d e rs) w as
3 6 ± 3 .8 c o u n ts d u rin g 15 m in a n d 2 1 6 ± !5 .2 fo r th e se co n d
(stro n g re sp o n d e rs ). T h e re sp o n s e o f th e se tw o g ro u p s to
sa lin e a d m in istra tio n did n o t diffe r m a rk ed ly . It w as 1168+98
c o u n ts d u rin g 30 m in fo r w e a k r e s p o n d e rs a n d I 2 2 4 ± I I5
cou nts for s tro n g re s p o n d e rs . S im u lta n eo u sly w ith b e h a v ­
io ral in v e stig a tio n s 3H -sp ip e ro n e in v iv o b inding stu d ie s
w e re p e rfo rm e d . N P A (5 an d 50 Mg/kg) an d ca eru lein (100
Mg/kg) w e re u se d a s d isp la c in g d ru g s. T w o d o se s o f N P A
w e re a d m in iste re d to d e m o n s tra te tw o d istin c t b in d in g sites
for N P A on d o p a m in e ,- a n d s e ro to n in ,-receptors. In h ib itio n
o f ’H -spiperone b in d in g b y 5 Mg/kg N P A exp ressed th e
am ount o f hig h -aflin ity site s fo r N P A , w hereas the differe n ce
b e tw e e n th e inh ib itin g a c tio n o f 50 an d 5 Mg/kg N P A d e m o n ­
s tra te d the n u m b e r o f lo w -aflin ity site s. C a tec h o la m in erg ic
neurotoxin 6-O H D A (60 д а ) w a s in je c ted into th e right lat­
e ra l cerebral v e n tric le in a v o lu m e o f 5 м I d u rin g 3 m in u n d er
eth er anesth esia. S ev en d a y s w ere allow ed fo r re c o v e ry from
m tn ventricu lar intervention. A fter com pletion o f behavioral
exp erim en ts the site o f m icroinjection w a s detected h istolog­
ic»» у .
RESULTS
E ffe c t o f C aerulein u n d N P A on E x p lo ra to ry M o to r A c tiv ity
C ae ru lein in a d o se d e p e n d e n t m a n n e r d e p re sse d the e x ­
p lo ra to ry a ctiv ity in m ale m ice (Fig. 1A). T w o д а /kg c a e ru ­
lein c a u se d th e m inim al sig n ific an t re d u c tio n o f m o to r a c tiv ­
ity a n d 20-50 д а /kg th e m axim al e ffect. L o w d o se s o f N P A
also re d u c e d th e an im a ls' sp o n ta n e o u s lo c o m o to r ac tiv ity .
0.5 Mg/kg N P A c a u se d re m a rk a b le a n d 10 д g/kg N P A in­
d u c e d th e m axim al re d u c tio n o f m ice e x p lo ra to ry b e h a v io r
(Fig. 1B). T h e fu rth e r e le v a tio n o f N P A d o se did n o t en h a n c e
th e se d a tiv e a c tio n , b u t o n th e c o n tra ry 50 д а /kg N P A h ad a
m ild stim u la tin g e ffec t o n m o to r ac tiv ity o f m ice. A fter
c o a d m in istra tio n o f N P A an d c a e ru le in the re d u c tio n o f
m o to r activ ity w as o b v io u sly h ig h er co m p a re d to th e tre a t­
m e n t o f bo th d ru g s alo n e (T ab le I). O n e Mg/kg ca e ru le in ,
w hich did n o t significantly a ife c t th e m ice b e h a v io r, p o te n ­
tiated the m o to r d e p re s s a n t e ffec t o f N P A (10 Mg/kg). T h is
co m b in a tio n o f dru g s c a u se d n e arly c o m p le te s u p p re ssio n o f
lo c o m o to r ac tiv ity . In m ice , s e le c te d a c c o rd in g to th e ir
m o to r re sp o n se a fte r a d m in istra tio n o f 100 ^ g /k g N P A , 1
д а /kg N P A in a sim ilar m a n n e r su p p re sse d e x p lo ra to ry a c ­
tiv ity in stro n g a s w ell a s in w ea k r e s p o n d e rs (T a b le 2).
H o w e v e r, th e se d a tiv e e ffec t o f c a e ru le in (2 д а /k g ) w a s d e ­
p e n d e n t o n th e m ice sen sitiv ity to 100 д а /k g N P A . In s tro n g
re s p o n d e rs th e s e d a tiv e effe ct o f c a e ru le in w as lo w er. S ignif­
ic an t d iffe re n c e s w e re fo u n d a lso in ’H -sp ip e ro n e b inding
p e rfo rm ed in " i n v iv o " c o n d itio n s (T ab le 2). In w e a k re ­
sp o n d e rs c a e ru le in (100 д а /kg) stim u lated ’H -sp ip e ro n e b in d ­
ing in b o th b ra in reg io n s stu d ie d , w h e re a s in stro n g re s p o n ­
d e rs it had th e o p p o site effe c t, in h ib itin g ’ H -sp ip e ro n e b in d ­
ing (T a b le 2). F iv e д а /kg N P A a lso in c re a se d ’ H -sp ip e ro n e
b in d in g in w ea k re sp o n d e rs, w hile th e d isp lac in g p o te n c y o f
50 д а /kg N P A in w ea k re sp o n d e rs w a s lo w e r th a n th e e ffec t
o f 5 д а /kg N P A in s tro n g re sp o n d e rs.
E ffe c t o f 6-O H D A o n L o c o m o to r E ffe c ts o f C a erulein a n d
N P A , u n d *H -Spiperone B in d in g
In tra v e n tric u la r a d m in istra tio n o f 6-O H D A (60 д а ) in­
d u c e d m o re th a n 60% re d u c tio n o f d o p a m in e an d its m e ta b o ­
lite 3 .4 -d ih y d ro x y p h e n y la c e tic ac id (D O PA C ) le v e ls in
s tria ta l slic es (d o rsa l c o r te x , stria ta a n d m e so lim b ic s tru c ­
tu re s) o f m ice b rain w ith o u t ch an g in g m a rk ed ly s e ro to n in
le v els. S im u lta n e o u sly th e re d u c tio n o f s p o n ta n e o u s lo co m o ­
to r a c tiv ity w as se e n in m ice a fte r 6-O H D A tre a tm e n t (F ig.
CAERU LEIN AND DOPAMINERGIC MECHANISMS
TA BLE 4
TH E EFFECT O F HALOPERIDOL AND CAERULEIN ON MICE
EXPLORATORY ACTIVITY AFTER 14 DAYS
HALOPERIDOL TREATMENT
Saline
Haloperidol
M otor activity counts during 30 min
%
Drug/dose
Saline
Caerulein 2 ^g/kg
Haloperidol 50 /xg/kg
Caerulein + Haloperidol
FIG. 3. The influence o f p-chloroamphetamine and 5,7dihydroxytryptamine pretreatm ent on motor depressant effect of
caerulein and N-propylnorapomorphine. White bars—caerulein 2
Mg/kg, striped bars— NPA 0.5 pg/kg and black bars—
caerulein + NPA. The mean value of motor activity for saline treated
group was I180±122 in case o f long-term saline administration,
1020± 140 in case of 5,7-DHT and 1270± 178 counts during 30 min in
case of PCA. •/><0 05; * V < 0.02; •••p < 0 .0 1 ; ••••p < 0 .0 0 1 , com­
pared to saline pretreatm ent (Student's /-test).
2). N P A c o m p le te ly lo s t its s e d a tiv e a c tio n a n d stim u la te d
th e m ice e x p lo ra to ry a c tiv ity a fte r a d m in istra tio n o f
6 -O H D A , w h ile th e a c tio n o f ca e ru le in rem a in e d u n c h an g e d
(Fig. 2). In b in d in g e x p e rim e n ts 6 -O H D A c a u se d a signifi­
c a n t in c re a se in d isp la c in g a c tio n o f 5 д а /kg N P A , b u t re­
d u c e d th e p o te n c y o f 50 fxg/kg (T a b le 3). T h e in h ib itin g a c ­
tion o f cae ru le in (5 0 /ig /k g ) o n ‘H -sp ip e ro n e b inding w as a lso
s o m e w h a t lo w e r a fte r 6-O H D A tre a tm e n t. A d m in istra tio n o f
6 -O H D A a lte re d 'H -s p ip e ro n e b inding m ore re le v a n tly in
su b c o rtic a l s tr u c tu r e s th a n in d o rsa l c o rte x .
DISCUSSION
C a e ru le in an d N PA in low d o s e s c a u se d sim ilar s u p p re s ­
sio n o f e x p lo ra to ry a c tiv ity o f m ice. C o a d m in istra tio n o f
N P A an d c a e ru le in e v id e n tly p o te n tia te d th e ir d e p re ssiv e
a c tio n o n b e h a v io r. T h e re is c le a r e v id e n c e fo r c o e x iste n c e
o f d o p a m in e an d C C K -8 in th e sa m e m e se n ce p h a lic
d o p a m in e rg ic n e u ro n s [31). It w as d e m o n stra te d th a t C C K -8
and c a e ru le in p o te n tia te d a p o m o rp h in e -in d u c e d inhibition o f
d o p a m in e rg ic n e u ro n s in m e se n c e p h a lo n [30]. L esio n o f
p re sy n a p tic d o p a m in e rg ic term in a ls by 6-O H D A c o m p lete ly
re v e rse d th e m o to r d e p re ss a n t a c tio n o f N P A , d e m o n stra tin g
th e p re v a le n t ro le o f p re s y n a p tic m ech an ism s in the a c tio n o f
N P A . T h e m o to r d e p re s s a n t effect o f c ae ru le in w as re sistan t
to th e a d m in is tra tio n o f 6 -O H D A . T h e d iffere n t ac tio n o f
c a e ru le in in se le c te d m ice a c c o rd in g to th e ir re sp o n se to 100
/Ag/kg N P A re v e a le d th a t th e s e d a tiv e e ffec t o f c a e ru le in w as
m o re p ro b a b ly re la te d to p o s ts y n a p tic d o p a m in e re c e p to rs.
T h e se d a tiv e e ffect o f c a e ru le in w as hig h e r in w ea k N PA
re s p o n d e rs , w h ich ev id e n tly had low er d e n sity o f
dopam ine^- an d se ro to n in r re c e p to rs in fore b rain stru c tu re s.
It w as e sta b lish e d th a t d o p a m in e .j-re ce p to rs had on e high*
affinity site fo r n e u ro le p tic d ru g s , but tw o sites— low - and
h ig h -affin ity — fo r d o p a m in e , ap o m o rp h in e and N PA 118.27].
In w eak re s p o n d e rs c a e ru le in a n d 5 Mg/kg N PA stim u la te d
'H -sp ip e ro n e bin d in g , bu t in h ib ited it in s tro n g re sp o n d e rs.
N PA had sim ilar se d a tiv e a c tio n in b o th g ro u p s o f sele cted
m ice, re v e a lin g th a t d o p a m in e " a u to r e c e p t o r s " w ere not re ­
la te d to h ig h-affinity d opam ine.:-re c e p to rs [27]. C o stall [ 14J
1180
680
880
620
♦
±
±
-
188
78*
96
64*
100
58
75
53
%
1054
920
1280
520
±
±
±
±
143
89
160
56t
100
90
122
49
The investigation was performed 72 hours after cessation of halo­
peridol or saline treatment. The mean values of three independent
studies are advanced. *p<Q.05; tp < 0 .0 1, compared to saline treated
animals (Student’s /-test).
h a s found th a t in s tro n g re sp o n d in g ra ts to N P A (50 tig /k g )
th e c o n te n t o f d o p am in e (in n u c le u s a c c u m b e n s) w as a p p ro x ­
im ately tw ice hig h e r th a n in w e ak re sp o n d e rs. I t a p p e a rs th a t
d isp la cin g p o te n c y o f ca e ru le in ag a in st ^H -sp ip ero n e b in d in g
is d e p e n d e n t on d o p am in e c o n te n t in b rain s tr u c tu re s an d
c a e ru le in on ly m o d u la te s th e in te ra c tio n o f e n d o g e n o u s
d o p a m in e w ith d o p a m in e ^ -rec ep to rs. It is q u ite p o ssib le th a t
th e se d iffe re n c e s in the a c tio n o f c a e ru le in o n ‘H -sp ip e ro n e
b inding in tw o se le c te d g ro u p s o f m ice a re lin k ed to th e
d iffere n t se d a tiv e e ffe c ts o f c a e ru le in in th e se an im a ls.
In c o n c lu sio n , e x p e rim e n t I e v id e n c e s th a t th e se d a tiv e
e ffect o f ca e ru le in is re la te d , differe n tly fro m N P A a c tio n , to
p o stsy n a p tic d o p am in e re c e p to rs. C a eru lein se e m s to a c t as
a fun ctio n a l a n ta g o n ist o f b e h a v io r stim u latin g e ffec t o f
d o p a m in e.
E X P E R IM E N T 2: T H E E F F E C T O F S E R O T O N IN E R G IC
L E S IO N S A N D L O N G -T E R M H A L O P E R ID O L
T R E A T M E N T O N M O T O R D E P R E S S A N T A N D ЛНS P IP E R O N E B IN D IN G IN H IB IT IN G E F F E C T S O F
C A E R U L E IN A N D N -P R O P Y L N O R A P O M O R P H IN E
E x p erim e n t I sug g este d d iffe re n c e s in th e m ec h a n ism o f
se d a tiv e ac tio n o f c a e ru le in an d N P A . T h e aim o f e x p e rim e n t
2 w as to stu d y fu rth e r th e m ec h a n ism s o f a c tio n o f ca eru le in
an d N P A using s e ro to n in e rg ic lesio n s a n d lo n g -te rm
ad m in istra tio n o f ha lo p e rid o l.
METHOD
S e ro to n in e rg ic n e u ro to x in 5 ,7 -D H T (60 *tg) w »s in jec ted
in to th e right latera l v e n tric le in a vo lu m e o f 5 /xl d u rin g 3 min
u n d e r e th e r an e sth esia . S ev e n d a y s w ere allow ed for recovery
from in tra v e n tric u la r in te rv e n tio n . A fte r co m p letio n o f b e ­
h a v ioral e x p e rim e n ts the site o f m icro in jec tio n w as d e te c te d
h istologically. A c co rd in g to so m e a u th o rs [5, 28, 35), a d m in ­
istra tio n o f p -ch lo ro a m p h e ta m in e (P C A ) in high d o se s c a u se s
d e g e n e ra tio n o f se ro to n in erg ic term in a ls in fo re b ra in s tru c ­
tu re s . PCA w as in je cte d tw ice in a d o se o f 15 m g/kg, 8 a n d 7
d a y s b e fo re th e b e h a v io ra l a n d b inding e x p e rim e n ts. T h e
a c tio n o f N P A a n d c aeru le in w as a lso stu d ie d a f te r 14 d a y s
474
VASAK. M AlM liT S. NU K K , StK )SAA R A N D ALLIKM HT S
FIG. 4. The changes tn motor inhibiting action of
N-propylnorapomorphine and caerulein after cessation of 14 days
haloperidol medication. While bars—caerulein 2 Mg/kg. slriped—
NPA 0.5 and 5.0 /ig/kg. black—the combination of caerulein and 0.5
^g/kg NPA. The mean value o f motor activity for saline treated
group was J180±!47 counts during 30 min. *p<0.05; **/><0 02;
***/><0.01 (Student’s M est).
a d m in istra tio n o f h alo p erid o l (0.25 m g/kg. tw ic c d a ily ), in­
c re a sin g th e s e n sitiv ity o f pre- an d p o stsy n a p tic d o p a m in e
re c e p to rs [11,53]. S e v e n ty -tw o h o u rs a fte r c e ssa tio n o f tw o
w e e k s h a lo p e rid o l tre a tm e n t th e b e h a v io ral e x p e rim e n t with
a p p ro p ria te d o s e s o f c a e ru le in , N P A a n d h a lo p e rid o l w as
p e rfo rm e d . S im u lta n e o u sly w ith th e b e h a v io ral ex p erim e n t
th e in v iv o 'H -sp ip e ro n e bin d in g s tu d ie s w e re c a rrie d out
a f te r lo n g -te rm a d m in istra tio n o f P C A a n d h a lo p e rid o l. A fter
lesio n in g o f s e ro to n in e rg ic te rm in a ls o f b ra in by PC A and
5 ,7 -D H T th e s p e c tro flu o rim e tric m e th o d w as u se d fo r d e ­
te c tio n o f d o p a m in e , s e ro to n in an d th e ir m ajo r m e ta b o lite s
[ 20].
RESULTS
E ffe c t o f P C A a n d 5.7-1УН Т o n L o c o m o to r E ffe c t o f
C a eru lein a n d N P A
T h e p re tre a tm e n t w ith PC A a n d 5 ,7 -D H T d e c re a se d o b ­
v io u sly (50 -6 0 % ) th e le v e ls o f s e ro to n in an d its m a jo r
m e ta b o lite 5 -h y d ro x y in d o le a c c tic acid in striatal slices,
w ith o u t ch a n g in g d o p a m in e c o n c e n tra tio n s . T h e ad m in istra ­
tio n o f b o th s e ro to n in e rg ic n e u ro to x in s ev id en tly po ten flated
th e m o to r in h ib itin g e ffe c t o f N P A . T h e a ctio n o f s im u lta ­
n e o u s a d m in istra tio n o f N P A and c a e ru le in w as a lso a u g ­
m e n te d , w h e re a s th e se d a tiv e e ffe c t o f ca e ru le in in gro u p ed
m ice w as so m e w h a t e n h a n c e d o n ly a f te r ad m in istra tio n o f
P C A (Fig. 3). T h e p re tre a tm e n t w ilh PC A (2 x 1 5 m g/kg)
in h ib ite d th e d isp la c in g p o te n c y o f 5 fi%/kg N P A an d 50 jxg/kg
c a e ru le in (T a b le 3). w hile th e p a rt o f 'H -sp ip e ro n e b inding
d isp la c a b le o nly b y 50 Mg/kg N P A re m a in ed u n c h a n g ed .
Effect, o f N P A a n d C a e n d e in o n L o c o m o to r A c tiv ity
a n d 3H -S p ip e ro n e B in d in g A fte r L o n ^ -T er/n
H a lo p e rid o l T re a tm e n t
T h e m ild se d a tiv e e ffe c t o f 50 jig /k g h alo p erid o l w a s re ­
v e rs e d to stim u la tio n o f e x p lo ra to ry a c tiv ity a fte r ce ssa tio n
o f lo n g -te rm h a lo p e rid o l (0.25 nig/kg tw ice daily d u rin g tw o
w e e k s) tre a tm e n t (T a b le 4). T o le ra n c e d e v e lo p e d a lso to the
m o to r d e p re s s a n t a c tio n o f 2 /xg/kg c a e ru le in . In salin e preI re a te d m ice th e s e d a tiv e a c tio n o f s im u lta n e o u s tre a tm e n t o f
и
FIG. 5. The action of caerulein on :lH-spiperone binding after cessa­
tion of 14 days haloperidol treatment. Black squares—the action of
caerulein after saline pretreatm ent. white squares—after two weeks
haloperidol administration. Abscissa: the dose of c&emlein in /xg/kg,
ordinate: radioactivity counts per gram tis s u e .-----inhibition, and
+ —stimulation of ‘H-spiperone binding. *p<Q.05; **/><0.01 vs,
saline pretreated animals (Student's /-test).
h a lo p e rid o l an d c a e ru le in did not d iffer from th e a c tio n o f
c a e ru le in a lo n e . H o w e v e r, a fte r w ith d raw a l o f lo ng-term
a d m in istra tio n o f h alo p erid o l th e c o n c o m ita n t tre a tm e n t o f
ca eru le in an d h alo p erid o l co m p le te ly re v e rse d th e to le ra n c e
to th e a c tio n o f b o th d ru g s (T a b le 4). T h e c h a n g e s in m o to r
d e p re s s a n t ac tio n o f N P A w ere d e p e n d e n t o n the d o se o f
N P A . 0.5 Mg/kg N P A had m o re p ro n o u n c e d inhibiting effect
a fte r tw o w ee k s h a lo p e rid o l m e d ica tio n (Fig. 4), w hile th e
ac tio n o f 5 ^ g /k g N P A w as sig n ific an tly re d u c e d . T w o w e ek s
h a lo p e rid o l tre a tm e n t a lso re d u c e d th e in te ra c tio n b etw e en
N P A an d c a e ru le in (F ig. 4). S om e a n im als b e c a m e h y p ere x c tta b le a fte r sim u lta n e o u s a d m in istra tio n o f N P A a n d c a e r u ­
lein to h a lo p e rid o l p re tre a te d m ice. T h e dim in u tio n o f 5
Mg/kg N P A in hibiting a c tio n o n ;,H -sp ip e ro n e b in d in g w as
see n a fte r 14 d a y s h a lo p e rid o l m e d ic atio n (T able 3), w h ere as
th e a c tio n o f 50 p.g/kg N P A w as ev id en tly in c re a se d . T h e
in h ib itin g a c tio n o f 50 ^-g/kg ca e ru le in w as tu rn e d to stim u la ­
tion o f 'H -sp ip e ro n e b inding a fte r c e ssa tio n o f lo ng-term
n e u ro le p tic tre a tm e n t l i a b l e 3). M ore de ta iled a n a ly sis o f
ca e ru le in inhibiting actio n re v e a le d (Fig. 5) the m ore
p r o n o u n c e d effe ct o f c a e ru le in on ^H -sp ip ero n e b in d in g in
su b c o rtic a l s tru c tu re s , w ith m axim al inhibition a fte r a d m in ­
istra tio n o f 100 ftg/kg c a e ru le in . A fte r c e ssa tio n o f tw o w e ek s
h a lo p e rid o l tre a tm e n t the in h ib itio n cu rv e o f ca e ru le in w as
shifted to stim u la tio n o f 3H -sp ip e ro n e b inding (F ig. 5).
DISCUSSION
E x p e rim e n t 2 e v id e n tly s u p p o rts o u r op in io n th a t the
se d a tiv e e ffe c ts o f ca e ru le in an d N PA a re m e d ia te d th ro u g h
d issim ila r m e c h a n ism s. L e sio n s o f se ro to n in e rg ic te rm in als
C A E R U L E I N A N D D O P A M IN E R G IC M E C H A N IS M S
by PCA ar.d 5 ,7 -D H T d e m o n s tra te the in v o lv em en t o f se ro to n in e ig ic m e c h a n ism s in th e in h ib ito ry ac tio n o f N P A . T h is
o p in io n w a s su p p o rte d b y o u r p re v io u s in v e stig a tio n (52],
w h e re th e p o te n tia tio n o f a p o m o rp h in e s e d a tiv e e ffec t by
lo w d o se o f p ire n p e ro n e . a se le c tiv e a n ta g o n ist o f
se ro to n in 2-re c e p to rs . w as sh o w n . T h e se d a tiv e e ffe c t o f
c a e ru le in w as in flu e n c e d o n ly by p re tre a tm e n t w ith PC A .
b u t n o t by m ic ro in je c tio n o f 5 ,7 -D H T . T h e p o ssib le e x p la n a ­
tion fo r th e s e d iffe re n c e s m ay b e th e d issim ila r a c tio n o f
5 ,7 -D H T a n d PC A o n p o s ts y n a p tic sero to n in ^ -re ce p to rs
s e n sitiv ity . It w a s fo u n d th a t 5 ,7 -D H T c a u se d b e h av io ral
h y p e rs e n s itiv ity o n s e ro to n in re c e p to rs 16, 49. 501. while
PC A in d u c e d s u b s e n sitiv ity to se ro to n in a g o n ists (5). T h e se
findings m ay s u p p o rt th e in v o lv e m e n t o f p o stsy n a p tic
s e ro to n in .-re c e p to rs in th e a c tio n o f c a e ru le in , b u t to a le sse r
e x te n t th an d o p a m in e 2-re c e p to rs. T h is o p in io n is in a g re e ­
m e n t w ith b in d in g s tu d ie s w h e re h ig h er d o s e s o f ca eru le in
w ere n e e d e d fo r in h ib itio n o f ;JH -sp ip e ro n e b inding to
sero to n in ., r e c e p to rs in d o rs a l c o rte x th a n to dopam ine*re c e p to rs in s u b c o rtic a l s tr u c tu re s .
I n v e s tig a tio n s p e rfo rm e d a fte r c e ssa tio n o f tw o w e ek s
h a lo p e rid o l tre a tm e n t s u p p o rt th e h y p o th e sis o f P ro ta is [42]
th a t th e s e d a tiv e e ffe c t o f m o d e ra te d o s e s o f N P A is relate d
to o th e r ty p e s o f d o p a m in e re c e p to rs th a n th e a c tio n o f low
d o se s . L o n g -te rm h a lo p e rid o l m e d ic a tio n in d u c e d to le ran c e
to th e s e d a tiv e e ffe c t o f 5 p-g/Jcg N P A , b u t in c re a se d th e
ac tio n o f 0.5 jxg/kg N P A . In th e b in d in g e x p e rim e n ts the
r e d u c tio n o f d isp la c in g p o te n c y o f 5 Mg/kg N P A a fte r w ith ­
d ra w a l o f 14 d a y s h a lo p e rid o l w as a lso seen . It is p ro b a b le
th a t N PA in m o d e ra te d o s e s in te ra c ts w ith p o stsy n a p tic
d o p a m in e 2- re c e p to rs h a v in g high-affinity fo r d o p a m in e
a g o n ists a n d n o t o n ly w ith s o -c alled d o p a m in e “ a u to re c e p to r s .” T w o w e e k s h a lo p e rid o l tre a tm e n t c a u se d to le ra n c e to
b o th e ffe c ts o f c a e ru le in — se d a tiv e a n d in h ib itio n o f :|Hsp ip e ro n e b in d in g . T h e in te ra c tio n b e tw e e n N P A an d c a e ru ­
lein w as a lso d e c re a s e d a f te r 14 d a y s n e u ro lep tic a d m in istra ­
tio n , w h ile c o a d m in is tra tio n o f h alo p erid o l a n d c a e ru le in in
low d o s e s r e v e rse d th e to le ra n c e to th e s e d a tiv e e ffe c ts o f
b o th d ru g s. It is p ro b a b le th a t the stim u la tio n o f :'H s p ip e ro n e b in d in g to d o p a m in e 2- a n d se ro to n in 2-re c e p to rs
a fte r lo n g -term n e u ro le p tic m e d ica tio n p lays a role in the
a n tip s y c h o tic a c tio n o f n e u ro le p tic d ru g s. T h e re w as d e ­
s c rib e d th e s u b s ta n tia l d o s e d e p e n d e n t in c re a se o f C C K -8
c o n te n t in s u b c o rtic a l f o re b ra in s tru c tu r e s a fte r tw o w ee k s
a d m in is tra tio n o f d iffe re n t n e u ro le p tic d ru g s (h a lo p e rid o l,
c h lo rp ro m a z in e , c lo z a p in e ) f23}. T h e d e n sity o f C C K b in d ­
in g site s w as a ls o e le v a te d a f te r lo n g -term n e u ro le p tic m e d i­
c a tio n [12].
In c o n c lu s io n , e x p e rim e n t 2 s u p p o rts th e idea a b o u t th e
in v o lv e m e n t o f p o s ts y n a p tic d o p a m in e .,-re c e p to rs a n d to a
le s se r e x te n t s e ro to n in 2- re c e p to rs in th e a ctio n o f c a e ru le in .
It is p ro b a b le th a t th e a c tio n o f c a e ru le in on a n im als b e h a v io r
a n d 'H -s p ip e ro n e b in d in g is re la te d to th e fu n c tio n a l a c tiv ity
o f dopam ine.,- a n d s e ro to n in r r e c e p to r s , b u t a lso to th e lev els
o f e n d o g e n o u s n e u ro tra n s m itte rs .
G E N E R A L D IS C U S S IO N
T h e re a re tw o o p p o s ite c o n c e p ts ex istin g a b o u t th e site o f
a c tio n o f C C K -8 an d c a e ru le in a fte r s y ste m ic a d m in istra tio n .
T h e first g ro u p o f in v e s tig a to rs [ 17,34) has d e m o n s tra te d the
re la tio n o f s e d a tiv e e ffe c ts o f C C K -8 and c a e ru le in to the
affe re n t s y s te m of n c rv u s v ag u s. V ag o to m y [34J o r lesio n s o f
n u c le u s tra c tu s s o lila riu s 117), th e c e n tra l te rm in a tio n o f
vagal se n s o ry fib e rs , a b o lis h e d th e d e p re ssio n o f som atic
475
fu n c tio n in d u c e d by C C K -8 o r ca e u rle in . H o w e v e r, th e
p h a rm ac o lo g ic al e x p e rim e n ts d e sc rib e d by Z e tle r (56, 5 7 ,5 8 )
sug g est th a t C C K -lik e p e p tid e s p o sse ss m a rk e d e ffe c ts in
anim al b e h a v io r m o d els k n o w n to re lia b ly re fle c t th e efficac y
o f w ell-k n o w n c e n tra lly a ctiv e d ru g s su c h as an a lg e sic s,
n e u ro le p tic s an d tra n q u iliz e rs.
T h e p re se n t in v e stig a tio n re v e a ls th a t a t le a st p a rtly th e
c e n tra l m o n o am in e rg ic m e c h a n ism s a re in v o lv ed in th e d e ­
p re ssiv e a c tio n o f c a e ru le in on m ice b eh a v io r. T h is id ea is
su p p o rte d by th e follow ing findings: (1) C a eru lein in h ib its in
viv o 'H -sp ip e ro n e b inding in th e b ra in , in lo w e r d o se s to
d o p a m in e ,-re c e p to rs in su b c o rte x an d in s o m e w n a t h igher
d o se s to se ro to n in 2-r tc e p to r s in d o rsa l c o rte x . T h is finding is
in a g re e m e n t w ith the in v itro in v e stig a tio n s [4] sh o w in g th at
10 nM C C K -8 sig n ific an tly m o d u la te s :,H -sp ip e ro n e binding
to d o p a m in e .-re c e p to rs in s tria tu m an d m o d e ra tle y to
s e ro to n in 2-re c e p to rs in d o rsa l c o rte x ; (2) T h e se d a tiv e e ffec t
o f ca e ru le in w a s in n eg ativ e c o rre la tio n w ith re a c tio n o f m ice
to m o to r stim u la tin g a c tio n o f N P A (100 /xg/kg) a n d d e n sity
o f ;lH -sp ip e ro n e b in d in g site s in fo re b ra in stru c tu re s ; (3 )T w o
w ee k s h a lo p e rid o l a d m in istra tio n in d u c e d th e to le ra n c e to
th e m o to r d e p ie s s a n t e ffe ct o f c a e ru le in a n d re v e rse d th e
in hibiting ac tio n o f c a e ru le in in to stim u la tio n o f *Hs p ip e ro n e binding.
T h e p o te n tia tio n o f a p o m o rp h in e -in d u c e d in h ib itio n o f
d o p am in e n e u ro n s by C C K -8 an d c ae ru lein w as d e m o n ­
s tra te d in m e se n c e p h a lo n (30]. B u t, th e p re s e n t investig atio n
in d ic a te s th e d iffe re n c e s in th e m ec h an ism o f m o to r d e­
p re ssa n t a c tio n o f N P A an d ca e ru le in . It a p p e a rs th a t N P A
re le a se s its inh ib itin g a c tio n o f m ice b e h a v io r th ro u g h th e
p re sy n a p tic d o p a m in e r e c e p to rs , w hile ca eru lein m ainly in­
te r a c ts w ith p o stsy n a p tic d o p a m in e 2-re c e p to rs. In trav e n tric u la r a d m in istra tio n o f 6 -O H D A , d e stru c tin g p re sy n a p tic
d o p am in e rg ic te rm in a ls, shifte d th e se d a tiv e e ffe c t o f N P A
into stim u latio n o f m ice e x p lo ra to ry a c tiv ity , w h e re a s the
a c tio n o f c a e ru le in re m a in e d u n c h an g ed . In fa c t, th e sed a tiv e
e ffect o f ca eru lein w a s in n eg a tiv e re la tio n w ith the
p o stsy n a p tic effect o f N PA — to stim u la tio n o f lo c o m o to r a c ­
tiv ity . S im ilar c o rre la tio n w a s fo u n d b etw ee n the b e h av io ra l
e ffe c t o f c ae ru le in and d e n s ity o f ’H -sp ip ero n e b inding site s
in fo re b ra in . T h e se findings a re in a g re e m e n t w ith in v e stig a ­
tio n s [4.25] sh o w in g th a t C C K -8 m ore read ily in te ra c te d w ith
‘H -sp ip e ro n e th a n ‘H -N P A b inding in “ in v itro ” co n d itio n s.
T h e re w as d e sc rib e d [18,27) th e e x iste n c e o f tw o binding
site s fo r d o p a m in e ag o n ists o n d o p a m in e 2-re c e p to rs (highan d lo*v-affinity) an d on ly h igh-affinity site for n e u ro le p tic
d rugs. It w as fo u n d [43] th a t th e se tw o site s fo r d o p a m in e
a g o n ists had d iffe ren t lo c a liz a tio n in s tria tu m — h ig h -affinity
site s w ere lo c ated p re d o m in a n tly on in trin sic n e u ro n s and
lo w -affinity s ite s on c o rtic o s tria ta l fib e rs. T h e h igh-affinity
site s w e re reg u lated by g u a n in e n u c le o tid e s: G T P o r its
a n alo g s sig n ific an tly re d u c e d the in te ra c tio n o f d o p a m in e
a g o n ists w ith d o p a m in e 2- re c e p to rs [27,43]. It se e m s th a t
c a e ru le in m o re p ro b a b ly in te ra c ts w ith high-affinity binding
s ite s fo r d o p am in e a g o n ists on d o p a m in e 2-r e c e p to rs ,
a n ta g o n iz in g the stim u latin g a c tio n o f d o p a m in e an d its
a n alo g s on a n im a ls ' b eh av io r. C aeru le in (75 Mg/kg an d h igher
d o se s) e ffec tiv ely r e v e rse d th e m o to r stim u la tin g ac tio n o f
d l-a m p h e ta m in e (5 nig/kg), b u t did not affect q u ip a z in e (5
m g/kg), se ro to n in .,-re c e p to rs a g o n ist, h e a d -tw itc h e s (o u r u n ­
p u b lish e d d a ta ) an d cage clim b in g b e h a v io r in d u ced by
h igher d o s e s o f a p o m o rp h in e in m ice [57]. T h e s e le c tio n o f
m ice a c c o rd in g to th e ir re sp o n se a fte r a d m in istra tio n o f 100
Mg/kg N P A a lso su p p o rt th e in v o lv e m e n t o f high-affinity
d o p a m in e 2-re c e p to rs in the ac tio n o f ca e ru le in . T he cle arc u t
476
VASAK. M AIM ET S. N U R K . SO O SA A R A N D A LL IK M E TS
p o sitiv e c o rre la tio n b e tw e e n ih e c o n te n t o f d o p a m in e in nu­
c le u s a c c u m b e n s an d th e re s p o n s e to m o to r stim u la tin g ef­
fect o f N P A w as d is c o v e re d in ra ts [ 14]. In stro n g re sp o n d e rs
th e c o n c e n tra tio n o f d o p a m in e in n u c leu s ac c u m b e n s w as
a p p ro x im a te ly tw o tim e s h ig h e r c o m p are d to w eak re sp o n ­
d e rs [14}. In th e p re s e n t stu d y , c a e ru le in a n d 5 p g /k g N PA
stim u la te d :,H -sp ip e ro n e b in d in g in w eak resp o n d in g m ice,
w h ile in s tro n g re sp o n d e rs bo th d ru g s ha d th e o p p o site e f­
fect. It a p p e a rs th a t th e a c tio n o f ca e ru le in on MH -sp ip e ro n e
b in d in g is d e p e n d e n t o n the lev els o f d o p am in e an d affinity
o f d o p a m in e r r e c e p to rs to d o p a m in e . T h e long-term infusion
o f d o p a m in e in to n u c le u s a c c u m b e n s c a u se d the o p p o site
c h a n g e s in d o p a m in e 2-re c e p to rs se n sitiv ity in se le c te d rats
[15]. In w eak re s p o n d e rs d o p a m in e d e m o n stra te d d o p am in e
r e c e p to r an ta g o n ist like p ro p e rtie s , in c re asin g th e se n sitiv ity
o f d o p a m in e 2-re c e p io rs , w h ile in s tro n g re sp o n d e rs it h a d the
o p p o s ite e ffe c t, d e c re a sin g th e affin ity o f d o p am in e re c e p ­
to rs . It is p ro b a b le th a t N P A , sim ilar to d o p a m in e , has
d o p a m in e an ta g o n ist p ro p e rtie s in w eak re sp o n d e rs in m o d ­
e ra te d o se (stim u la tio n o f 3H -sp ip e ro n e binding) and in high
re s p o n d e rs it a c ts as a re c e p to r a g o n ist (in h ib itio n o f ‘Hs p ip e ro n e binding). T h e m ix e d a g o n ist-a n ta g o n ist p ro p e rtie s
o f ap o m o rp h in e a n d N P A seem to h av e th e c linical re le ­
v a n c e , b e c a u se a p o m o rp h in e re d u c e s the p sy c h o tic sy m p ­
to m a to lo g y o n ly in o n e su b g ro u p o f sc h iz o p h re n ic p a tie n ts,
su fferin g m ain ly fro m th e p a ra n o id s c h iz o p h re n ia , receiv in g
n c u ro le p tic m e d ic a tio n , b u t n o t w ith o u t n e u ro lep tic d ru g s [1,
19, 21, 37, 47}. P ro b a b ly , th is a c tio n o f a p o m o rp h in e is d if­
fe re n t fro m th e s e d a tiv e a c tio n o f a p o m o rp h in e , w hich w as
a n ta g o n iz e d by n e u ro le p tic d ru g s [13]. It is possib le th a t in
th e s e p a tie n ts ap o m o rp h in e c a u se d th e sh o rt-lastin g stim u­
latio n o f n e u ro le p tic s b in d in g to dopam ine,,- and serotonin-»re c e p to rs .
T h e d iffe re n c e s in th e a c tio n o f N PA an d ca eru lein also
in v o lv e th e se ro to n in e rg ic m ec h a n ism s. It seem s that the
in h ib itin g a c tio n o f ca e ru le in o n m ice b e h a v io r is m ainly d e ­
p e n d e n t o n d o p a m in e rg ic m e c h a n ism s, w hile N PA a lso
in te ra c ts w ith s e ro to n in r e c e p to rs . T h e re w as d e m o n stra te d
th e d isp la c e m e n t o f :,H -k e ta n se rin fro m se ro to n in 2-re c e p to rs
b y ap o m o rp h in e [36]. In th e p re se n t stu d y N PA in hibited
sim ilarly 3H -sp ip e ro n e b in d in g in d o rsal c o rte x (m ainly
se ro to n in 2-re c e p to rs) as w ell a s in su b c o rtic a l fo reb ra in
stru c tu re s (p re v a ilin g d o p a m in e r re c e p to rs). C a eru lein in
lo w er d o s e s in te ra c te d w ith d o p a m in e r re c e p to rs , w h e rea s
th e h ig h e r d o se s w e re n eed ed fo r in te ra c tio n with
s e ro to n in .-re c e p to rs . It w as found th a t to su p p re ss do p am in e
tu rn o v e r lo w e r c o n c e n tra tio n s o f C C K w ere n ee d ed th an to
in hibit se ro to n in tu rn o v e r [51]. D e stru c tio n o f sero to n in e rg ic
te rm in a ls by PCA an d 5 ,7 -D H T sig n ificantly in c rea se d th e
m o to r d e p re s s a n t e ffect o f N P A , w hile only P C A , d ec rea sin g
also s« ro to n in 2-re c e p to rs se n sitiv ity [5], m o d e rately p o te n ­
tia te d th e a c tio n o f ca e ru le in . T he inv o lv em en t o f
s e ro to n in e rg ic m e c h a n ism s in th e b e h av io ral e ffe cts o f
ap o m o rp h in e w as a lso s ta te d by o th e r a u th o rs. T h e ad m in is­
tr a tio n o f d iffe re n t s e ro to n in ag o n ists into m edian ra p h e n u ­
clei. in n e rv a tin g m esolim bic a re a , p o te n tia te d in ra ts the
m o to r stim u la tio n in d u c e d by a p o m o rp h in e [22]. Л рош огphine in high dose* (o v e r 4 m g/kg) in d u c ed in c a ts b e h aviorai
effects sim ilar to L S D , an ag o n ist o f se ro to n in .-re c e p to rs
[50]. In th e clinical s tu d ie s [33], it w as e sta b lish e d th at
ap o m o rp h in e had p ro n o u n c e d se d a tiv e a ctio n only in p a ­
tien ts w ith en la rg ed c e re b ra l v e n tricles. In th is su b g ro u p o f
sc h iz o p h re n ic
p a tie n ts
the
d e c re a se d
c o n te n t
of
5-h y d ro x y in d o le a c e tic a cid , the m ajo r m eta b o lite o t s e ro to ­
n in . in c e re b ro sp in a l fluid w as d e sc rib e d [41]. T h e se clinical
o b se rv a tio n s a re in ag re e m e n t w ith o u r stu d y sh o w in g the
in c re a se d se d a tiv e effe ct o f a p o m o rp h in e an d N P A in the
c a se o f d eficien c y o f c e n tra l se ro to n in e rg ic m e ch a n ism s.
S p ec ia l a tte n tio n w as d raw n to the in te ra c tio n b e tw e e n
h a lo p e rid o l, the classica l n e u ro le p tic d ru g , a n d c a c ru ie in . In
th e pharm ac o lo g ica l e x p e rim e n ts sim ila ritie s w e re fo u n d in
th e b e h a v io ra l e ffec ts o f c a e ru lc in a n d h a lo p e rid o l, b u t a
po sitiv e in te ra c tio n b etw een th e se d ru g s w as n o t fo u n d [55,
57, 58]. S im ilar a b se n c e o f in te ra c tio n in in ta c t anim als w as
e sta b lish e d in the p re se n t stu d y . T h e in te ra c tio n b etw ee n
ca eru lein and halo p erid o l b ec am e e v id e n t a fte r tw o w eeks
h a lo p e rid o l a d m in istra tio n . C a eru lein re v e rse d the to le ra n c e
to th e s e d a tiv e effect o f h a lo p e rid o l an d in c re a se d :,Hsp ip e ro n e b inding a fte r long-term n e u ro le p tic m ed ica tio n .
T he in c re a se d n u m b e r o f C C K bin d in g site s w as d e m o n ­
stra te d a fte r lo n g -term h a lo p e rid o l tre a tm e n t [12]. D ifferen t
n e u ro lep tic dru g s (h alo p erid o l, c h lo rp ru m a z in e , c lo z ap in e)
in d u ce d d o se d e p e n d e n t ele v a tio n o f C C K -8 co n te n t in forcbra in su b co rtic al s tru c tu re s a fte r tw o w e ek s a d m in istra tio n
123]. It is p o ssib le th e m ec h an ism s d e sc rib e d a b o v e a re in­
volved in the beneficial a c tio n o f C C K -lik e p e p tid e s in
n e u r o le p tic -re sista n t sch iz o p h re n ic p a tie n ts [39,40].
In c o n c lu sio n , it is pro b ab le th a t ap o m o rp h in e an d N PA
h ave at le a st th re e d istin c t levels o f a ctio n : ( i ) l h e stim u la ­
tion o f d o p a m in e " a u to r e c e p to rs ’' c a u se s th e se d a tiv e effect
in anim a ls an d hu m an s [37}; (2) T h e in te ra c tio n w ith highaffinity d o p a m in e ,- an d se ro to n in r re c e p to rs in d u c e s the
stim u latio n o f ‘H -sp ip e ro n e b inding in anim als re sp o n d in g
w eakly to m o to r stim u lan t ac tio n o f N P A . T h e b e n eficial
clinical effec t o f a p o m o rp h in e a n d N P A [47,48] m ight be
related to th e se m o n o a m in erg ic m e c h a n ism s; (3) T h ro u g h
the stim u latio n o f low affinity dopam ine;,- an d se rotonin.,re c e p to rs a re m ed iate d the typical b e h a v io ra l e ffe c ts o f
a p o m o rp h in e an d N PA in hig h er d o se s (ste re o ty p e d b e h a v ­
ior. cage clim bing b e h a v io r, ag g re ssiv e n e ss, e tc .).
C a e ru le in , a fte r sy stem ic a d m in istra tio n , m ore probably
in te ra c ts w ith high-affinity d o p a m in e .-re c e p to rs an d to a les­
se r e x te n t w ith high-affinity se ro to n in ^ -re c e p to rs, inhibiting
the stim ulating effect o f do pam ine and its analogs on anim als'
b e h av io r.
ACKNOWLEDGEMENTS
W e w ould like to ih a n k F a rm ita lia C a rlo lir b a t'ta ly ) and
S terlin g -W in th ro p (U S A ) for th e g e n ero u s gifts o f d rugs.
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IS S N 0365-9615
БЮЛЛЕТЕНЬ
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Э. Э. Васар, A . X. Соосаир, М. О. М ай метс.
Л. X. Алликмет с
П О Н И Ж Е Н И Е ЧУ В С ТВ И ТЕЛ ЬН О С ТИ ХОЛЕЦ И С Т О К И Н И Н О В Ы Х РЕ Ц Е П Т О РО В В М О З­
ГЕ ПОД В Л И Я Н И Е М Д Л И Т Е Л Ь Н О Г О ВВЕ­
Д Е Н И Я Г А Л О П Е РИ Д О Л А
Л аб о р ато р и я психофармакологии Н И И обшей и м олекуляр­
ной патологии Т артуского университета
Представлена акад. АЛШ СССР А. В Нальдманом
П о имеющ имся данным, длительное применение
нейролептиков оказы вает сущ ественное влияние
на
холецистокинин-8-(Х Ц К -8)-ергические про­
цессы мозга. У становлено, что 2-недельное вве­
дение галоперидола или резерпина повыш ает
плотность ХЦ К-8-рецепторов в переднем мозге
мышей [2J. Применение разных нейролептиков
(клозапина, хлорпромазина и галоперидола) уве­
личивает содерж ание ХЦК.-8 в подкорковых ли м ­
бических структурах и хвостатом ядре [5]. В н а ­
ших предыдущих исследованиях было показано,
что длительное введение галоперидола вы зы вает
извращ ение ингибирующего влияния церулеина,
агониста ХЦК-8, на связы вание :,Н-спироперидола в опы тах in vivo [7]. Церулеин стимулировал
связы вание :|Н-сппроперндола с дофам пн2- и серогонин2-рецепторами после 15-дневного приме­
нения галоперидола [7 |. Сущ ествует мнение, что
снижение содерж ания ХЦ К-8 в некоторых струк­
турах переднего мозга м ож ет л еж ать в основе ре­
зистентности к нейролептикам у психических
больных 13]. В настоящ ем исследовании изуче­
но влияние длительного введения типичного ней­
ролептика галоперидола на связы вание 3Н -\олецистокннииа в переднем мозге мышей и па по­
веденческие
эффекты
церулеина.
агониста
ХЦК-8-репепторов
М е т о д и к а и с с л е д о в а н и я . Опыты про­
ведены па белых мы ш ах-сам нах массой 25—30 г.
Физиологический раствор
или галоперидол
(0,25 м г/к[. «Oerieon Richter». П Н Р) пнодилн
2 раза в день па протяжении 15 диен. П оведен­
ческие опыты п опыты по радиолнгандном у св я ­
зы ванию ставили через 48 72 ч после послед­
ней инъекции га ю пери дола, В опытах но св язы ­
ванию
II ХЦК Н (КО К и/м mix. ii,.
Лин-i .li.iiiu.
Англии) и переднем мо.не мышеи пенп.и, ишалп
моД||(|н|| 1нрои. 1Пную методику
| -I | . >\и и <ту » ►
' к а т . гоммтеш нирпвалн в Ю о б ье м ах холодного
грис 11(11 6yi|)epiiorii раствора (pH 7,4) при 2 0 "С.
с помощью гомогенизатора Н и г е р а
Гомогени­
зированную тк ан ь цептрифугироиалп п р и 4 8 ООО ц
в течение 15 мни, после чего полученный осадок
снова гомогенизировали в 10 объем ах тр и с-Н О буферпого раствора и гомогенат центриф угиро­
вали п р и 48 ООО и в течение 15 мни. О кончатель­
ный осадок гом огенизировали в 100 объем ах ин­
кубационного буфера, состоящ его из 10 мМ
H L P E S , 130 мМ хлорида натрия, 5 мМ хлорида
калия, 5 мМ хлорида магния и 1 мМ Э Д ТЛ (pH
доводили до 7,4 с помощью гидроксида к а л н я ).
■Н-ХЦК 8 добавили в инкубационную смесь в
р азны х концентрациях от 50 нМ до 3 нМ. Н е­
специфическое связы вание определяли д о б а в л е ­
нием I мкМ церулеина ( « - F a r r n i t a l i a » , И т ал и я ).
Пробы инкубировали при 24 "С в печение 90 мин.
П осле инкубации пробы центриф угировали при
12 00 g в течение 2'/г мин. Супернатант вы ли ва­
ли и осадок осторожно промывали несколько раз
с помощью холодного инкубационного буфера.
Р ад иоактивность проб (4 пар алл ел ей ) опреде­
л яли в сцинтилляторе Брея на счетчике ß -частиц
«ЛС-6800» («B cckm an», С Ш А ). Опыты п овторя­
ли 3—4 р аза. Полученные данны е о брабаты вали
с помощью ан али за С кетчарда.
П ар ал л ел ьн о с опытами по радиолнгандном у
связы ванию проводили исследование изменений
в поведенческих эфф ектах агониста Х Ц К -8-peцепторов
церулеина.
Влияние
церулеина
(10 м кг/кг подкож но) на ориентировочно-иссле­
довательскую активность мышей регистрировали
с помощью фотоэлектрического актом етра. С р а ­
зу после введения церулеина или физиологиче­
ского паствора животных помещ али в индивиду­
альны е клетки актом етра и определяли их д в и га ­
тельную активность в течение 30 мин. По м ето­
дике «лектроболевого разд р аж ен и я изучали д ей ­
ствие 40 м кг/кг церулеина на агрессивное пове­
дение мышей после 2-педелыю й инъекции галоп ерпдола. Через 20 мип после введения церулеина 2 ж ивотных помещ али в кам еру электроболеного р азд р аж ен и я, где они на протяж ении 2 мин
полумили 48 электрических ударов напряжением
40 В П арам етром интенсивности агрессивного
поведения служ и ло число агрессивных контактов
меж ду животными. П ротнвосудорож ное действие
церулеина (125 мкг/кг подкож но) исследовали
lui модели пикротокенновых судорог. У станов­
лено, что Х Ц К-8 и его аналоги и м алы х
д озах
антагонплнруют
пикротокенноным
су­
дорогам как при
виутрнж елудочковом .
так
н при
системном введении [6]. Ц ерулеин
применяли за 10 мин до введения 10 мг/кг пикротоксина. О пределяли 3 основных п арам етра
ппкротоксиновых судорог: латентны е периоды
клинических судорог, латентны е периоды тони­
583
судорог и продолжительность ж и з н и мы­
шей. Все полученные в поведенческих опытах
данные обработаны статистически с помощью
критерия t Стьюдента.
Р е з у л ь т а т ы и с с л е д о в а н и я . Примене­
ние галоперидола в течение 15 дней (0,25 мг/кг
2 раза в день) в наших опытах повышало плот­
ность высокоаффннных мест связы вания ХЦК-8
(табл I). Число низкоаффинных мест связы ва­
ния уменьшалось, однако их аффинность к ХЦК-8
зам етно повыш алась. П араллельн о изменениям
на местах связывания ХЦ К-8 наблю далось ос­
л абление или извращ ение поведенческих эфф ек­
тов церулеина, агониста ХЦК-8.
Церулеин
(10 мкг/кг) у мышей, получавших предваритель­
но в течение 15 дней галоперидол. в значитель­
ной степени утратил свою способность подавлять
ориентировочно-исследовательскую реакцию, в
то время как антиагрессивное действие церулеи­
на извращ алось (табл. 2). Церулеин в дозе
40 м кг/кг значительно повышал число агрессив­
ных контактов между мышами (см. табл. 2).
Длительное предварительное введение галоперидола достоверно ослабляло антагонистическое
влияние церулеина (125 м кг/кг) на пикротокси­
новые судороги (табл. 3).
Таким образом, результаты настоящ его иссле­
дования свидетельствуют о том, что длительное
применение типичного нейролептика галоперидолл, несмотря на повышение числа высокоаффшшых и увеличение аффинности низкоаффннных мест связывания ХЦК-8, вы зы вает ослабле­
ние или извращ ение поведенческих эффектов це­
руленна. О пониженной чувствительности ХЦК-8рецепторов свидетельствуют такж е данные на­
ших предыдущих исследований [1 , 7 ] , в кото­
рых было установлено, что длительное введение
галоперидола возвращ ает ингибирующее влия­
ние церулеина на связывание 3Н-спироперидола
в опытах in vivo. В то ж е время длительное при­
менение галоперидола существенным образом
сн и ж ает число высокоаффинных дофаминг- и серотонинг-рецепторов [1 , 7 ) . Понижение плотно ческих
't а б л н ц а I
Влияние длительного введения галоперидола на связывание
3II-холецистокинина в переднем мозге мышей
вещество
Ф изиологи­
ческий рас*
тпор
Г алоперидол
Высоколффинные места
снизывания
к,
Свмакс
0..45:-: 0.05
7 ,3 ± 0 .8
0 ,4 5 ± 0 ,0 5 11,7 ± 1.0*
Ниэкоаффин
ч
СвМ1яе
2,6 6 ± 0 ,2 5
3 1 ,5 ± 2 ,5
1,14±0,12** 21,4± 2.0*
11 р и м с ч а н н е . К — константа диссоциации (в н.'\),
^-вма«с “ ■максимальная плотность мест связывания (в фмоль
на 1 мг белка). Здесь и в табл. 2 и 3 одна звездочка — Р <
< 0 ,0 5 , две — /><0,01.
•84
Влияние длительного применения галоперидола на седягвяп
и антиагрессивное действие церуленна
Длительное вагдемкс
г& лопгрмда»
Орнен ти ровоч но-нсследовател ъс кая
активность мышей
число импульсов в течение
15 ннн
30 иян
15 мнк
30 мин
Физиологический
раствор
Церулеин
(10 мкг/кг)
187±17
3 2 5± 39
160±12
2 7 6 ± !7
142±23 2S2±27
115±19 198±23
Электроболевая агрессивность мышей
число агрессивных контактов
в течение 2 м ин
Физиологический
раствор
Церулеин
40 м кг/кг
13,5±0,95
8 ,6 ± 0 ,9 3
5 ,8± 0,82
21,8 ± 2 .9 9
Таблица
3
Изменение протнвосудорожного действия «ерудеина после
длительного введения галоперидола
П нкротоссал
(10 ыт/кг)
физиологически* р*ет-
Длительное
введение
физиологического
Латентное время клонических
судорог, с
Латентное время тонических
судорог, мин
Продолжительность жизни, мин
1 6 ± 1 ,5
17,4± 1,7
Пнкротокснв
(10 мг/«г) +
nepyjetiM
(123 мкг/кг)
раствора
24,9±2,0**
2 6 ,i± i.e * *
Длительное введение галоперидола
Латентное время клонических
судорог, с
Латентное время тонических
судорог, мин
Продолжительность ж изни.
427±32
776±65**
15,8±1,4
20,5± 1,5*
18,1± 1,5
2 1 ,5 ± 1 .5
сти этих моноамннергических рецепторов, по-ви­
димому. н определяет
гипочувствительность
ХЦК*8-рецепторов. Учитывая тесную морфофункциональную связь между ХЦК-8, дофамином и
серотонином, а такж е значительны е адаптаци­
онные изменения на ХЦК-8-рецепторах при дли­
тельном введении типичного нейролептика галоперндола, можно полагать, что ХЦК-8 имегт
значение для реализации как антипсихотическо­
го [3], так и побочных эффектов нейролептиков.
.Л И Т Е Р А Т У Р А
1. Васар Э. Э., Н урк А А!,
Майметс М . О.,
.4x»i*метс Л . X . / / Б юл. эксиср. биол. — 1985.— Л* !• —
С. 7 2 - 7 4 .
2. C hung R. S. L.. L otti V. /„ M artin G. £ . et a l.//L * » e
Sei. — 1983. — Vol. 32. — P. 871—878.
3. F ern er I. N.. R oberis G. ft7.. G row T. J. et al. / / Ib id .—
Vo!. 33. - P 475—482.
4. F in kvlstein J. A.. S te p le s s A. W.. M arline? P. Л.. Pruissm an M- / / P e p tid e s .— 1984. — Vol 5. — P. 11 — 14.
5. Frey P. 11 E urop. J. Pharm aco l. — 1983. — Vol. 95. —
P 87—92
6. K adar Т.. P e sti A., P enke В. e t a I. / / N europharm acolo­
gy. — 1984. — Vnl. 23 — P. 955—961.
7. V asar E.. M a im e ts M., N u rk A ., A llik m e ts L. / / Psychopharm acol. Bull. — 1984. — Vol. 20. — P. 691—692.
Поступила 12.I2.8S
R E D U C E D S E N S IT IV IT Y O F T H E B RA IN CH O L ECY STO ­
K IN IN R E C E P T O R S U N D ER T H E E FF E C T O F H A LO ­
P E R ID O L P R O L O N G E D A D M IN IST R A TIO N
E. E. Vasar, A. Kh. S o osaar, M. O. M aim ets, L. K. A llikm e ts
T a rtu U n iv ersity , T a rtu
The a u th o rs have used behav io ural and radioreceptor
m e th o d s of in v e stig a tio n , th a t helped to find co rre la te s of
th e beh av io u ral p henom ena on the re ceptor level.
УДК «1в.127-в05.4-0в5.23-Юв.в
Ключевые
слова:
натрия оксибутират; пирацетам;
п иридоксинил-глиоксилат ;
креатинфосфокинаэа;
инфаркт миокарда.
И. Б. Цорин, Г. Г. Ч ичканов
Д ЕЙ С ТВ И Е ПРЕПАРАТОВ С АНТИГИПОКСИЧЕСКИМИ СВОЙСТВАМИ НА ИШЕМИЧЕ­
СКОЕ П О ВРЕЖ ДЕ Н И Е М ИОКАРДА
Л аб о р ато р и я
ф арм акологии
антиангинальных
средств
( з а в .— доктор мед. наук Г. Г. Чичканов) И нститута ф а р ­
макологии АМ Н С С С Р, М осква
П редставлена акад. АМН СССР А, В. Вальдманом
В последние годы ш ироко об су ж д ается вопрос
о возм ож ности ограничения разм еров инфаркта
м иокарда (И М ) с помощ ью ф арм акологических
вещ еств. В ы сказы ваю тся противополож ны е то ч­
ки зрения д а ж е в отношении таких тради ц ион ­
ных «антиинф арктны х» средств, к ак ß -адреноблокаторы [8, 9, 13].
В настоящ ем исследовании изучено влияние
препаратов с антигипоксическим действием —
п иридокеннил-глиоксилата (гли о-6), натрия окс ибутирата и пи раиетам а — на развитие иш е­
мического повреж дения и конечный р азм ер ИМ
в условиях окклю зии коронарной артерии.
М е т о д и к а и с с л е д о в а н и я . Д л я су ж д е­
ния о влиянии препаратов на разви ти е иш ем иче­
ского повреж дения в сердце при окклю зии ко р о ­
нарной артерии -жепернменты проводили на кош ­
ках массой 3— 4 кг, анестезированны х этам инаIOM натрия (40 м г/кг внутривенно), в условиях
искусственного ды хания. У животных п ер евязы ­
вали переднюю нисходящ ую ветвь левой коро­
нарной артерии в средней ее трети. В нутривен­
но вводили гепарин в дозе 1000 1-Д/кг. Пробы
крови б рали из коронарного синуса перед о ккл ю ­
зией венечного сосуда, а та к ж е через 20 и 60 мин
после нее. П роведены 4 серии экспериментов.
П реп араты вводили ср азу после окклю зии коро­
нарной артерии внутривенно: натрия оксибути­
р а т — 200 мг/кг,
пирацетам — 400
мг/кг.
глио-6 — 100 мг/кг. В контрольной серии ж и вот­
ным вводили эквивалентны й объем ф изиологи­
ческого раствора. Активность креатинфосфокнназы (К Ф К ) в п лазм е крови оп ределяли по м ето­
ду [10]. П олученны е данны е о б р аб аты вал и с т а ­
тистически. Коэффициенты линейных регрессий
определяли с помощ ью непарам етрического кри ­
терия Тейла. Д остоверность различий м еж ду уг­
л ам и наклон а регрессионных прямых о п р ед ел я ­
ли с помощью одностороннего н еп арам етри че­
ского критерия Холлендера.
Д л я изучения влияния препаратов на р а зм е ­
ры ИМ проводили опыты на беспородных белы х
кры сах-сам ц ах массой 180— 200 г. Всего в эк сп е­
риментах использованы 44 крысы (5 серий, по
8— 10 животны х в каж д о й ). ИМ воспроизводили
по м етоду [11]. У животны х регистрировали
Э К Г в 3 стан дартн ы х отведениях до окклю зии
венечной артерии, через 5 мин после нее и перед
забоем животных. Крыс заб и в ал и через 72 ч
после операции. С ердца извлекали и готовили в
криостате срезы . Д е л а л и 5 срезов толщ иной
25 мкм через каж д ы е 2 мм, начиная от в ерхуш ­
ки левого ж елудочка. Срезы о кр аш и вали с по­
мощ ью
нитротетразолиевого синего, вы являя,
таким образом , активность сукцинатдегидрогеназы. Д л я определения разм еров ИМ и спользо­
в али м атем атическую м одель [4]. Р азм ер ИМ
рассчиты вали по объему некротической массы,
в ы раж енном у в процентах от объем а м иокарда
всего левого ж елудочка. П реп араты вводили
внутрибрю ш инно в разовы х дозах: натрия окси ­
б у т и р а т — 200 мг/кг, пирацетам — 400 мг/кг.
гл и о -6 — 100 мг/кг. В качестве п р еп ар ата с р а в ­
нения использовали пропранолол в разовой д о ­
зе 1 м г/кг. Схема введения препаратов бы ла
следую щ ей: 1-е введение — за 15 мин до о кк л ю ­
зии коронарной артерии, 2 - е — через 2 ч после
окклю зии, затем по 2 введения еж едневн о в те­
чение следую щ их 2 сут. В контрольной серии
животным вводили эквивалентны й объем ф изио­
логического раствора. Полученные результаты
о б р аб аты вал и статистически. Д остоверность р а з­
личий определяли с помощью критерия t Стьюдента.
Результаты исследования.
Хорошо
известно, что степень увеличения активности
КФ К в крови является важ ны м п оказателем т я ­
жести иш емического повреж дения |6 , 12| Но
д инам ике н ар астан и я активности КФ К и у с л о ­
виях острой ишемии м иокарда м ож но судить о
скорости перехода обратимы х ишемических по­
вреж дений п необратимы е. Установлено, что
при окклю зии коронарной артерии препараты с
протпноншемичсским действием сниж аю т актив
Г.8Г,
гг
ЖУРНАЛ ВЫСШЕЙ НЕРВНОЙ ДЕЯТЕЛЬНОСТИ
ТОМ X X X V II
1987
ВЫ П. 4
У ДК 6 12.821.6+ 612.8.015
ИЗМЕНЕНИЕ ПОВЕДЕНЧЕСКИХ И БИОХИМИЧЕСКИХ
ЭФФЕКТОВ ЦЕРУЛЕИНА, АНАЛОГА ОКТАПЕПТИДА
ХОЛЕЦИСТОКИНИНА, ПОСЛЕ ДЛИТЕЛЬНОГО ВВЕДЕНИЯ
ГАЛОПЕРИДОЛА
В А С А Р Э. Э.,
АЛЛИКМЕТС
Л . А '., С О О С А А Р A . X . , Л А Н Г А . Э.
Л аб ор а т о р и я п с и х о ф а р м а к о л о ги и Тарт уского го суд а р ст вен но го университета
По существующим представлениям октапептид
холецистокинина
(Х Ц К -8) имеет тесные морфофункциональные связи с дофаминергическими системами переднего мозга [13, 21]. ХЦ К-8, являясь комедиатором
дофамина в нейронах центральной части покрышки, влияет на высво­
бож дение и метаболизм дофамина [10, 20] и изменяет аффинность и
плотность доф амин2-рецепторов [1 1 ]. Имеются данные, что дофаминсргические механизмы участвуют в реализации некоторых поведенческих
эффектов ХЦ К -8 и его аналога церулеина: седативного действия, подав­
ления фенаминового возбуждения, антиагрессивного действчя и т. д.
[15, 22, 2 3 ]. С другой стороны, установлено модулирующее влияние д о ­
фамина на холецистокининергические механизмы. Длительная блокада
дофаминовых рецепторов нейролептиками приводит к повышению со­
держания ХЦ К -8 и количества его рецепторов в переднем мозге под­
опытных животных [5, 9 ].
Однако функциональное значение влияния дофаминергических м еха­
низмов на холецистокининергические процессы изучено в меньшей сте­
пени. В связи с этим целью настоящей работы было изучение
влияния длительной блокады дофаминовых рецепторов галоперидолом
на поведенческие эффекты церулеина,
агониста ХЦК-8-рецепторов,
а такж е выявление возможных механизмов изменения поведенческих
эффектов церулеина под влиянием галоперидола.
М ЕТ О Д И К А
Опыты проведены на 320 мышах-самцах массой 20— 25 г и 250 крысах-сам цах массой 200— 250 г, разделенных на группы по 10— 12 ж ивот­
ных в каждой. Галоперидол, как правило, вводили в течение 15 дней в
до зе 0,25 мг/кг 2 раза в день. Через 72 ч после последней инъекции га­
лоперидола определяли поведенческие и биохимические эффекты церу­
леина (производство «Фармиталия— Карло Эрба», И талия). Седативное
действие церулеина у мышей исследовали с помощью фотоэлектричес­
кого актометра. Животных помещали сразу после подкожного введения
церулеина (15 мкг/кг) в актометр, где в течение 30 мин определяли дви­
гательную активность. П ротивосудорожное действие церулеина иссле­
довали в модели пикротоксиповых судорог. Ц ерулеин (125 мкг/кг) вво­
дили за 10 мин до внутрибрюшинного введения пикротоксина (8 мг/кг).
Определяли три параметра пикротоксиповых судорог: латентные перио­
ды клонических и тонических судорог, а такж е продолжительность ж и з­
ни мышей после введения пикротоксииа. При исследовании седативного
и антипикротоксинового действия церулеина у крыс им под эфирным нар­
козом за 7— 8 дней до опыта имплантировали унилатеральные канюли
для микроинъекций в латеральный желудочек мозга по координатам
атласа мозга крысы [4]. Канюли прикрепляли к черепу зубным цемен­
том. Церулеин разводили в стерильном физиологическом растворе и в
течение 30 с вводили с помощью микроинъектора в правый латеральный
желудочек мозга в объеме 5 мкл (в дозах 5— 500 нг). Контролем сл уж и ­
ли мнкроинъекции физиологического раствора в том ж е объеме. Н а 60-й
секунде после введения церуленна или физиологического раствора крыс
помещали и центр открытого поля или им вводили пикротоксин (4 мг/кг).
В открытом ноле (размером 1 0 0 х 100X 40 см) в течение 5 мин с по­
мощью пяти независимых фотоэлектрических каналов определяли дви­
гательную активность крыс, а такж е число вставаний на задние лапы и
число обнюхиваний гнезд. П осле введения пикротоксина изучали
латентные периоды возникновения тремора и клопических судорог, изме­
ряли продолжительность жизни животных. В поведенческих исследова­
ниях установлено, что церулеин при однократном введении с галоперидолом вызывает у крыс двухнедельное подавление возбуж даю щ его д ей ­
ствия фенамина 115]. В настоящем исследовании оценивали влияние
хронического введения галоперидола на такой длительный эффект це­
рулеина. Д ля этого через 72 ч после последней инъекции физиологичес­
кого раствора или галоперидола части крыс вводили подкожно церулеин
(40 мкг/кг). Контролем служ ило подкожное введение физиологического
раствора в том ж е объеме. Опыты с d-фенамином (2,5 мг/кг) проводили
трижды: через 1 сут, на 5-й и 12-й день после однократного введения
церулеина. На 30-й минуте после введения фенамина определяли интен­
сивность стереотипного поведения [6] и через 45 мин в течение 5 мин
изучали действие фенамина на двигательную активность крыс в модели
открытого поля. И сследовали такж е связывание 3Н-спироперидола и 3НХЦК-8. В опытах in vivo наблюдали влияние разных доз церулеина
(20— 250 мкг/кг) на связывание 3Н -спироперидола (5 мкг/кг, 17 Ки/
/ммоль) в переднем мозге мышей. Церулеин вводили за 15 мин до вве­
дения меченого нейролептика, а через 20 мин после введения 3Н-спироперидола животных декапитировали. Опыты связывания проводили по
методике, описанной нами ранее 12, 19]. Опыты связывания 3Н -ХЦК-8
(86 Ки/ммоль) проводили в переднем мозге крыс и мышей по модифи­
цированной методике 1317]. М озговую ткань гомогенизировали в 10
объемах холодного трис-НС1-буферного раствора (pH 7,4 при 20° С) с
помощью гомогенизатора Поттера-С («Браун М елсунген» Ф Р Г ). Гомо­
генизированную ткань центрифугировали при 48 000 g в течение 15 мин,
после чего полученный осадок снова гомогенизировали в 10 объемах
трис-НС1-буферного раствора и гомогенат центрифугировали
при
48000 g в течение 15 мин. Окончательный осадок гомогенизировали в
100 объемах инкубационного буфера, состоящего из 10 мМ ХЕПЕС
(М-2-гидрокси-этилпиперазин-М'-2-этансулфоновая кислота;
«Сигма»,
СШ А), 120 мМ хлорида натрия, 5 мМ хлорида калия, 5 мМ хлорида
магния и 1 мМ этилендиаминтетрауксусной кислоты (pH было доведено
до 7,0 с помощью гидроксида калия). 3Н-ХЦ К-8 добавляли в инкуба­
ционную среду в разных концентрациях — от 50 пМ до 3 нМ. Н еспеци­
фическое связывание определяли добавлением 1 мкМ церулеина. Пробы
были инкубированы при 24° С в течение 90 мин. П осле инкубации пробы
центрифугировали при 12000 g в течение 2 мин. Супернатант выливали
и осадок осторожно промывали с помощью холодного инкубационного
буфера. Радиоактивность проб (четыре параллели) определяли в сцин­
тилляторе Брея на счетчике бета-частиц JIC-6800 («Бекман», СШ А).
Опыты повторяли 3— 4 раза. Полученные данные подвергали анализу
Скэтчарда.
РЕ ЗУ Л Ь Т А Т Ы И С С Л Е Д О В А Н И Й
У контрольных мышей церулеин в дозе 15 мкг/кг вызывал торм ож е­
ние двигательной активности (рис. 1). Однако после длительного приме­
нения галоперидола (0,25 мг/кг 2 раза в день в течение 15 дней) церулеип не изменял моторную активность подопытных животных, торм озя­
щее действие исчезало. П осле галоперидола ослаблялось также антаго­
нистическое действие
церулеина (125 мкг/кг) на пикротоксиновые
(8 мг/кг) судороги у мышей (табл. 1). У животных, получавших преднарительио галоперидол, церулеин значительно слабее удлинял латент697
12*
Таблица I
Влияние длительного введения галоперидола на противосудорожное действие
церулеина у мышей
ГЬр::\ц'тры пикротоксиповых су д о р о г
Пикротоксии 8 мг/юЧ фиэно- Пикротрксип8 мг/кг-f церулеин
л о г и ч е с к и й рас тв о р
125 м к г /к г
Длительное введение физиологического раствора
Латентное время клонических судо­
рог, с
Латентное время тонических судо­
рог, мин
Продолжительность жизни, мнн
440+ 25
780+72**
1 6 + 1 ,5
25 + 2 ,0 * *
1 7 + 1 ,7
26 + 1 ,6 * *
Длительное введение галоперидола
Латентное время клонических судорог, с
Латентное время топических судорог, мин
Продолжительность жизни, мин
4 30+ 32
780+75**
1 6 + 1 ,4
2 0 + 1 ,5 *
1 8 + 1 ,5
2 1 + 1 ,5
П р и м е ч а н и е . • — р < 0 ,0 5 ; •* — /><0,01 по ср авн ен и ю с вв ед ен и ем
т о к е н н а (по t -те с т у С тью деи та; т о ж е д л я т а б л .З ) .
ф и зи о л о г и ч е ск о г о
раствора 4 - п и кро-
ный период тонических судорог и продолжительность жизни по сравне­
нию с контрольной группой. Сходное ослабление или извращ ение дейст­
вия церулеина наблюдалось такж е у крыс при внутрижелудочковом его
введении. Если у контрольных крыс малые дозы церулеина (5 и 50 нг)
угнетали ориентировочно-исследовательскую активность, то у животных,
получавших галоперидол, церулеин не подавлял (5 нг) или д а ж е усили­
вал (50 нг) активность животных. Вве­
дение 50 нг церулеина в латеральный
ж елудочек мозга не только усиливало
двигательную активность животных,
но повышало и число вставаний на
задние лапы и обнюхиваний гнезд
(табл. 2 ). П осле галоперидола разви­
валась такж е толерантность к противосудорожному действию церулеина у
крыс (табл. 3 ). П осле длительного
применения галоперидола церулеин
(5 нг) не вызывал достоверного осл аб­
ления пикротоксиновых (4 мг/кг) судо­
рог. Единственным поведенческим эф ­
Рис. 1. Влияние длительного в в еде­
ния галоперидола на седати вное д е й ­ фектом церулеина, который существен­
ствие церулеина у мышей. П о оси
ным образом усиливался после дл и ­
ординат— число импульсов в течение
тельного введения галоперидола, было
30 мин. Светлые столбики — эффект
длительное
угнетение возбуж даю щ его
введения физиологического раствора;
действия d-фенамина (2,5 м г/кг), при­
заш трихованны е— церулеина (15 мкг/
/к г). • — р < 0 ,0 5 (по U -тесту М ан­
чем интенсивность фенаминовоб сте­
на — Уитни)
реотипии при этом у крыс не изменя­
лась. У контрольных крыс церулеин
достоверно угнетал эффект фенамина только на 5-й день после однократ­
ного введения 40 мкг/кг церулеина (рис. 2 ), в то время как у животных,
получивших галоперидол, такое действие церулеина было заметно уж е
через 24 ч после его однократного введения. Угнетающее влияние церу­
леина на возбуж даю щ ее действие фенамина было заметно на 15— 20-й
день после однократного введения церулеина.
У контрольных мышей церулеин достоверно угнетал специфическое
связывание 3Н-спироперидола в опытах in vivo, однако при длительном
введении галоперидола очевидным стал противоположный эффект церу­
л еи н а — стимуляция связывания 'Н-снироперидола (рис. 3 ). В опытах
Г,98
Таблица 2
Влияние внутрижелудочкового введения церулеина на ориентировочно-исследовательскую
активность крыс, получавших предварительно на протяжении 15 дней галоперидол
Ч и сл о им пульсов
В ещ ество и д о за
Физиологический
раствор
Церулеин, 5 нг
Церулеин, 50 нг
Церулеин, 500 нг
Ч и сло вставан и й
ф изиологи­
ческий
р а с тв о р
гал о п е р н дол
ф и зи о л о ги ч е ­
ск и й р а ств о р
4 0 + 3 ,1
5 8 ± 7 ,5
2 ,2 + 0 ,3 8
29 + 4 ,4 * 45-ЬЮ, 9 2 ,7 + 0 ,7 0
2 7 + 3 ,6 * 93 £ 9 ,2 * 0 ,8 р ),2 6 *
5 2 + 6 ,6
3 ,0 Й , 3 9
5 3 ± 5 ,6
Ч и с л о обню хипаннА г н е з д
га л о п е р и д о л
ф и зи ологи ­
чески А
рас тн о р
галоперидол
4 ,2 + 1 ,0 7
5 ,9 Н ) ,9 2 4 ,8 + 0 ,7 7
4 ,5 + 0 ,8 7
1 2 + 2 ,8 6 *
3 ,7 + 0 ,5 5
4 ,8 + 0 ,6 9 8 ,2 Н1,4 5 4 ,2 Й , 03 1 1,3 И , 83**
6 ,2 + 1 ,1 0 5 ,0 + 0 ,С 1
П р и м е ч а н и е : * — р < 0 ,0 5 ; •* — *><0,0! п о с р ав н ен и ю с вн у тр и ж е л у д о ч к о в ы м вв ед ен и ем ф нзиол огическ о*
го р а с тв о р а (по U -те с т у М анна — У и тн и ).
Т а б л и ц а .3
Влияние длительного введения галоперидола на противосудорожное действие
церулеина у крыс
П и к р о т о к е и и , 4 м г к г - f ф изио­
л о г и ч е с к и й р а ств о р
П а р а м е т р ы п и к р о то к с и н о в ы х с у д о р о г
П н к р о то к еи н ,
4 м г /к г + ц е р у л с н п 5 и г
Длительное введение физиологического раствора
Латентное время тремора, мин
Латентное время клонических судо­
рог, мин
Продолжительность жизни, мин
9 ,7 + 0 ,3 5
1 3 ,1 + 0 ,4 6
1 2 ,1 + 0 ,5 5 * *
1 5 ,2 + 1 ,0 4 *
3 4 ,5 + 1 ,3 5
4 0 ,6 + 1 ,7 8 *
Длительное введение галоперидола
Латентное время тремора, мин
Латентное время клонических судорог, мин
Продолжительность жизни, мин
1 0 ,9 + 0 ,6 3
1 5 ,3 + 1 ,0 5
1 1 ,8 + 1 ,8 0
1 5 ,3 + 1 ,5 4
3 7 ,0 + 1 ,6 3
3 9 ,2 + 2 ,3 8
Таблица 4
Влияние длительного введения галоперидола на связывание ’Н-холецистокинина
в переднем мозге
Н и зко я ф ф н н н ы е
В ы сок оаф ф н н н ы е м е с т а с в я з ы в а н и я
В е щ ество
<-'в м а к с
КД
Кд
^ “ м ак с
Крысы
Физиологический раствор I
Галоперидол
j
0 ,1 9 + 0 ,0 4
0 ,2 6 + 0 ,0 6
Физиологический раствор |
Галоперидол
|
0 ,3 5 + 0 ,0 5
0 ,4 5 + 0 ,0 5
I
|
1 1 ,7 + 1 ,8 8
1 3 ,1 + 2 ,0 8
I
|
0 ,6 6 + 0 ,0 8
0 ,4 0 + 0 ,0 4
I 2 1 + 2 ,0 8
| 1 5 + 1 ,2 2 *
I
|
2 ,6 6 + 0 ,2 5
1 ,1 4 + 0 ,1 2
I 3 1 + 2 ,5
| 2 1 + 2 ,0 *
Мыши
П р и м е ч а н и е . К д — к о н с т а н т а д и сс о ц и ац и и ,
б е л к а : • — р < 0,05 (но 1-тесту С тью д е н та).
I 7 ,3 -г О ,80
| 1 1 ,7 + 1 ,0 0 *
н м ол ь;
С в м а к с - п л о тн о с ть м ест с в я зы в а н и я , ф ч о л ь /м г
связывания in vitro выяснилось, что ХЦ К -8 имеет два места связывания
в переднем мозге как у мышей, так и у крыс. Разница в аффинности этих
двух мест связывания была более значительной в переднем мозге мы­
шей, чем крыс (табл. 4 ). Длительное введение галоперидола несколько
уменьшало аффинность и повышало плотность высокоаффинных мест
связывания ХЦК-8, в ю время как аффинность иизкоаффинпых мест
повышалась с одновременным понижением их плотности. Указанные из­
менения были одинаковыми в переднем мозге крыс и мышей.
О Б С У Ж Д Е Н И Е РЕЗУЛ ЬТАТОВ
Проведенный анализ поведения свидетельствует о том, что длитель­
ное применение галоперидола изменяет все основные эффекты церулсина, агониста ХЦК-8-рецепторов. В основном эффекты церулеина осл аб­
ляются или извращаются, только антифенаминовое действие церулеина
усиливается после 15-диевпого применения галоперидола. Ослабление
или извращение поведенческих эффектов церулеина одинаково как при
подкожном введении мышам, так и при внутрижелудочковом введении
крысам. Полученные данные свидетельствуют в пользу мнения, что се­
дативное и противосудорожное действия церулеина в основном реали­
зуются через центральные механизмы, а не только через афферентную
систему блуждаю щ его нерва, как утверждают некоторые авторы [7,
1 4 ]. Длительное введение галоперидола изменяет не только состояние
моноаминергических (дофамин, серотонин и ГАМК) процессов мозга
Р и с. 2.
Р и с. з.
Рис. 2. Д л и те л ь н о е угн етен ие ц ер у л еи н о м в о зб у ж д а ю щ е го д е й ст ви я ф ен ам и н а у кры с,
п олучи вш их п р е д в ар и те л ьн о гал о п ер и д о л . П о оси о р д и н а т — число и м п ульсов в течение
5 м ин; по оси абсци сс — дн и после о д н о к р а т н о го в в е д ен и я ц ер у л еи н а (40 м кг/кг) или
ф и зи ол оги ч еск ого р а с тв о р а. С в етл ы е к в а д р а ты — д ей стви е ф ен ам и н а (2,5 м г/кг) у
кры с, п олучи вш их ф и зиологи чески й р аств о р + ф и зиологи чески й р а с тв о р ; тем ны е — ф и ­
зи ологи ческий р а с т в о р + ц е р у л е и н ; с в етл ы е тр еу го л ьн и ки — га л о п ер и д о л + ф и зи о л о ги ч е­
с кий р а с тв о р ; тем ны е — гал о п ер и д о л + церулеи н . * ■
— р < 0,05; * * — р < 0,01; * » * —
р < 0 , 0 0 5 по срав н ен и ю с группой ф и зиологи ч ески й раство р -I-ф и зи о л о ги чески й р а с тв о р
(по U -тесту М анн а — У итни)
Р и с. 3. В л и ян и е ц е р ул еи н а на с в язы в ан и е 3Н -сп и р о п ер и д о л а в оп ы тах in v iv o в п е р е д ­
нем м озге м ы ш ей , п олучи вш их п р ед в ар и тел ьн о гал о п ер и д о л . П о оси о р д и н а т — и зм е н е ­
ние сп еци ф ич еского св я зы в ан и я 3Н -сп и р о п ер и д о л а (5 м кг/кг) под вли ян и ем ц е р у л еи н а
(20— 500 м к г /к г ), число р а с п ад о в на 1 г ткани. «— » — угнетение с в я зы в ан и я 3Н -спнроп ер и д о ла; « + » — сти м у л и р о в ан и е с в я зы в ан и я . П о оси аб сци сс — д о за ц ер у л еи н а , м кг/
/кг. С в етл ы е треугол ьн и ки — эф ф е к т ц е р у л еи н а п осле в в е д ен и я ф и зио л о ги ч еск о го р а с т ­
в о р а ; тем ны е — га л о п е р и д о л а . • * — р < 0 , 0 1 (по t -тесту С ты о д е н та )
[1 ], но вмешивается и в регуляцию центральных холсцистокшшнергических механизмов. Однако, вероятно, такое действие галоперидола не
является непосредственным, так как галоперидол не взаимодействует с
ХЦК-8-рецепторами, его влияние опосредуется через те дофаминергические механизмы, комедиатором которых ХЦК-8 является [13]. По су­
ществующим представлениям, угнетающее влияние церулеина на доф аминергические процессы реализуется через два разных механизма [21].
Во-первых, церулеин, подобно ХЦК-8, устраняет ингибирующее влияние
дофамина на постсинаптических мембранах и прилегающем ядре и, вовторых, как и ХЦК-8, подавляет вызванное ионами калия кальций-зависимое высвобождение дофамина [2 1 ]. В постсинаптичсском действии
церулеина, по-видимому, ведущее значение имеет его взаимодействие с
высокоаффинными дофамин .-рецепторами на вставочных нейронах в пе­
реднем мозге [12, 18]. Впутрнмозговое введение каиновой кислоты р аз­
рушает именно ХЦК-8-рецепторы и высокоаффинные дофамшь-рецепторы [8, 18]. Диалогичное снижение высокоаффннпых дофамины-рецепто­
ров [2, 3, 19] и ХЦК-8-реиеиторов наблюдается после длительного вве­
дения галоперидола, и и связи с этим в опытах in vivo церулеин не инги­
бирует связывание :'Н-снироперидола. а, наоборот, даж е стимулирует
связывание 3Н-нейролептик«. Па основе привеченных данных можно
700
полагать, что длительное введение галоперидола приводит к функцио­
нальному выключению определенной части вставочных нейронов в пе­
реднем мозге, на которых и взаимодействуют дофамин и ХЦК-8. Именно
функциональным выключением части вставочных нейронов и объясняет­
ся ослабление или извращение поведенческих и биохимических эф ф ек­
тов церулеина.
В отличие от других эффектов церулеина длительное антифенаминовое действие, вызванное однократным введением, значительно усилива­
ется после длительного введения галоперидола. Этот факт свидетельст­
вует о том, что антифенамнновое действие церулеина реализуется через
другие механизмы по сравнению с изложенными выше. Существует мне­
ние, что указанный эффект церулеина реализуется через пресинаптические дофаминергические механизмы в прилегающем ядре [1 5 ], церулеин
подавляет высвобождение доф амина, вызванное фенамином, причем
промежуточным звеном здесь является бета-эндорфин [1 6 ]. О различи­
ях меж ду постсинаптическим и пресинаптическим действием ХЦ К -8 и
церулеина на уровне дофаминергической системы говорит и факт, что
антагонист ХЦК-8-рецепторов проглумид устраняет только постсинаптическое действие церуленна и ХЦК-8, не влияя при этом на их действия
на высвобождение дофамина [2 1 ].
Итак, длительное введение галоперидола оказывает неодинаковое
влияние на поведенческие эффекты церулеина, сильного агониста ХЦК8-рецепторов. В основном поведенческие и биохимические эффекты ц е­
рулеина ослабляются или извращаются, усиливается только длительный
антагонизм с фенаминовым возбуждением у крыс. Ведущ им во многих
эффектах церулеина является его влияние на высокоаффинные доф амин2-рецепторы, находящиеся на вставочных нейронах хвостатого ядра
и лимбических структур. Длительное введение галоперидола вызывает
функциональное выключение этих нейронов, чем обусловлено и осл аб­
ление или извращение эффектов церулеина. Длительное антифенаминовое действие церулеина реализуется через пресинаптические доф ам инер­
гические механизмы, причем бета-эндорфины выполняют здесь роль про­
межуточного звена м еж ду ХЦК-8- и дофамином [1 6 ].
ВЫ ВО Д Ы
1. Длительное введение галоперидола в основном ослабляет или из­
вращает поведенческие эффекты церулеина, агониста ХЦ К-8-рецепторов. Только длительный антагонизм церулеина с возбуждающ им дей ст­
вием фенамина у крыс усиливается под влиянием галоперидола.
2. В основе ослабления или извращения поведенческих эффектов
церулеина после отмены длительного введения галоперидола лежит
функциональное выключение части вставочных нейронов в подкорковых
структурах переднего мозга, что выражается в понижении числа высокоаффннпых дофамины-рецепторов и низкоаффинных ХЦК-8-рецепторов.
3. Усиление антагонизма церулеина с возбуждаю щ им действием ф е­
намина у крыс обусловлено более выраженным угнетающим влиянием
церуленна па высвобождение дофамина из пресинаптических доф аминергических терминален после длительного введения галоперидола.
Л итература
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Поступила в редакцию
30. V I. 1986
C H A N G E OF BE H A V IO U R A L A N D BIO C H EM IC A L EFFE C T S OF C A E R U L E IN .
AN A N A L O G U E O F C H O LEC Y ST O K IN IN O C T A P E P T ID E (C C K -8),
FO LLO W ING LO NG -TERM A D M IN IST R A T IO N OF H ALO PER ID O L
V A S A R E „ A L L I K M E T S L. , S O O S A A R A . , L A N O A.
U n iv e rs ity , T a r tu
In experim ents on m ale m ice and rats, long-term haloperidol adm ini­
stration (0,25 m g /k g tw ice a day during 15 days) sign ifican tly changed
behavioural effects of caerulein, an agon ist of CCK-8 receptors. As a rule,
the effects of caerulein were reduced or inverted; only long-term an tago­
nism w ith am phetam ine motor excitation in rats increased after the cessa ­
tion of haloperidol adm inistration. The decrease or inversion of caerulein’s
effects w a s connected with reduction of high-affinity dopamine*- and lowaffinity CCK-8 rcceptors’ density, reflecting the inhibition of som e inter­
neurons’ activity in subcortical forebrain structures after haloperidol treat­
ment. A more pronounced inhibition of dopam ine’s release by caerulein w as
the reason for the increased antiam phetam ine action after long-term halo p u id o l treatm ent. It seem s p ossible that both above m echanism s are
involved in the antipsychotic action of haloperidol.
702
ISSN 0365-9615
БЮЛЛЕТЕНЬ
ЭКСПЕРИМЕНТАЛЬНОЙ
БИОЛОГИИ И МЕДИЦИНЫ
Li ю л . -лм iii jMiM. О и о л с и и и и \ к д и к и м и , ЮНН, ( Л ', I , ! - Г2Н
вании значительных различий в свойствах серо­
тониновых С|д-рецепторов в мозге человек! по
сравнению с мозгом крысы и быка (например,
об их гетерогенности по сродству к буспнрону),
либо о выраженных возрастных изменениях этих
рецепторов, приводящих к снижению сродства
для буспирона. Возможность таких возрастных
изменений в свойствах серотониновых рецепто­
ров подтверждается данными литературы [9].
В заключение можно сделать вывод, что анксиолитик буспирон взаимодействует в микромолярных концентрациях с серотониновыми Q - и
Cj-рецепторами мозга человека.
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Vol. 61. — P. 373— 378.
8. R ib let L. A ., T o ylo r D. P , E iso n M. S ., S ta n to n H. C. / /
J. d i n . P sy ch iat. — 1982. — Vol. 43. — P. 11— 16.
9. S c h tn / ., Y o u n g H . 11 Life Scl. 1978. — Vol. 23. —
P. 1441— 1448.
10. S k o ln ic P ., P a u l S . / / J. clin. P sychiat. — 1982. —
Vol. 43. — P . 4 0 - 4 2 .
11. T em ple D., Yevich /., N e w / . / / I b i d . — P. 4—9.
12. Y o ung A. B., S n y d e r S . H. / / P roc. n a t. A cad. Sei. USA. —
1974. — Vol. 71. — P. 4002— 4007.
Поступил# 02.09,86
IN T E R A C T IO N O F AN A N X IO LYTIC A G EN T BU SP1RO N ,
W IT H HUM A N BR A IN SE R O T O N IN AND SO M E O TH ER
R E C E PT O R S
A
Ya. K orneev, М. I. Factor, C han T k h i B in A n
U SS R R esearch C e n tre fo r M en tal H ealth, A cadem y of M e­
d ical S ciences of the U SS R , M oscow
B usp iro n ad d M j 138-05 (u p to 0.1 m M ) did n o t disp la ­
ce specifically bound (’ H ) trip tam in e , (*H) strychnine.
(*H) flu n itra z e p a m an d (*H) im ipram ine in hu m an cortical
a n d h ippocam pal m em b ran e p re p a ratio n s. A t th e sam e tim e
both co m p o u n d s d isp lay ed sim ila r to se ro to n in affin ity
(1C,« in the ra n g e of 2-6 цМ) fo r (m I)-L S D specific binding
s ite s in the h u m an co rtex an d hippocam p. 1C» of serotonin
a n d b uspiron an d M i 138-05 for (*H) LSD (2 nM ) specific
b in d in g site s in the h ippocam p w as determ ined a s 0.14 )iM,
2.3 цМ and 6.1 цМ, respectively; and for (’ H ) sero to n in spe­
cific b in d in g site s in th e h ippocam p a s 0.005 цМ, 3.8 |iM
an d 21 |iM . respectively. The affin ity for h u m a n cortex
(*H) LSD b in d in g site s w a s 10-fotd low er in ca se of se ­
ro to n in an d 4 -fold low er in c a se of b u spiron a n d M j 138-05
th a n in the hippocam p. H ow ever, the affin ity for (*H) sero­
tonin b in d in g site s in the co rtex w as the sam e a s in the
hippocam p in case of se ro to n in an d 12-15-fold low er th a n
in the h ippocam p in case of b u spiron a n d M j 138-05. It is
concluded th a t in h u m an b ra in b uapiron and M j 138-05 in teract
w ith m icrom olar affin ity w ith 5 H Tj an d a re cap a b le of
b in d in g to a su b p o p u latio n of 5 HT, re ceptors in the hippo­
cam p.
УДК eiS.tl!.7:t<7.571.0IS.4:tl3.82l.!/.1.0l4.M :eiS .ll3
Ключевые
слова:
церулеин ; кетамин; стереотипное
поведение; двигательное возбуж дение; атаксия;
амнезии
Э. Э. Васар, Л . X. Алликметс, A. X. Соосаар
АНТАГОНИЗМ
ЦЕРУЛЕИНА,
АГОНИСТА
ХЦК-8 РЕЦЕП ТО РО В К ПОВЕДЕНЧЕСКИМ
ЭФФЕКТАМ КЕТАМИНА У МЫШЕЙ И КРЫС
Л а б оратори я психоф армакологии Тартуского университета
П редставлена акад. АМН СССР А. В. Вальдманом
Ф енциклидин и д ругие арилциклогексилам ины в
субамнестических д о зах о казы ваю т психотомнметическое действие на человека [1 ]. П рим ене­
ние фенциклидина или его более слабого а н а­
лога кетам ина соп ровож дается амнезией у че­
л о в ека [2, 6 ]. У крыс и мышей фенциклидин и
кетам ин вы зы ваю т усиление двигательной а к ­
тивности и стереотипное поведение — поведен­
ческие эф ф екты , напоминаю щ ие во многом д ей ­
ствие фенамина и других дофам ином иметиков у
этих ж ивотны х [9 ]. А ктивация моторики при вве­
дении ф енциклидина или кетам ина соп р о во ж д а­
ется атаксией [1 4 ]. П о казан о , что введение ф ен ­
циклидина м ы ш ам полностью п од авляет в ы р а ­
ботку защ итного р еф лекса по м етодике пасси в­
ного и збегания [1 1 ]. С ущ ествует мнение, что
стереотипное поведение, вы званное фенциклиди­
ном, обусловлено его взаим одействием с серотонин2-рецепторам и [9 ], в то время к а к амнестическое действие ф енциклидина реализуется че­
рез опиоидные рецепторы [1 1 ]. О ктапептид хо­
лецистокинина (Х Ц К -8) и его близкий аналог
церулеин о казы ваю т
антидофаминергическое
действие, антагонизирую т возбуж даю щ ем у дей ­
ствию фенамина на поведение кры с и мышей
[1 5 ]. П оявились т а к ж е данны е, что Х Ц К -8 вы ­
зы вает у кры с вы раж енны й антиамнестический
э ф ф е к т '[7].
Ц елью н астоящ его исследования бы ло изуче­
ние влияния церулеина, агониста Х Ц К -8 рецеп­
торов, на поведенческие эф ф екты кетам ина у
кры с и мышей, при этом специальное внимание
о б р ащ ал и на изм енения в опиоид- и дофам инергической системах.
Методика
и с с л е д о в а н и я . Все пове­
денческие опыты были проведены на мышахс ам ц ах массой 25—30 г и на кры сах-сам ц ах м ас­
сой 220— 270 г. В эксперим ентах на м ы ш ах изу­
чали влияние церулеина на основные поведенче­
ские эф ф екты кетам ина — усиление двигательной
активности, стереотипное поведение и атаксию .
Кетамин "Gedeon R ich ter” , В енгрия) в д озе
15— 30 мг/кг вводили мыш ам подкож но за 5 мин
до подкож ного введения церулеина в д озе 75—
375 м кг/кг f 'F a r m ita lia -C a r lo ЕгЬа” , И т ал и я ).
Галоперидол (0.1— 1,5 м г/м кг внутрибрюш инно,
•'O edeon R ich ter” , В ен гри я), антагонист доф а43
мнн2-рецепторои, вводили за 30 мин до введения
кетам ика. Интенсивность стереотипного поведе­
ния исследовали по методике [4] на 10-й мину­
те после введения кетамина.
Интенсивность
атаксии оценивали такж е на 10-й минуте после
введения по условно выработаннс(й ш кале [3 ].
С 10-й по 15-ю минуты после введения кетам и ­
на исследовали его влияние на ориентировочно­
исследовательскую активность мышей в откры ­
том поле. Открытое поле (3 0 X 3 0 X 1 5 см) р а зд е ­
лял и линиями на 16 секторов (в центре каж дого
бы ло гнездо). В течение 5 мин определяли чис­
ло секторов, пройденных мышами, число вста­
ваний на задние лапы и число обследо­
ванных
гнезд. У
крыс
изучали
влияние
церулеина
на амнестическое действие к ета­
м ина по методике пассивного избегания в чел­
ночной камере. В 1-й день исследования ж и вот­
ных адаптировали к обстановке опы та. Н а 2-й
день проводили обучение животных. После пе­
рехода крысы в темный отсек дверь меж ду д ву ­
мя частями челночной кам еры закр ы вал и и ж и ­
вотное получало 4 электрических у дара через
пол кам еры напряж ением 40 В. И н тервал м еж ­
ду электрическими ударам и 45 с. Д л я опыта
отбирали только тех животных, которые в тече­
ние 20 с переходили из светлого отсека кам еры
в темный. С разу после обучения животным вво­
дили кетамин (7,5—30 мг/кг п одкож но), церуле­
ин (10 м кг/кг подкожно) и налоксон (5 мг/кг
подкожно, “ Endo L abs” , СШ А ), а та к ж е ц еруле­
ин и налоксон в сочетании с кетамином. Через
24 ч после обучения определяли латентное вр е­
мя перехода животного из светлой в темную
часть челночной кам еры , а та к ж е время пребы ­
ван ия крысы в темном отсеке. З а поведением
каж дого ж ивотного наблю дали в течение 3 мин.
П арал л ел ьн о с поведенческими опы тами иссле­
довали влияние кетам ина на связы вание 3Н-спи-
роперидола во фронтальной коре крыс в присут­
ствии 5 мкМ сульпирида (" R a v iz z a ", И т ал и я ),
избирательного ан тагониста дофаминг-рецепторов, и в хвостатом ядре в присутствии 1 мкМ
пиренперона (" Ja n s s e n P h a rm a c e u tic a" , Б е л ь ­
ги я ), антагониста серотонин2-рецепторов. С в я ­
зы вание
3Н -спироперидола
(16
Ки/ммоль,
"A m ersh am In te rn a tio n a l”, Англия) исследовали
по методике [5 ]. Влияние кетам ина на св язы ­
вание 3Н -эторф ина (36 Ки/ммоль, “A m ersham
In te rn a tio n a l” изучали в переднем мозге кры с по
методике [12].
Р е з у л ь т а т ы и с с л е д о в а н и я . П одкож ное
введение кетам ина в д озах 15 и 30 мг/кг в ы зы ва­
ло отчетливое усиление двигательной активно­
с т и — двигательное возбуж дение у мышей в о т­
крытом поле, у животны х отм ечались интенсив­
ные стереотипные принюхивания, а т а к ж е а т а к ­
сия (табл. 1). И з-за атаксии у мышей, получав­
ших кетамин, отсутствовали вставан и я на задние
лапы . Ц ерулеин в д озе 75 м кг/кг антагонизировал
двигательному возбуж дению , вы званному к е т а ­
мином (30 м г/к г), и лиш ь в д озе 360 м кг/кг
полностью п одавлял кетамнновую стереотипию.
П ри этом церулеин слабо влиял на кетаминовую атаксию (см. табл. 1). Галоперидол в дозе
0,5 мг/кг достоверно о слаб лял дви гательн ое во з­
буж дение и стереотипное поведение, вы званны е
кетамином (30 м г/кг). Н а кры с кетамин о к а зы ­
вал амнестическое действие (табл. 2 ). В д озах
15 и 30 мг/кг п реп арат достоверно н аруш ал обу­
чение крыс. В д озе 10 мкг/кг церулеин не влиял
на обучение крыс в челночной кам ере, однако
полностью устранял амнестическое действие ке­
там ина. Аналогичное действие о казы в ал а н т а ­
гонист опиоидных рецепторов налоксон в дозе
5 мг/кг (см. табл. 2 ). В опы тах по изучению радиолигандного связы вания кетам ин д а ж е в кон­
центрации 100 мкМ не влиял на связы вание 3НТаблица
1
Влняние церулеина и галоперидола ка лоаеденческк аффекты кетамина у мышей
Доза
Физиологический
раствор
Кетамин
То же
Кетамин -J- церулеин
Кетамин + церулеин
Кетамин + церулеин
Кетамин + церулеин
Кетамин 4- галоперидол
Кетамин -j- галоперидол
Кетамин + галоперидол
30
30
30
30
15 мг/кг
30 мг/кг
м г/кг+75 мкг/кг
мг/кг+150 мкг/кг
мг/кг-j-225 мкг/кг
мг/кг-j-375 мкг/кг
30+0,1 мг/кг
3 0 + 0 ,5 мг/кг
3 0 + 1 ,5 мг/кг
Стереотипное
баллы
0±0
1,7 5 ± 0 ,15
1,92±0,12
1,75± 0,20
1,2 0 ± 0 ,18*
0 ,8 3 ± 0 ,12*
О.ЗЗгЬО,15**
1,50±0,20
1,2 0 ± 0 ,16*
0 ,3 3 ± 0 ,15**
Ориентировочно-исследовательская
активность в течение S мин
Атаксия, баллы
число прой­
денных сек­
торов
0±0
1,20 ± 0 ,2 0
1,83± 0,25
1,7 5 ± 0 ,15
1,50± 0,20
1.40±0,20
1,3 2 ± 0 ,15
1,42±0,20
1,17±0,25
1,83±0,20
40± 3,2
60± 5 ,8
85± 6,6
48±4,2»
3 5 ± 4 ,0 24±5,2**
И ± 1 ,6 » «
58± 5,2
21±4,2<*
4±0,2***
число обсле­ число вста­
дованных
вания на задгнезд
иие лапы
9 ± 1 ,2
8 ± 1 ,8
8 ± 0 ,9
6 ± 0 ,8
6 ± 0 ,9
4± 0,5*
3± 0,6**
6 ± 0 ,9
4 ± 0 ,5 0±0***
7 ± 1 ,6
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
Примечание
Звездочки — достоверность различия У тест Манна-Уитни показателей по ' сравнению с введена м
кетамина: одна— р< 0 ,0 5 , две — р< 0 ,0 1 , три — р < 0 ,0 0 1 .
13*
Таблица
Влияние ц ер у л е и н а и налоксона на
кетамина у крыс
ам н ести ческо е
2
д е й с тв и е
lii
Кетамин
Кетам mi
Кетямин
Кетамин -|- церулеин
Кетамин + церулеин
Церулеин
Кетамин + налоксон
Налоксон
11 к - V , .
~
Звездочки—достоверность различия ( t ~тест
Стьюдента) показателей по сравнению с введением кетамина; о д ­
н а —р с О . 0 5 . две —р < 0 . 0 1 .
спироперидола (0,25 нМ) в хвостатом ядре (дофа мин2-рецепторы) и во фронтальной коре (серотонин2-рец еп торы ). Кетамин вы зы вал полуингибирование связы вания 3Н -эторф ина (0,25 нМ)
при концентрации 30 мкМ. При дальнейш ем по­
вышении концентрации кетамина его влияние на
связы вание 3Н -эторф ина не изменялось.
Таким образом , полученные данные свидетель­
ствую т о том, что агонист ХЦ К-8 рецепторов
церуленн способен антагоннзировать определен­
ным поведенческим эф ф ектам кетам ина — стиму­
л ятора фенциклидииовых рецепторов. В ы сказано
предположение, что двигательное возбуж дение,
вы званное фенциклидином, у крыс и мышей ре­
ализуется через серотонин2-рецепторы [9 ]. О д ­
нако по данным настоящ его исследования, ке­
там ин такое действие не оказы вал. Кетамин не
взаим одействовал с серотонии2-рецепторами и не
вы зы вал поведенческих эффектов, характерны х
для серотониномиметиков (встряхивание голо­
вой, встряхивания «мокрой собаки» и т. д .). По
всей вероятности, стереотипное поведение и д в и ­
гательное возбуждение, наблю даем ы е после вв е­
дения кетам ина, обусловлены , как и в случае ф е­
намина, усилением высвобождении доф ам ина из
пресинаптических терм иналей в хвостатом ядре
ii мезолимбических структурах. К ак известно,
церулеин
оказы вает
антидофамннергичеслое
действие [13, 15]. С антицофаминергическим
действием церулеина связан, вероятно, и ан таго­
низм церулеина со стереотипным поведением и
двигательны м возбуж дением, вы званны х кетамином. В пользу этого предположения свидетельст­
вует и ф акт, что галоперидол, преимущ ественный
антагонист доф ам ин2-рецепторов, о казы вает а н а ­
логичное с иерулеином угнетаю щ ее влияние на
поведенческие эфф екты кетам ина у мышей. По
данным C o n trera s и соавт [3 ], атакси я, н аблю ­
д аем ая после введения кетамина и ф енциклиди­
на, реализуется по сравнению со стереотипным
поведением через другие механизмы. Этим о б ­
стоятельством , по-видимому, обусловлено и с л а ­
бое влияние церулеина на кетам иновую атаксию .
В амнестнческом действии кетам ина взаи м о д ей ­
ствие п реп ар ата с опиоидными рецепторам и име­
ет ведущ ее значение, как и з случае с ф ен ц и к­
лидином [11]. Опиоидный антагонист налоксон
является эфф ективны м антагонистом амнестического действия кетам ина. У становлено, что ц е ­
рулеин в д озе 10 м кг/кг и меньш е блокирует
аналгезию , вы званную морфином у кры с [8 ].
М ожно п олагать, что ф ункциональны й ан таго ­
низм с опиоидными рец епторам и находится в
основе антагон изм а церулеина с амнестическнм
действием кетам ина.
ЛИТЕРАТУРА
1978.
1. Allen R М., Young S. 1.Ц Amer. J. Psychiat.
Vol. 135. — P. 1081 — 1089.
2. Burns R, S., Lerner S. E. //C lin . Toxicol. — 1976. —
Vol. 9. — P. 477—501.
3. Contreras P. C., Rice К■ C.. Jacobson A. E., 0 ‘Dono­
hue T. L. // Europ. J. Pharmacol. — 1986. — Vol. 121. —
P. 9— 18.
4. Costall S., Naylor R. I. / / Ibid. — 1974. — Vol. 29. —
P. 206—222.
5. Creese 1., Schneider R., Snyder S. H. / / Ibid. — 1977. —
Vol. 45. — P 377—381.
6. Ghoneim M. M., Hinrichs J. V. Mewaldl S. P., Peter­
sen R. C. j j J. clin. Psychopharmacol. — 1985. — Vol 5. —
P. 70—77.
7. Katsuura G., Itoh S. 11 Drug Dev. Res. — 1986. —
Vol. 7. — P. 269—276.
8. Magnani M., Mantovani P., Pepeu G. / / Neuropharmaco­
logy. — 1984. — Vol. 23. — P. 1305— 1309.
9. Nabeshima Т., Yamaguchi K . Yamada K. et al.//E u ro p .
J. Pharmacol. — 1983. — Vol. 93. — P. 229—234.
10. Nabeshima Т., Noda Y M a ts u n o K. et a l ./ / Res. Commun. Subst. Abuse. — 1984. — Vol. 5. — P. 81—87.
11. Nabeshima Т., Kozawa Т., Furukawa H., Kameyama Т. П
Psychopharmacology. — 1986. — Vol. 89. — P. 334—337.
12. Owen F, Bourne R. C., Poulter M. et al. / / Brit. J. Piychiat. — 1985. — Vol. 146. — P. 507—509.
13. Vasar E., Maimets M., Nurk A. et a l ./ / Pharm. Biochtm.
Behav. — 1986 — Vol. 24. — P. 469—478.
14. Vincent J. P., Kartalovski B.. Geneste P. et al / / Proc
nat. Acad. Sei. USA — 1979 — Vol. 76. — P. 4«7t—
4682.
15. Zetler G. / / Peptides. — 1985 — Vol. 6. — P. 3» 4«
П остун м м Ш Ш -V
CAERULEIN, AN AGONIST OF CCK-8 RECEPTORS, AN­
TAGONIZES THE BEHAVIOURAL EFFECTS OF KETAMI NE IN MICE AND RATS
E E. Vasar, L Kh. Allikmets. A. Kh. Soosaar
Tartu State University
It has been established in experiments on m *k mice
and rats that caerulein antagonized the behaviour*! effects
of ketamine. an agonist of phenAyclidine receptor». Caeru
lein (75-375 (ig/kg) and haloperidol (0.1-I.5 m g/kg) »uppressed the stereotyped behaviour and motor excitation induced by ketamine (30 m g/kg) in mice. Caeruloia and ha­
loperidol failed to affect ketamine-induced ataxia. Caerulein
(10 HgAg) and the opioid antagonist naloxone (& e f / k g ;
completely blocked the amnestic action of kttim ine
(30 m g/kg) in passive avoidance experiment» on rat* It
seems likely that the suppression of the behavioural effects
of ketamine by caerulein is related to its functional an ta­
gonism with dopamine and opioid receptors.
45
ISSN 0365-9615
БЮЛЛЕТЕНЬ
ЭКСПЕРИМЕНТАЛЬНОЙ
БИОЛОГИИ И МЕДИЦИНЫ
ft
h* I
■»k i Kiv'iHiM f i i n j. i u im i u mi . ( п ш ш м . И М *. » Л . ‘J . Il*»
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24 !i t.l llir i n | i‘<‘ J j o i I, h e lo ie I m* ним* I o l I h r le i'.i s le r a b le
p a n r .i te n i u
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n e u tro p h ils a n d
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T i o p i e a l M e d ic i n e , M in i s tr y o f H e a lth o M Iie U S S R . M o sc o w
NBT-test l<*r (irn il.itint; neutrophils and monocytes
in I lit* blood of шин1 inimcutated with Plasmodium berghei.
Ф А Р М А К О Л О ГИ Я "^
^
^
^
К л ю ч е вы е слова:
церулеин, фенамин,
хинолиновая кислота, двигательное возбуждение,
судороги
Э .Э . Васар, J 1 .X . Алликметс, И. В. Рыжов,
И. Б. Прахье, A . X . Соосаар, С. Мирзаев
РАЗЛИЧИЯ В ПОВЕДЕНЧЕСКИХ
ЦЕРУЛЕИНА — АГОНИСТА РЕ­
ОКТАПЕПТИДА ХОЛЕЦИСТОКИ­
БЕЛЫХ МЫШЕЙ И КРЫС
Л а б о р а то р и я психоф арм акологии 111 111 общей и моле­
к у л яр н о й патологии Т а р ту ск о го университета, л аб о р а­
то р и я
п сихоф арм акологии Л е н и н гр ад с к о го иаучио-иссл ед о ва те л ьс к о го
психоневрологического
институ­
та им. В. М. Б ехтерева
Представлена акад. АМН СССР. А Б. Ьальдыат-м
Октапептпд холецистокинина (ХЦК-8) и его близ­
кий аналог церуленн обладают широким спектром
фармакологического действия 115]. Они угнетают
спонтанную двигательную активность, противо­
действуют фенаминовому двигательному возбужде­
нию, блокируют стереотипное поведение, вызван­
ное дофаминомиметиками, оказывают противосудорожиое действие п т .д . [3, 8, 14, 151. Однако не
всегда исследователям удавалось в своих опытах
воспроизвести результаты, полученные другими
авторами. Т ак, в одних исследованиях церулеин
и ХЦ К-8 угнетали поведенческие эффекты апомор­
фина [12, 151, в других наблюдалось противополож­
ное действие
усиление эффектов этого дофамнномиметнка 12, 131.
В настоящем исследовании была поставлена
цель выяснить причину такой разноречивости
данных. В связи с этим мы изучили видовые р аз­
личия в действии церулеина — агониста рецепто­
ров ХЦ К-8 В опытах на мышах-самцах и крысах-самцах в сравнительном аспекте были изуче­
168
и и и -д - o f
Ни
-.и Ь ч с ц и .ц !
..x
.< .'»<>
days
Ml(l%
c r i/ v iiie
s y s te m
m o n o c y te s of
o l N U T te s l c o u ld
in
n i e i i l a l in i!
the host
b lo o d
p h a g o c y te s ,
I lie r e s u l ts
he u t i l i z e d lo r th e I n v e s t i t i o n
ol im -
m u n o h u i k a l d i s o r d e r s a n d a ls o f o r th e d if f e r e n t i a l d ia tf u o
—~
УДК в 12 .8 2 1.01 4.4 в :6 15.357.34:577.175.734
ВИДОВЫЕ
ЭФФЕКТАХ
ЦЕПТОРОВ
НИНА — У
th e
v l i a i n ti .cil lo r in n o t til a li o ii)
ie d ti< | io n in N H T p o s i t i v e c e l l . \va.-. o b s e r v e d . T h is d e m o n
• Ir.ile -, I he .( b ill I v o l fit.-il.ii i.i p a r a s i l e I n M ip p ie sv t h e o x y
\ I.л к I \
/:. (illpin Juckwn,
,m
rd i-p rn (liH ): o l
ны длительнее антнфенаминовое действие церулеи
на 171 и антагонизм церулеина с эндогенным кон
вульсантом хинолиновой кислотой (Х И К) 11, 111.
Методика
исследования.
В опы­
тах использовали белых беспородных мышейсамцов массой 18—24 г и беспородных крыссамцов массой 180—220 г из питомника «Рапполово» АМН СССР (Ленинградская область) в весенне-летний период.
Антагонизм с фенаминовым двигательным вез
буждением изучали по следующей схеме: в пер­
вый день опыта одной группе крыс или мышей
внутрибрюшннно вводили физиологический рас­
твор, другой — подкожно церулеин (крысам —
40 м кг/кг, мышам — 50 и 100 мкг/кг), третьей—
внутрибрюшннно галоперидол (0,25 мг/кг), чет­
вертой — галоперидол совместно с церуленном.
Опыты с фенамином были проведены трижды:
через 1 сут, на 7-е и 14-е сутки после однократно­
го введения галоперидола («Gedeon
Richter»,
В Н Р) и церулеина («Farm italia — Carlo Erba»,
И талия).
Возбуждающее
действие фенамина
(3 мг/кг) на моторику мышей определяли с помощью
фотоэлектрического актометра. Ж ивотных поме­
щали в актометр через 15 мин после внутрибрюшинного введения фенамина и в течение 30 мин
определяли двигательную активность животных.
Влияние фенамина (2 мг/кг) на двигательную ак­
тивность крыс определяли через 45 мин после
внутрибрюшннного введения в течение 5 мин в
тесте открытого поля (регистрация фотоэлектри­
ческим способом с помощью 5 независимых ка­
налов). В отдельной серии опытов определяли дей­
ствие совместного введения проглумида (50 мг/кг,
«Rotta Research Labs», Италия) — антагониста
рецепторов ХЦ К-8 — с галоперидолом и церулеином на длительное антнфенаминовое действие церуленна у крыс.
Влияние церулеина на эффекты различных конвульсантов у мышей определяли следующим об-
Влияние однократного предварительного подкожного введе­
ния церулеина (50--КЮ мкг/кг) и внулрибрюшинного »веде­
ния галоперидола (0,25 мг/кг) на двигательное возбужде­
ние, вызванное фенаминам (3 мг/кг) у мышей (М \ т)
Чж’ЛП иипулм-пм в Т.чейне 3U мим
Вещество
ЬОд.нь
Влияние однократного предварительного подкожного вве­
дения церулеина (40 мкг/кг) и вмутрибрюшинного вве­
дения галоперидола (0,25 мг/кг) на двигательное возбуж­
дение, вызванное фенамином (2 мг/кг) у крыс.
По оси абсцисс — срок после однократного введения церулеина
или галоперидола (в сут), по оси ординат — число импульсов в те­
чение 5 мин. / — контроль (действие фенамина у крыс, получивших
предварительно физиологический раствор); 2 — церулеин: 3 — га
лоперидол; 4 — галоперидол + церулеин. Одна звездочка — р <
< 0 .0 5 . две — р< 0.01 по сравнению с контрольными животными,
получавшими физиологический рвствор (U -тест Манна — Уитни).
разом: Х И К (5 мкг), L-каиновую кислоту (0,2 мкг),
L-кинуренина
сульфат (50 мкг) и Н-метил-Dаспартат (0,1 мкг; все — фирмы «Sigma», США)
вводили в латеральный желудочек мозга с помощью
полуавтоматического аппарата по ранее описан­
ной методике [5] в судорожных дозах (ЭД1М) в
постоянном объеме 2 мкл. Проглумид вводили мы­
шам внутрибрюшннно за 5 мин до внутрижелудочкового введения церулеина или за 10 мин до коивульсанта. Церулеин (1—50 нг в желудочек мозга
и 100—500 м кг/кг подкожно) вводили за 5 мин
до введения конвульсантов. Длительность на­
блюдения после введения конвульсанта составля­
ла 10 мин.
В опытах на крысах изучали судороги, вы з­
ванные ХИК, и церулеин вводили подкожно в до­
зе 200 м кг/кг или в желудочек мозга в дозах 2—
20 нг за 5 мин до ХИК (30 и 60 мкг в ж елудочек
мозга). В ж ивление какюли в левый боковой ж е ­
лудочек крыс проводили под нембуталовым нар­
козом (40—50 мг/кг). Детально метод вж ивления
описан ранее (6). В эксперимент животны х бра­
ли через 4—5 сут после операции. Растворы пре­
паратов вводили с помощью шприца Гамильтона
и полиэтиленовой трубочки. За поведением крыс
наблюдали в течение 90 мин после введения кон­
вульсанта. Во всех группах определяли 4 пока­
зателя: латентный период наступления клони­
ческих судорог, частоту клонических и тониче­
ских экстензий и летальность в группе.
Р е з у л ь т а т ы и с с л е д о в а н и я . У крыс
однократное совместное введение церулеина и
галоперидола и в меньшей степени введение од-
Физиологический раствор 4* физиоло­
гический раствор
Физиологический раствор -j- фенамин
(3 мг/кг)
Церулеин (50 мг/кг) 4- фенамин
(3 мг/кг)
Церулеин (100 мг/кг -f- фенамин
(3 мг/кг)
Галоперидол (0,25 мг/кг 4 фона мин
(3 мг/кг)
Галоперидол (0,25 мг/кг) 4 церулеин
(50 мг/кг) 4- фенамин (3 мг/кг)
Галоперидол (0,25 мг/кг) 4 церулеин
(100 мг/кг) 4 фенамин (3 мг/кг)
314 ± 30
7 Я А' "»
35С 1 34
589 ± 69
4%-*-Гь
510±61
ÜI5.LÜ2
854 ±98*
баз ± 70
717±58
578±(W
815±80*
799±80*
814±76*
747±7С*
П р и м е ч а н и е . Звездочка— р < 0 ,0 5 (тест U Ман­
на—Уитни) по сравнению с группой мышей, получивших
физиологический раствор 4 фенамин (3 мг/кг).
ного церулеина оказы вало длительное ингиби­
рующее влияние на возбуждающее действие 2 мг/кг
фенамина (см. рисунок). У же через 1 сут после
совместного введения церулеина и галоперидола
четко проявилось их антнфенаминовое действие.
Существует мнение, что длительное ингибирующее
влияние совместного введения галоперидола и це­
рулеина на фенаминовое возбуждение моторики
реализуется через ß-эндорфин в прилегающем яд­
ре 17). Наши данные указы ваю т на то, что вл и я ­
ние церулеина на ß-эндорфинергнческие процес­
сы опосредуется через рецепторы ХЦК-8. В п оль­
зу этого предположения свидетельствует тот факт,
что антнфенаминовое действие развивается и после
введения одного церулеина. Однако проглумид
(50 м г/кг), известный антагонист ХЦК-8, не устра­
нял эффекта, вызванного совместным введением
галоперидола и церулеина. Э го позволяет пола­
гать, что проглумид не взаимодействует с рецеп­
торами ХЦК-8. Р оль галоперидола заклю чается
в повышении чувствительности опиоидных рецеп­
торов к ß -эндорфин у в мезолимбических структу­
рах [10]. В опытах на мышах церулеин и галопе­
ридол такого антифенаминового действия не о ка ­
зывали. Предварительное введение церуленна (50
п 100 мкг/кг) н совместное введение церулеина
и галоперидола даж е усиливало аффект фенамина,
т. е. наблюдалась гиперчувств!ярельнс1сть к воз­
буждающему дейстиию фенамина у мышёП Тем
таблицу).
Введение церулеина (1 нг) в ж елудочек мозга
предупреждало у мышей судороги, вызванные
ХИК. Проглумид (50 мг/кг) полностью устранял
защитный эффект церуленна. В ченыпей до«(25 мг/кг) проглумид потенцировал судорожный
эффект подпороговой дозы Х И К (2.5 мкг)
унг-
личнвал число животных с судорогами от 0 до
5 в группе ич 6 мышей. Следует отметить, что противосудорожное действие церулеина в данной
модели имеет, по-видимому, довольно избиратель­
ный характер Церулеин предупреждал вызван­
ные только ХИ К и Н -м ет и л -D-acnapTaTOM судо­
роги. Это подтверждает предположение о том,
что данные вещества действуют на один общий
Н-метил-О-аспартатный рецептор [111. Церулеин
был неактивен против каиновой кислоты и кинуренина. При подкожном введении мышам церу­
леин в большом диапазоне доз (100—500 мкг/кг)
слабо влиял на ХИК-судороги, удлинив лишь
латентный период их наступления. Предвари­
тельно!; подкожное (200 мкг/кг) или внутрижелудочковое введение (2—20 нг) церулеина не пре­
пятствовало развитию ХИК-судорог у крыс, не
изменялись ни количество судорожных присту­
пов, ни латентные периоды клонических и тони­
ческих судорог. Продолжительность жизни ж и­
вотных была даже короче в опытной группе по
сравнению с контролем (соответственно 52 и
92 мин).
Таким образом, полученные данные свидетель­
ствуют о значительных различиях в действии це­
рулеина на крыс и мышей. Неодинаковое влияние
церулеина на возбуждающее действие фенамина,
по-вндимому, обусловлено различиями во взаимо­
действии между ХЦК-8 и дофаминергическими
системами у крыс и мышей. Если в опытах на мы­
шах церулеин при подкожном введении в дозе
75 мкг/кг и более устранял повышенную двига­
тельную активность, вызванную фенамином [131,
то у крыс доза 40 мкг/кг церулеина (при подкож ­
ном введении) не изменяла эффект фенамина [71.
На основании этих данных можно полагать, что
у мышей церулеин при системном введении ока­
зывает непосредственное угнетающее влияние на
дофаминергические процессы в
лимбических
структурах, что обусловливает повышенную чув­
ствительность мышей к возбуждающему действию
фенамина после однократного введения церулеина.
У крыс взаимодействие между дофамином и ХЦ К-8
является более сложным и, возможно, опосреду­
ется через усиление высвобождения ß-эндорфина
в прилегающем ядре [71, что в конечном счете
приводит к длительному понижению чувствитель­
ности крыс к возбуждающему действию фенамина.
В опытах на мышах церулеин при внутрижелудочковом (но не при системном) введении был
сильным и избирательным антагонистом эндоген­
ных конвульсантов — Х И К и Н-метил-D-acnapтата. Следует отметить, что у травяной лягуш ки
(R ana tem poraria), в коже которой обнаружено
большое количество церулеина, его предваритель­
ное введение (1—5 нг) такж е предупреждало
ХИК-судороги, а проглумид (50— 100 мг/кг) сни­
мал этот эффект. В опытах на крысах церулеин
такого действия не оказывал. Кроме того, у крыс
при внутрижелудочковом его введении ХИ К-су­
дороги развивались значительно медленнее, чем
170
у мышей. Вероятным объяснением этих различий
у крыс и мышей является неодинаковое простран­
ственное расположение латеральных желудочка
и гиппокампа в мозге 14, 6, 91, что обусловли­
вает неодинаковое
проникновение исследуемых
веществ в гиппокамп при их внутрижелудочковом
введении.
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1985 — № 6. — С. 13 -1 7 .
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4. König J . F. R., Klippel R. A. The rat brain: A ste­
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5. Lapin I. P. II J . Neural Transm. — 1978. — Vol. 42.—
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7. Matsubara K.. Matsushita A. II Jap. J . P h arm ac o l.—
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8. Matsushita A ., Itoh S. II World Psychiatric Accoclation
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9 Slolnick В. M ., Leonard C. M. A Stereotaxic Atlas of
Ihe Albino Mouse fore Brain. — Rockville, 1975.
10. Stinus L., Nadaud D., Juuregui J., Kelley A. E. II
Biol. Psychiat. — 1986 — Vol 21. - P 34—38.
11. Stone T. W., Connick J. H. II Neurosci. — 1985. —
Vol. 15. — P. 597—617.
12. Van Ree J . M ., Gaffori О. , De Wied D. II Europ. J.
Pharmacol. — 1983. — Vol. 93. — P. 63—78.
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Pharm a­
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478.
14. ZetlerG. '
Neuropharmacology. — 1981. — Vol 20.
15. ZetlerG il Peptides. — 1985. — Vol. 6, Suppl. 3. —
P. 3 3 -4 6 .
Поступила 25.11.86
IN TERSPECIES DIFFERENCES IN THE BEHAVIOU­
RAL EFFECTS OF CAERULEI N, AN AGONIST OF
CCK-8 RECEPTORS, IN MICE AND RATS
E E. Vasar, L. Kh. Allikmets, I. V. Ryzhov, I. B. Prakhye,
A. Kh. Soosar, S. M inaev
Tarty State University; V. M. Bekhterev Institute of Psy­
choneurology, Leningrad
It has been shown in the behavioural experiments that
combined pretreatm ent with haloperidol (0,25 mg/kg)
and caerulein (40 |ig/kg). and to a lesser extent pretreatment with caerulein alone caused long-term reversal of
am phetam ine (2 mg/kg) induced hyperexcitability in rats.
Administration of proglumide (50 mg/kg), an antagonist
of CCK-8 receptors, did not reverse long-term antiam phe­
tam ine effect ol caerulein. In mice pretreatm ent with caeru­
lein (50 and 100 \iajkg) alone or in combination with ha­
loperidol (0.25 mg/kg) caused hypersensitivity to the be­
havioural effect of amphetamine (3 mg/kg). Intraventri­
cular (I ng), but not systemic (100—500 (xg/kg) adm inistra­
tion of caerulein selectively antagonized seizures in mice
induced by intraventricular adm inistration of quinolinic
acid (5 jig) and N-methyl-D-aspartate (0,2 pg). P retreat­
ment with proglumide (50 mg/kg) reversed the anticonvuisive effect of caerulein in mice. In rats, caerulein failed to
affect the seizures caused by intraventricular adm inistration
of ^ u tn o lm ic дс!о1.
EESTI NSV TEADUSTE AKADEEMIA TO IM ETISED . B IOLO OGIA
И ЗВЕСТИЯ АКАДЕМ И И НАУК ЭСТОНСКОЙ ССР. БИ О Л О ГИ Я
P R O CEED IN G S OF TH E ACADEMY O P SCIEN C ES OE TH E ESTONIAN SSK
BIOLOGY
1УНН, 37, 2
У Д К 615.767.22
Э эро B A C A P , А н д р ес С О О С А А Р , А а во Л А Н Г
УЧАСТИЕ ХОЛЕЦИСТОКИНИНОВЫХ РЕЦЕПТОРОВ В
РЕАЛИЗАЦИИ ПОВЕДЕНЧЕСКИХ И БИОХИМИЧЕСКИХ
ЭФФЕКТОВ ДЛИТЕЛЬНОГО ВВЕДЕНИЯ ГАЛОПЕРИДОЛА
Д л и тельн о е введение нейролептиков, эф ф ективн ы х и р асп ростран ен ­
ных антипсихопатических вещ еств, вы зы вает весьм а разн он ап равлен ны е
изм енения в активности нейром едиаторпы х систем м озга (А лликм етс
и др., 1984). С реди этих изменений н аи более значим ы м и являю тся
сдвиги в плотности н ей рональны х рецепторов центральной нервной
системы (А лликм етс и др., 1984, 1986). У становлено, что длительное
введение нейролептиков вы зы вает увеличение числа доф ам и н 2- и глутам ати ы х рецепторов в переднем м озге (Ж ар ко вски й , А лликм етс, 1986),
в то врем я как плотность ГАМ Ка- и бен зоди азеп и новы х рецепторов
п он и ж ается (А лликм етс и др., 1986). О траж ен и ем этих изменений на
м олекулярном уровне яв л я ется гиперчувствительность подопытных
ж ивотны х к поведенческим эф ф ектам доф ам ином им етиков ( Ж а р к о в ­
ский, А лликм етс, 1986), поведенческие ж е эф ф екты агониста ГАМ Кдрецепторов м усцимола и бензодиазеп и н ового ан тагони ста Ro 15-1788
и звр ащ аю тся (В асар и др., 1986). В последние годы появились данны е,
что д ли тельн ое применение различны х по химической структуре н ейро­
леп ти ков (гал о п ер и д о л а, хл орп ром азин а и кл о зап и н а) вы зы вает за м е т ­
ное увеличение со д ер ж ан и я октапеп тид а холецистокинина (Х Ц К -8) в
п одкорковы х стр у кту р ах мозга (F rey, 1983). У становлено, что д л и тел ь ­
ное введение типичного ней ролептика га лоп ери д ол а пон и ж ает п лот­
ность Х Ц К -8 рецепторов в переднем мозге (В асар и др., 1986) и о сл а б ­
л яет поведенческие эф ф екты агониста Х Ц К -8 рецепторов церулеи н а,
вы зы вая к ним гипочувствительность. П о к азан о , что Х Ц К -8 явл я ется
сом едиатором до ф ам и н а в м езолим бических структурах (H ö k felt и др.,
1980), а ГАМ К — в гиппокам пе и корковы х структурах больш их п олу­
ш арий (K osaka и др., 1985). Ц елью н астоящ его исследования бы ло
изучение роли Х Ц К -8 рецепторов в тех поведенческих и нейрохим иче­
ских изм енениях, которы е н абл ю даю тся после дли тельн ого введения
гало п ер и д о л а.
Методика
О пыты проводили на кры сах (сам цы массой 220—270 г) и м ы ш ах
(сам цы массой 20—25 г). Н а кры сах провели два исследован и я: пер­
вый в октябре, второй — п д ек а б р е 1986 г. С ледует отм етить, что вто­
рой опыт был зав ерш ен непосредственно перед резким похолоданием .
В течение 15 дней кры сам вводили впутрнбрю ш инно галоп ери дол
(0,5 м г/кг в ден ь, «G etleon R ichter», В енгрия) или ф изиологический
раствор. Ч ерез 72 ч после отмены галоп ери д ол а ж ивотны х разд ел и ли
на две группы: одни бы ли использованы дл я поведенческих иссл ед ов а­
ний, другие — д л я опы тов радиол и гап дн ого связы ван и я. П еред н а ч а ­
лом поведенческих опы тов половине крыс подкож но ввели церулеин
(40 м кг/кг, « F a rm ila lia -C a rlo lürha», И т а л и я ), остальны м — ф и зи ол оги ­
14
ческий раствор. После введения д важ ды — через 24 ч и через 7 дней —
определяли основные поведенческие эф ф екты иидн рентного доф аминомпметика ф енам ина (2 м г/кг): стереотипное поведение по условной
ш кале {C ostа 11, N aylor, 1074) и усиление орнептировочпо-нсследоиательской активности. П ервое исследовали через 30 мин после введения
ф енам ина, второе — через 15 мни. О риентировочно-исследовательскую
активность оценивали на открытом поле (1 0 0 X 1 0 0 X 4 0 см ), где в теч е­
ние 5 мин с помощью независимых ф отоэлектрических кан ал ов оп ред е­
ляли двигательную активность крыс.
Р ади оли ган дное связы вание определяли на основе п арам етров сия
зы вания 3Н -пен тагастрнн а (уд. активность 81 К и/м м оль, NHN, (ЛИЛ) и
3Н -ф луп и тразеп ам а (уд. активность 81 Ки/м моль, «A m ershain I n te r n a ­
tio n al» , А нглия) в коре больш их полуш арий, и 3Н -спиронеридола (уд.
активность 17 К и/м моль, «A m ersham In te rn a tio n a l» , А нглия) в хвоста
том ядре. С вязы ван ие 3П -п ен тагастри и а, л и ган д а центральны х ХЦК-8
рецепторов, проводили по методике Л\. П рен зм ани а (P ra is s m a n и др.,
1983), связы ван и е 3Н -ф лун н тразеиам а и 3Н -сииропсридола — по м ето­
дике, описанной нами ранее (Н урк и др., 1984). Д ан ны е опытов о б р а ­
баты вали с помощью ан ал и за С кетчарда.
В отдельной серии опытов на м ы ш ах-сам цах в течение 15 дней оп р е­
деляли влияние длительного введения галопери д ола (0,5 мг/кг в день)
и церулеина (0,1 м г/кг в день) на поведенческие эф ф екты ф ен ам ина,
м усцимола (« S erva», Ф РГ ) и Ro 15-1788 («M offm ann-L a Roche», Ш вей­
ц ар и я). Ф енамин (3 м г/кг), мусцимол (1 мг/кг) и Ro 15-1788 (10 м г/кг)
вводили за 15 мин до помещения мышей в ф отоэлектрический актом етр
(диам етр 40 см ). П ар ал л ел ь н о с поведенческими опытами исследовали
связы вание 3Н -сппроперндола, 3Н -пеитагастри на, 'Н -ф л ун н тразеп ам а и
3Н -эторф ина (уд. активность 36 Ки/м моль, «A m ersham In te rn a tio n a l» ,
Англия) по м етодике Owen и др., 1985.
Р езультаты исследования и обсуж дение
Р езультаты двух независимы х исследований сущ ественны м образом
разл и ч ал и сь (табл. 1). Если в октябре ф енамин у контрольных крыс
вы зы вал характерн ое усиление двигательной активности, то в декабре,
как не парадоксальн о, он таким действием не о б л ад ал . В октябре д л и ­
тельное введение галоперидола усиливало поведенческие эф ф екты ф е н а ­
мина: двигательную активность больш е, стереотипную — меньше. В
декабр е действие ф енам ина ослабилось (табл. 1). Н еодинаковы м было
в этих двух опытах п действие однократно введенного церулеина: в
октябре он устранял повыш енную чувствительность к ф енам ину, вы зв ан ­
ную длительны м введением галоп ери дола, в декабре, наоборот, восста­
н авли вал (табл. 1).
Зам етн о отличалось и действие длительного введения галоперидола
на связы ван и е различны х радиолигандов (табл. 2). В октябре отм е­
чали достоверное увеличение плотности мест связы ван и я 'Н -сипропсридола в хвостатом ядре (доф ам и н 2-рецситоры) и пониж ение :'Н -ф лунитразепам а и 3Н -пеитагастрииа (Х П К -8 рецепторы) в коре больш их
полуш арий. Эти изменения характерны длительном у введению галоиеридола (В асар и др., 1986). В декабре установили лиш ь умеренное
увеличение числа дофамины-рецепторов, в то врем я как плотность бепзодиазепиновы х и ХПК-8 рецепторов имела д аж е тенденцию к повышению
(табл. 2).
В опытах на мышах длительное введение галоперидола и церулеина
о к азы вал о весьма сходное влияние на поведенческие эффекты ф ен а­
мина, м усцимола и Ro 15-1788 (табл . 3). П осле их длительного ниеде132
Таблица /
Влияние длительного введения галоперидола (0,5 мг кг в день, в течение 1л дней)
на поведенческие эффекты фенамина (2 м г/кг) и церуленна (40 мкг кг)
I о п ы т (о к тя б р ь)
Ф и зи о л о ­
гический
р аств о р ~
ф нзиологическип
раство р
Ф и зи о л о ­
гический
р а с тв о р +
ф ен ам и н
Ф и зи о л о ­
гический
р а с тв о р ц ерулеи н —
ф ен а м и н
11 оп ы т (д е к а б р ь )
Г ал о гкр и до л —
ф ен ам и н
Г ал о п ер и лол ц ерулеи н ф ен ам и н
Фи Ш О Л О ги чески ii
раствор ф и зи о л о ги ­
ческий
р а с тв о р
Ф и зи о л о ­
гический
р а с тв о р —
ф ен ам и н
Ф>1 i l l О, it 1-
гический
р а с тв о р —
цер у л еи н ф ен ам и н
Г ал о п ер и до л -г
ф ен ам н н
Г а л о п ерн дол —
ц ер у л еи н -т
ф ен ам и п
1-й день
С тереоти п н ая
акти вн ость, бал л ы
Д в и га те л ь н ая
ак г ивность,
имп / 5 мин
0
1.28 ± 0 ,1 2
1. 13 ± 0.1 4
1,57 ± 0.15
3 8 ± 3 .õ
106 ± 7 ,2
1 0 5 ± 6 ,4
138 ± 8 .2
0
1,52 ± 0 ,1 5
1 ,0 9 ± 0 ,1 7 *
3 2 x 3 ,8
1 0 4 x 8 ,2
71 ± 6 .9 *
1 .2 0 x 0 ,1 3 "
7 5 x 6 .3 *
0
1 ,1 6 ± 0 .15
0.84 ± 0 .1 4
1.38 ± 0.12
1 ,4 4 ± 0 .2 0
44 ± 5 .2
5 7 ± 4 .8
5 5 + 5.3
3 9 ± 4 .0
81 ± 5 . 6 “
1,28 ± 0 ,1 3
1,21 ± 0 ,1 5
1.43 + 0,12
4 6 ± 4 ,5
4 0 ± 3 ,8
34 ± 3 ,0
7-й день
С тереоти п н ая
а кти в н ость, бал л ы
Д в и га т е л ь н а я
акти вн ость.
имп / 5 мин
* р < 0 .0 5 (по у-тесту М а н н а — У и тн и ).
1 .5 3 + 0 .1 8
1,11 ± 0 ,1 2 *
1 0 8 - 7 ,9
54 ± 5 ,6 *
0
38 ± 4 ,6
1 ,6 3 x 0 .1 5
91 ± 8 .0 *
Таблица 2
Влияние длительного введения галоперидола (0,5 мг/кг, в течение 15 дней) на
дофамин2-, бензодиазепиновые и ХЦК-8 рецепторы в мозге крыс
I о п ы т (о к т я б р ь )
Ф и зи ологи чески й
раство р
Г а л о п ер и д о л
11 о п ы т (д е к а б р ь )
%
Ф и зи о л о ги чески й
р а с тв о р
Г а л о п ер и д о л
о/
/0
2Н-спироперидол
К , нМ
0 ,4 7 ± 0 ,0 6
0 ,4 6 + 0 ,0 7
98
0 ,6 0 ± 0 ,0 7
0 ,5 8 ± 0 ,0 5
97
Свмакс, ф м о л е й /м г б е л к а
352±25
460±28*
131
382±24
425±20
111
2,56 + 0,20
3,09 + 0,25
121
1,84 ± 0 ,2 5
1,52 ± 0 ,1 5
83
2930+280
2380+270*
81
1 5 3 5 ± 180
1 6 9 0 ± 150
110
1,07± 0,10
4 0 ,2 + 2 ,0
1 ,0 1 + 0 ,1 2
3 3 ,2 ± 2 ,5 *
94
83
0 ,8 0 + 0 ,0 6
3 9 ,2 ± 2 ,5
0,69 + 0,07
4 2 ,0 + 2 ,7
86
107
3Н-флунитразепам
К
нМ
Свмакс. ф м о л е й /м г б е л к а
3Н-нентагастрин
К , нМ
С в м акс, ф м ол ей /м г б е л к а
* р < 0 ,0 5
(/ — тест
С т ь ю д е н т а). С в макс — число
м ест
связы ван и я;
К д — к о н с та н та д и ссо ц и ац и и .
Таблица 3
Влияние длительного введения (15 дней) галоперидола (0,5 мг/кг в день)
и церулеина (0,1 мг/кг в день) на действие фенамина, мусцимола и
Ro 15— 1788 при изучении двигательной активности мышей, имп/30 мин
В ещ ество, д о за
Ф и зи ологи чески й
раствор
Ф и зи ологи чески й
р а с тв о р
171 ± 1 5
Ф ен ам и н (3 м г/к г)
М усци м ол (I м г/к г)
Ro ( 1 5 - 1 7 8 8 (10 м г/к г)
409±30
89±10
201 ± 17
Г а л о п ер и д о л
188 ± 14
598 ± 4 5 *
203 ± 3 6 *
1 6 2 ± 15*
Ц ерулеин
1 8 4 ± 18
704 ± 6 2 *
17 0 ± 28*
1 9 3 ± 16*
* р < 0 ,0 5 ( у -тест М а н н а — Уйтни, по с р ав н ен и ю с д л и тел ьн ы м введ ен и ем ф и зи о л о ги ч е ­
с кого р а с т в о р а ).
ния ф енам ин (3 м г/кг) ещ е сильнее стим ули ровал дви гател ьн ую а к т и в ­
ность мышей, в то врем я к ак к эф ф ектам м усцим ола (1 м г/кг) и Ro
15-1788 (10 м г/кг) р а зв и в а л а с ь толерантность. М усцим ол не бы л спосо­
бен у гн етать дви гател ьн ую активность, a Ro 15-1788 больш е не о к а зы ­
вал стим улирую щ его вли яни я на поведение мыш ей (таб л . 3 ). Сходным
бы ло и влияние дли тельн ого введен и я га лоп ери д ол а и ц ерулеи на на
плотность разн ы х рецепторов в головном м озге мыш ей (таб л . 4 ). П од
их влиянием повы силось число доф ам инг-рецепторов в хвостатом яд р е
и опиоидны х рецепторов в лим бических структурах. Ч исло Х Ц К -8
рецепторов ум еньш илось в коре больш их п олуш арий к а к после д л и ­
тельного применения галоп ери д ол а, т а к и ц ерулеи н а (таб л . 4 ). П л о т­
ность бензодиазепиновы х рецепторов и зм ен ял ась в зависим ости от
исследованны х структур. Если в переднем м озге галоп ери д ол и ц ер у ­
леин у м еньш али их число, то в стволе м озга н абл ю д ал о сь достоверное
их повыш ение.
С равн ен и е данны х двух незави си м ы х исследований, п роведенны х в
о к тябр е и д ек а б р е, д ае т нам основание п ол агать, что чувствительность
доф ам и н овы х рецепторов во многом зав иси т от ф ункц ион альн ого
состояния Х Ц К -8 и бен зодиазепи н овы х рецепторов в переднем мозге.
Г и перчувствительность на доф ам и новы х рец еп торах в хвостатом яд р е и
м езолим бических стр уктурах р азв и в ается только при сущ ественном
пониж ении числа бен зоди азеп ин овы х и Х Ц К -8 рецепторов в коре б о л ь ­
ших полуш арий при дли тельн ом введении галоп ери д ол а. Н а ф оне н еко­
торого увеличения их числа в д ек а б р е н аб л ю д ается пониж ение чувстви ­
тельности доф ам и н овы х рецепторов на дви гател ьн ую акти вность кры с,
о чем сви детельствует осл абл ен и е стим улирую щ его вл и яни я индиректlioro д о ф ам и н ом и м етика ф ен ам ин а. С л ед ует отм етить, что поведенче­
ские и биохим ические изм енения, вы зван ны е галоп ери долом в октябре,
типичны, в то врем я к ак в д ек а б р е галоп ери д ол о к азы в ал п а р а д о к с а л ь ­
ное действие. По всей вероятности сдвиги последнего м ож но св я зы в ать
с м етеорологическими условиям и, а именно, с бы стры м и резким п охоло­
д ан и ем . От изм енения числа Х Ц К -8 и бен зоди азеп и новы х рецепторов
зав и си т и действие ц ерулеина после дл и тел ьн ого введения га л о п ер и ­
д о л а. О дн ак о в лю бом случае церулеин и зм ен ял чувствительность д о ф а ­
миновых рецепторов, вы званную галоп ери д ол ом . При развити и гиперчувствителы ю сти
доф ам ин овы х
рецепторов одн ократн ое введение
ц ерулеина полностью устран ял о усиление поведенческих эф ф ектов
ф ен ам ин а, вы званное галоп ери д олом , а при пониж енной чувстви тел ь­
ности — во сстан авл и вал о. Уменьш ение чувствительности доф ам и н овы х
рецепторов под влиянием ц ерулеина хорош о согласуется с клиниче1.35
Таблица 1
Влияние длительного введения (15 дней) галоперидола (0,5 мг/кг в день) и церулеина
(0,1 мг кг в день) на связывание дофамин.,-, бензоднаэепиновых, опиоидных и ХЦК-8 рецепторов в мозге мышей
Ф и зи ологи чески й р а с т в о р
Ц ерулеин
Г а л о п ер и д о л
Р а д и о л и га н д ы
Кд
^ вмаьс
Кд
Кд
^ в маис
3Н -сп и ропери дол в стр и ату м е
0.47 = 0,05
348 ± 3 0
0,62 ± 0 .0 5
450=25*
0,63 ± 0 ,0 5
492±32**
3Н -ф л у н и тр а зе п а м в коре
больш их п ол уш ари й
1,70 ± 0 ,2 5
1 9 8 0 ± 120
1,60 ± 0 ,2 5
1440 ± 1 5 0 '
1 ,5 0 = 0 ,1 8
1 3 8 0 ± 140*
3Н -ф л у н н тр а зе п а м в с тв о л е м о зга
2,42 = 0.20
1030 ± 8 0
1,92 ± 0 ,1 8
1250 ± 1 2 0
2 ,0 2 = 0 .1 7
1420 ± 1 6 0 *
3Н -эторф и н в ли м би ческих
с тр у к ту р а х
0.62 ± 0 ,0 5
328 ± 2 4
0,61 ± 0 ,0 5
420±25'
0,77 = 0,05
460±32*
3Н -п ен тагастр и н в ко р е больш их
п о л у ш ар и й
3 .5 0 ± 0 ,4 0
50±5
3,20 ± 0 ,3 0
35±3*
3 ,2 0 ± 0 ,3 2
32±3*
* р < 0 ,0 5 ; ** р < 0 , 0 1 ( f -тест С тью д ента по ср ав н ен и ю
С в мa i,v — число м ест с в язы в ан и я , ф м о л ей /м г Оелка.
с д л и тел ьн ы м введен и ем ф и зи о л о ги ч ес к о го р а с т в о р а ). К д - - к о н с та н т а д и с со ц и ац и и , нМ ;
скими н аблю дениям и, в которых церулеин о к азы в ает б лагоп риятн ое
влияние па сим птом атику побочного эф ф екта нейролептического л еч е­
ния — поздню ю ди ски н езп к' (N ishikasva и др., 1986). В лияние церулеина
на чувствительность доф ам иновы х рецепторов хорош о коррел и рует с
данными наш их преды дущ их исследований, где на м ыш ах, отселектированных с помощ ью Н -п рон нлнораи ом орф и на, устан овлен о п ротивопо­
лож ное влияние церуленна па связы ван и е 3Н -сннроперидола в опы тах
in vivo (V a sa r и др., 1980). У мышей, реагирую щ их на введение
100 м кг/кг Н -н ропилиорапом орф ппа сильным усилением д ви гательн ой
активности, церулеин зн ачи тельн о п он иж ал связы вани е 3Н -спироперидо л а, в то время как у мышей, реагирую щ их уменьш ением д в и г а т е л ь ­
ной активности, достоверно повы ш ал. М ож но п олагать, что именно эти
изменения в плотности доф ам иновы х рецепторов находятся в основе
м одулирую щ его влияния церуленна на поведенческие эф ф екты ф е н а ­
мина после длительного применения галоп ери д ол а. О сущ ествовании
двух подтипов ХЦК-8 рецепторов, оказы ваю щ и х п ротивополож ное в л и я ­
ние на доф ам пнергическне процессы, сви детельствую т и дан н ы е других
авторов (V oigt и др., 1980; Н о т ш е г и др., 1986). В прилегаю щ ем ядре
вы явлены два подтипа Х Ц К -8 рецепторов, о казы ваю щ и х проти воп ол ож ­
ное влияние па вы свобож дение д оф ам ин а из иресипаптических термнпалей (V oigt и др., 1980). В черном вещ естве сущ ествует т а к ж е два
подтипа Х Ц К -8 рецепторов, противополож но влияю щ их на электроф изиологпчсские парам етры доф ам иновы х нейронов
(H o m m er и др.,
1986).
О
сущ ествовании зам етного м одулирую щ его влияния со стороны
ХЦ К-8 на эф ф екты длительного введения галоп ери д ол а сви д етел ь ­
ствует и сравнительное изучение эф ф ектов дли тельн ого введения га л о ­
перидола н ц ерулеина. Их введение вы звал о ги перчувствительиость
доф ам иновы х рецепторов, о чем сви детельствует усиление ф енам инового двигательного возбуж ден и я и повыш ение числа д о ф ам и н гр сц еп торов в х в о ста г о м ядре. Под влиянием ц ерулеина и галоп ери д ол а повы ­
ш алось Ч1Н.Ю опиоидных рецепторов в лимбических структурах, п о н и ж а­
лось число беп.!одиазеннновых рецепторов в переднем мозге и увел и чи ­
валось в стволе мозга. О траж ен и ем этих м олекулярны х п реобразовани й
явл яется полное исчезновение поведенческих эф ф ектов м усцим ола и
Ro 15-1788. Д ли тельное введение как ц еруленна, так н галоперидол'а
вы зы вает значительное пониж ение числа
ХЦК-8 рецепторов в коре
больш их полуш арий. Н едавно бы ла вы двинута гипотеза, что д л и тел ь ­
ное введение нейролептиков вы зы вает ден оляризан и онн ую б л окаду
до<Ьа чиповых нейронов (C hiodo, B im ney, 1983). Ф арм акологически й и
эл 'ктр о ф н зи о л о гн ч ески й ан али зы п о казали , что Х Ц К -8 о к азы в ает
подобное нейролептикам действие, в то время как ан тагон ист Х Ц К -8
рецепторов проглумид полностью устран яет ден оляризан и он н ую б л о ­
каду, вы званную длительны м введением нейролептиков (B u n n ey и др.,
1985). М ож но п олагать, что одинаковое влияние дли тельн ого введения
галоп ери дола и церулеина па попечение ж ивотных и на разн ы е н ей ро­
нальные рецепторы о тр а ж а е т ден оляризан и онн ую б л окаду д о ф ам и н о ­
вых нейронов. По всей вероятности, часть эф ф ектов длительн ого введ е­
ния нейролептиков реал и зуется именно через Х Ц К -8 ергическне м ех а­
низмы.
Л И Т Г. F> Л Т У Г> Л
Л п п к .ш ч с Л V Ж н /к о и ск и й Л. М. П ур к Л. М.. Н агар Э. .9.. Майметс М. О., Р я гп Л . К.
И м ш и т ' I.iiiii-.ii.iniiи iiiir.'U'iiiiM iifiipo.H -iriикон ип п л асти ч н о сть р ец еп то р о в
Ul IC
ISiviiNih \Л Ш С О Р, 1УК-1, № II 37- —12
137
Ллликметс Л . X., В асар Э. Э., Н ур к А. М., Майметс М. О. А дап тац и о н н ы е изм ен ен и я на
ГА М К - и б о н зо д и азеп н н о в ы х р ец еп то р ах после д л и тел ьн о го в вед ен и я галопср и дол а. — В сб.: Ф а р м а к о л о ги ч е ск а я р е гу л яц и я состоян и й д е за д а п та ц и и М
1981), 30 — 43.
В асар Э. Э., Н ур к А. М.. Мийметс М. О., С оосаар А. А.. Ллликметс Л . X. Н ео д и н ако в о е
вл и ян и е дл и тел ьн о го » вед ени я г а л о п е р и д о л а на ГЛМКд- и б ен зо д и азеп и н о в ы е
рец епторы н р а зн ы х о т д е л а х м о зга. — 13юл. чксп. бнол. мед., 1986, № 4,
433— 436.
В асар Э. Э., Соосиир A. X.. Майметс М. О., Алликмстс Л . X. П о н и ж ен и е ч у в с т в и те л ь­
ности хол сц истоки н ин оп ы х р ец еп то р о в п м озге под влиянием д л и те л ьн о го в в е ­
ден и я гал о п е р и д о л а . — Бю л. эксп. бнол., м ед.. 1986, № 11, 583— 585.
Ж а рк овски й А. М.. Ллликметс Л . X. В ли ян ие н ей р о леп ти к о в на рец епторы Ц Н С при
од н о кр атн о м и повторн ом применении. — В сб.: Всесою з. симп. «Х им ия, ф а р м а ­
к ол оги я и кл и н и ка н ей ролеп ти ков». Т а р ту , 1986, 47— 49.
Н у р к А. М., Майметс М. О., Р я го Л . К., В а са р Э. Э. А д ап тац и о н н ы е и зм ен ен и я
в
Г А М К -ергн ческой систем е после отм ены х рон и ческого п ри м ен ен и я га л о п е р и ­
д о л а . — В сб.: М ех ан и зм д ей стви я и к л и н и ка п р о и зв о д н ы х г а м м а -а м и н о м а с л я ­
ной кислоты . Уч зап . ТГУ , вып. 687, Т а р ту , 1984, 17— 27.
B u n n ey, В. S., C liiodo, L. A., F reem an, A. S. F u r th e r s tu d ie s o n th e s p e cific ity of
p ro g lu m id e a s a se le c tiv e c h o le c y sto k in in a n ta g o n is t in th e c e n tra l n e rv o u s
sy s te m . — A n n . N. Y. A cad. Sei., 1985, 448, 435— 441.
Chiodo, I. A.. B u nney, B. S. T y p ical a n d a ty p ic a l n e u ro le p tic s : d iffe re n tia l e ffe c ts of
c h ro n ic a d m in is tr a tio n on th e a c tiv ity of A9 a n d A10 m id b ra in d o p a m in e rg ic
n e u ro n s . — J. N eu ro sci. Res., 1983, 3, 1607— 1619.
C o sta ll, B., N a ylor, R. J. S te re o ty p e d a n d c irc lin g b e h a v io u r in d u ced by d o p a m in e rg ic
a g o n is ts a fte r lesio n s of th e m id b ra in ra p h e n uclei, — E u r. J. P h a rm a c o l., 1974,
29, 206— 222.
F rey, P. C h o le c y sto k in in o c ta p e p tid e lev e ls in r a t b ra in a re c h a n g e d a fte r s u b c h ro n ic
n e u ro le p tic tre a tm e n t, — E u r. J. P h a rm a c o l.. 1983, 95, 8 7 —92.
H om m er, D. W., S to n e r, G., C ra w le y , J. N.. P au l, S. M ., S k irb o ll, L. C h o le c y sto k in in d o p a m in e c o e x isten c e : e le c tro p h y s io lo g ic a l a c tio n s c o rr e s p o n d in g to c n o lecy sto k in in re c e p to r s u b ty p e . — J. N eu ro sci. Res,, 1986, 6, 30 3 9 — 3043.
H okfelt, Т., S k irb o ll, L., R ehfeld, J. F., G o ld ste in , M., M a rk ey , K ., D ann, О. A s u b p o p u ­
latio n of m ese n c e p h alic d o p a m in e n e u ro n s p ro je c tin g to lim b ic a re a s c o n ta in s
a c h o le c y sto k in in -lik e p e p tid e : e v id e n c e fo r im m u n o e h e m is try co m b in ed w ith
r e tr o g r a d e tra c in g . — J. N eu ro sci. Res., 1980, 5, 20 9 3 — 2124.
K osaka, Т., K osaka, K , T ateishi, K ., H arnaoka, У., Yanaihara, N„ Wu, J.-Y., H am a, K.
G A B A ergic n e u ro n s c o n ta in in g C C K -8-like a n d /o r V IP -lik e im m u n o re a c tiv ie s
in th e r a t h ip p o c a m p u s a n d d e n ta te g y ru s. — J. C om p. N eu ro l., 1985, 239,
4 2 0 —430.
N ish ikaw a, Т., T anaka, M., T suda, A ., K u w aharu, H , K o g a , I., U chida, У. E ffe c t of
c e ru le tid e on ta r d iv e d y s k in e s ia : a p ilo t s tu d y of q u a n tita tiv e c o m p u te r a n a ly s is
o n e le c tro m y o g ra m a n d m ic ro v ib ra tio n . — P sy c h o p h a rm a c o l., 1986, 90, 5 — 8,
O w en , F., B ourne, R. C., P o u lter, M ., C ro w , T. / .. P a te rso n , S. I., K o s le rlitz , H. T ritia te d
e to rp h in e a n d n a lo x o n e b in d in g to opio id re c e p to rs in c a u d a te n u c le u s in
sc h iz o p h re n ia . — B rit. J. P s y c h ia t., 1985, 146, 5 0 7 — 509.
P r a iss m a n , M., M a rtin e z, P. A., S a la d in o , С. F B c r k o w itz , I M., S te g g le s , A W., Finkelste in , J. A. C h a r a c te riz a tio n of c h o le c y sto k in in b in d in g s ite s in r a t c e re b ra l
c o rte x u s in g a l25I-C C K -8 p ro b e r e s is ta n t to d e g ra d a tio n . — J. N eu ro ch em ., 1983,
40, 1406— 1413.
V asar, E., M a im e ts, M., N u rk, A ., S o o sa a r, A., A llik m e ts , L. C o m p a ris o n of m o to r
d e p re s s a n t e ffe c ts of c a e ru le in a n d N -p ro p y ln o ra p o m o rp h in e in m ice. — P h a r m a ­
col. B iochem . B ehav., 1986, 24, 4 6 9 — 478.
V oigt, M „ W ang, R. Y., W estfa ll, Т. С. C h o le c y sto k in in o c ta p e p tid e s a lte r th e re le a se of
e n d o g e n o u s d o p a m in e fro m th e r a t n u c le u s ac cu m b e n s in v itro . — J . P h a r m a ­
col. E xp. T her., 1986, 237, 147— 153.
Тартуский государст венны й университет
П о сту п и л а в р ед акц и ю
Eero V/4S/1/?, A n d re s S O O S A A R , A a v o L A N G
KOLETSOSTOK1NIIN1 RETSEPTO RITE O SAI.EM INE HALOPERIDOOLI
PIKA A JA LISE M ANUSTAM ISE K ÄITLM USLIK E JA B IO KEEM ILISTE
EFEK TID E REA LISEERU M ISEL
K a ts e te s v a lg e te isa s te r o ttid e g a oti leitu d tih e se o s d o p a m iin i re ts e p to r ite a fiin su s e
n in g k o lcts iis to k in iin i (C C K -8) ja b e n s o d ia s e p iin i re ts e p to r ite a rv u v a h e l e e sa ju s h alo perid o o li (0,5 rn g /кц p iiev as) 1 5 -p äev ase m a n u s ta m is e jä re l. C C K -8 ja b e n s o d ia s e p iin i
re ts e p to r ite v ä h e n em ise l tõ u sis d o p a m iin i r e ts e p to rite tu n d lik k u s, kuid n e n d e a rv u s u u re ­
138
n e m in e v iis d o p a m iin i r e ts e p to r ite a fiin su s e v ä h e n em ise le . C C K -8 re ts e p to r ite a g o n is t
tse ru le iin k õ r v a ld a s m õ le m ad h a lo p e rid o o li k ro o n ilis e m a n u s ta m is e e fe k tid : d o p a m iin i
r e ts e p to r ite tu n d lik k u se tõusu ü h te d e l r o ttid e l j a la n g u s e teiste l. H a lo p e rid o o li (0,5 tn g /k g
p ä e v a s ) ja tse ru le iin i (0,1 m g /k g p ä e v a s ) 1 5 -p äe v a n e m a n u s ta m in e p õ h ju sta s id a n a lo o g ­
seid k ä itu m u slik k e ja bio k eem ilisi e fe k te v a lg e te l isa s te l h iirte l. H a lo p e rid o o li ja ts e r u ­
leiini k ro o n ilis e m a n u s ta m is e jä r e l s u u re n e s fen a m iin i (3 m g /k g ) m o to o rik a t stim u le e riv
toim e, ku id a re n e s to le r a n ts u s m u sts im o o li (G A V H A-re ts e p to rite a g o n is ti) ja b e n so d ia se piin i a n ta g o n is ti Ro 15-1788 e fe k tid e s u h te s . P a r a lle e ls e lt s u u re n e s d o p a m iin i- ja o p io id r e ts e p to r ite a rv h iire a ju s u b k o rtik a a ls e te s s lr u k lu u rid e s , s a m a l a ja l kui C C K -8 r e ts e p ­
to rite tih e d u s v ä h e n e s e e sa ju k o rtik a a ls e te s o s a d e s . M u u tu s e d b e n s o d ia s e p iin i re ts e p to r ite
a rv u s o lid s õ ltu v u s e s u u ritu d a ju s tru k tu u r is t. K ui e e sa ju k o rlik a a ls e te s o s a d e s r e ts e p to ­
rite a rv v ä h e n e s , siis a ju tü v e s s u u re n e s see h a lo p e rid o o li ja tse ru le iin i m õ ju l. S a a d u d
tu le m u s te s t jä r e ld u b , et n e u ro le p tik u m i k ro o n ilis e m a n u s ta m is e b io k ee m iliste j a k ä itu m u slike m u u tu s te fo rm e e ru m ise l e te n d a v a d v ä g a o lu lis t o s a C C K -8 -e rg ilise d m e h h a n is m id
a ju s .
E ero V A SA R , A n d re s S O O S A A R , A a v o L A N G
THE JNVOLVEM ENT OF CHOLECYSTOKININ RECEPTORS IN TH E
REALIZATION OF BEHAVIOURAL AND BIOCHEM ICAL EFFECTS
OF LONG-TERM HALOPERID O L ADM INISTRATION
E x p e rim e n ts w ith m ale a lb in o r a t s h a v e s h o w n th e d e p e n d e n c e of d o p a m in e re c e p to rs
a ff in ity o n th e d e n s ity of c h o le c y s to k in in (C C K -8 ) a n d b e n z o d ia z e p in e re c e p to rs a f te r a
1 5 -d ay -lo n g h a lo p e rid o l (0.5 m g /k g d a ily ) tre a tm e n t. In c a se th e n u m b e r of C C K -8 a n d
b e n z o d ia z e p in e re c e p to rs d e c re a s e d , th e a ff in ity o f d o p a m in e re c e p to rs in c re a se d , b u t an
in c re a se in C C K -8 a n d in th e d e n s ity of b e n z o d ia z e p in e re c e p to rs led to th e re d u c tio n
of d o p a m in e re c e p to rs a ffin ity . A n a c u te a d m in is tr a tio n of c a e ru le in , a n a g o n is t of C C K -8
re c e p to rs , a n ta g o n iz e d b o th e ffe c ts o f th e lo n g -te rm h a lo p e rid o l m ed ic a tio n : th e in c re a se
of th e d o p a m in e re c e p to rs a ffin ity in o n e g ro u p a n d the d e c re a s e in th e o th e r. The
h a lo p e rid o l (0.5 m g /k g d a ily ) a n d c a e ru le in (0.1 m g /k g d a ily ) tre a tm e n t d u r in g 15
d a y s c a u se d s im ila r b e h a v io u ra l a n d b io ch e m ic a l e ffe c ts o n m a le a lb in o m ice. The m o to r
s tim u la n t effe c t of a m p h e ta m in e (3 m g /k g ) in c re a se d , b u t th e to le ra n c e d e v e lo p e d to th e
e ffe c ts of m u sc im o l (1 m g /k g ) , th e a g o n is t o f G A B A A-re c e p to rs, a n d Ro 15-1788
(1 0 m g /k g ) , th e a n ta g o n is t of b e n z o d ia z e p in e re c e p to rs , a fte r a lo n g -te rm a d m in is tr a tio n
of c a e ru le in a n d h a lo p e rid o l. S im u lta n e o u s ly th e n u m b e r of d o p a m in e 2-re c e p to rs in s t r i a ­
tu m a n d op io id rc c e p to rs in lim bic s tr u c tu r e s in c re a se d , w h e re a s th e d e n s ity o f C C K -8
re c e p to rs s ig n if ic a n tly re d u c e d in fo re b ra in c o rtic a l s tr u c tu r e s . The c h a n g e s in th e n u m b er
o f b e n z o d ia z e p in e re c e p to rs d e p e n d e d o n th e b r a in s tr u c tu r e s s tu d ie d . In fo re b ra in
c o rtic a l s tr u c tu r e s th e n u m b e r o f b e n z o d ia z e p in e re c e p to rs d e c re a se d , b u t in b ra in s te m
th e ir d e n s ity w a s in c re a se d by c a e ru le in a n d h a lo p e rid o l. In c o n c lu sio n , it se em s v e ry
p ro b a b le t h a t C C K -8 -e rg ic m e c h a n ism s in th e b ra in p la y a s ig n if ic a n t ro le in th e
fo rm a tio n of b e h a v io u ra l a n d b io ch e m ic a l e ffe c ts o f a lo n g -te rm n e u ro le p tic m ed ic a tio n .
Sov. Med. Rev. G. Neuropharm., Vol. 1.1990, pp. 101-126
Photocopying permitted by license only
© 1990 Harwood Academic Publishers GmbH
Printed in the United Kingdom
ADAPTATIONAL CHANGES IN GABA,
BENZODIAZEPINE AND
CHOLECYSTOKININ RECEPTORS
ELICITED BY LONG-TERM HALOPERIDOL
ADMINISTRATION
L.H. ALLIKMETS1 and E.E. VASAR2
1Department o f Pharmacology, Tartu University ^Laboratory ofPsychopharmacology,
Institute o f General and Molecular Pathology, Tartu University
Abstract
The authors’ investigations into the effects of prolonged haloperidol treatment on the GABAand CCK8-ergic systems of the brain and into the roles of these neurochemical systems in
adaptational changes in response to long-term administration of antipsychotic drugs are
reviewed. Chronic haloperidol treatment, while reducing the density of dopam inei receptors, is
shown to reduce the density of GABAa receptors and of the associated benzodiazepine (BZ)
receptors, without affecting GABAb receptor numbers. As a result, the ‘stimulatory’ GABAa
and benzodiazepine receptors become predominant, and the behavioral effects of muscimol and
Ro 15-1788 are reversed. Administration of Ro 15-1788, together with haloperidol, blocks the
development of dopamine 2 receptor hypersensitivity and the modification of GABA,» and BZ
receptors. Cerulein, a CCK8 receptor agonist, destabilizes the interaction of dopaminergic
ligands with dopamine 2 receptors. Long-term haloperidol treatment made the animals (mice)
tolerant to the behavioral effects of cerulein. Adaptational changes in the CCK8-ergic systems
under chronic haloperidol treatment have been found to be associated with alterations in
dopamine2-ergic and opioid receptors.
101
102
L.H. ALLIKMETS and E.E. VASAR
Contents
1 Introduction
2. Involvement o f GABA and Benzodiazepine Receptors in Haloperidol
Actions
2.1 Comparative Effects o f Haloperidol and 6-Hydroxydopamine (6OHDA) or GAB A and Benzodiazepine Receptors
2 .2 Effects o f Diazepam and Ro 15-1788 on Adaptational Changes in
the GABA-ergic System and Benzodiazepine Receptors During
Long-term Haloperidol Treatment
3. CCK8-ergic Mechanisms in Haloperidol Actions
3.1 Effect o f Cerulein on Dopaminergic Mechanisms
3.2 Adaptational Changes in the CCK8-ergic System on Long-term
Haloperidol Treatment
4. Conclusions
102
103
103
108
110
110
118
124
I. Introduction
Neuroleptics (drugs with antipsychotic activity), are known to be potent
antagonists of dopamine receptors. A direct correlation has been demon­
strated between a neuroleptic’s affinity for dopamine2 -receptors in the stri­
atum of experimental animals and the clinically effective daily dose o f the
neuroleptic (Seeman, 1980). Adaptational changes that occur in the dopa­
minergic system after prolonged treatment with various neuroleptics have
been fairly well investigated. Dopamine2 receptor density has been shown
to increase during prolonged dopamine receptor blockade with the result that
a hypersensitivity to behavioral effects o f dopamine agonists develops (See­
man, 1980). However, long-term administration o f a neuroleptic also causes
substantial alterations in other neurotransmitter systems, including among
others the serotonergic y-aminobenzoic acid (GAB A)-ergic and cholinergic
systems (Allikmets et al., 1984). Of particular interest are alterations in the
GABA-ergic and cholecystokinin (CCK8)-ergic systems which are closely
linked up morphologically and functionally with the dopamine systems of
the brain. CCK8 has been shown to act as a dopamine cotransmitter in
mesolimbic and mesocortical structures (Hökfelt et al., 1980), and to be
co-present with GABA in neurons of the hippocampus and cerebral cortex
(Wise, 1985). GABA- and CCK8- ergic mechanisms play important roles in
regulating dopaminergic processes (Haefely et al., 1983; Wang et al., 1984)
The present studies were designed to examine adaptational changes in the
LONG-TERM HALOPERIDOL ADMINISTRATION
103
GABA- and CCK8-ergic systems during prolonged neuroleptic treatment
and to see how such changes relate to the overall adaptation o f the organism
to the neuroleptic. Haloperidol was chosen because it is a typical neuroleptic
with potent antipsychotic activity and has gained wide use in clinical practice.
2. Involvem ent of GABA and Benzodiazepine Receptors in
Haloperidol Actions
In the forebrain and diencephalon, the dopaminergic and GABA-ergic
systems are closely interrelated morphologically and functionally, so that
both GABA and benzodiazepine receptors are observed to be considerably
altered after long-term administration o f various neuroleptics that block
dopamine receptors (Allikmets et al., 1984). Altered activity o f GABA-ergic
mechanisms contributes significantly to tardive dyskinesia, a severe side-effect of neuroleptic treatment. Under such treatment, GABAa- and benzodiazepine-receptor densities fall in many forebrain structures (Allikmets el al.,
1984), although the substantia nigra shows increased G AB А л-receptor num­
bers together with supersensitivity o f its neurons to GABA. The adaptation
o f GABA-ergic mechanisms and benzodiazepine receptors on long-term
neuroleptic treatment is not, therefore, a straightforward process. We have
examined adaptational changes in GABA and benzodiazepine receptors in
response to long-term haloperidol administration and the possibility of
preventing these changes with diazepam and the benzodiazepine antagonist
Ro 15-1788 (flumazepil; Hofmann-La Roche, Basel, Switzerland).
2.1 Comparative Effects o f Haloperidol and 6-Hyaroxydopamine
(6-OHDA) on GABA and Benzodiazepine Receptors
The effect of long-term intraperitoneal haloperidol administration on these
receptors in mice and rats was compared with that of single-dose intraven­
tricular injection of 6-OHDA (60 fig per mouse and 200 |ig per rat). 6-OHDA
was chosen because its actions after intraventricular administration are very
similar to those of neuroleptics after a single intraperitoneal dose: both
strongly inhibit various forms of behavior and considerably increase the
sensitivity of postsynaptic dopamine receptors (Seeman, 1980). As can be
seen in Figure 1, muscimol (GABА л-receptor agonist) and baclofen
(G АВАв-receptor agonist) caused significant decreases in the motor activity
of mice. Muscimol enhanced rather than depressed the activity o f mice
treated with haloperidol for 15 days and restored it to the control level in
6-OHDA-treated animals. The sedative action of baclofen was not altered by
L.H. ALLIKMETS and E.H. VASAR
104
240
Saline
Haloperidol
6-hydroxydopamine
FIGURE 1 Effects of muscimol (0.75 mg kg'1) and baclofen (3 mg kg'1) on motor activity of
mice after long-term haloperidol treatment (0.25 mg k g '1, i.p., twice daily for 15 days) or a single
intraventricular 6-hydroxydopamine (6-OHDA) injection (60 (ig). Assays were carried out 48
h after the last haloperidol injection and 7 days after the 6-OHDA injection. Each mouse was
placed in a photoelectric actometer 15 min after an intraperitoneal injection of the GABA
mimetic, and the motor activity was measured over a 15-min period. White bars, physiologic
saline; hatched bars, baclofen. * P < 0.05; ** P < 0.01 vs the control (saline + saline-treated)
group by Mann-Whitney U test.
haloperidol but was significantly enhanced by 6-OHDA.
In rats, we examined the behavioral effects of Ro 15-1788 (5 mg kg'1),a
benzodiazepine antagonist (Table 1). In control animals, the drug had stimu­
latory effects, while in those treated with haloperidol it antagonized the
increase in orienting exploratory activity following the discontinuation of
prolonged haloperidol treatment. Preinjecting rats with 6-OHDA (200 jig)
intraventricularly resulted in a significant diminution of this activity. Ro
15-1788, like muscimol in mice, reversed the effect o f 6-OHDA, most likely
by accelerating dopamine metabolism, as is indicated by the observations
that Ro 15-1788 (5 mg k g 1) raises the level of 3,4-dihydroxyacetic acid, a
LONG-TERM HALOPERIDOL ADMINISTRATION
105
T able 1 Effect of a single dose (5 mg k g '1) of the benzodiazepine antagonist Ro
15-1788 on behavioral responses of rats after 15 days’ treatment with haloperidol
(0.25 mg kg’1 twice daily) or a single dose (200p.g; intraventricular) of 6-OHDA.*
Motor activity
Rearings
Counts
%
No.
Saline + saline
Saline + Ro 15-1788
30 ± 4 .2
100
39 ± 4.4
Haloperidol + saline
Haloocridol +
Ro 15-1788
6-OHDA + saline
42 ± 4.8*
131
142
26 ± 3.8
88
Head dips
Group
6-O H D A + Ro 15-1788
14.2 ± 2.6** 46
32 ± 4.8
110
%
No.
%
5.2 ± 1 .6
100
6.7 ± 0 .7
100
8.5 ±1.7*
163 11.5 ±1.6** 172
98 11.8 ±1.5** 176
6.0 ± 0.7
90
92
5.1 ±1.3
4.8 ± 1 .4
1.6 ± 0.6**
31
2.6 ±0.5**
3.2 ± 1.2*
62
4.8 ± 0.8
39
72
*Bchavior was assessed by ihe open field method. The test animal was placed in the center of
an open field (measuring 100 x 100 x 40 cm) at 30 min after i.p. injection of Ro 15-1788.
Motor activity was measured using five independent photoelectric channels. All behavioral
assays were for 5 min periods.
In comparison with the saline + saline group p < : 0.05; 0.01 (Mann-W hitney U test).
dopamine metabolite, in the rat caudate nucleus (Allikmets and Rägo, 1983)
and, when preinjectcd into rats, attenuates the sedation caused by a low dose
of apomorphine (Vasar et al., 1984ö).
In parallel with the behavioral experiments outlined above we carried out
radioligand-binding tests— 3H-muscimol, 3H-G ABA, and 3H-flunitrazepam
binding to G ABA a , G ABA b and benzodiazepine receptors, respectively. It
has been found (Table 2) that long-term haloperidol treatment decreases
GABA a - and benzodiazepine receptor numbers in the forebrain without
affecting G A B A b receptors but that, in contrast, intraventricularly adminis­
tered 6-OHDA has no significant effect on GABA a and benzodiazepine
receptor numbers while reducing those of GABA b receptors.
Such contrasting adaptational changes produced by haloperidol and 6OHDA notwithstanding their rather similar behavioral and biochemical
effects, may be attributed to differences in their mechanisms o f action on the
GABA and benzodiazepine receptors. Intraventricularly injected 6-OHDA
appears to reduce G ABAb receptor density by destroying presynaptic termi­
nals, as is attested by our and other studies where these receptors have been
shown to occur, in particular, on monoaminergic nerve endings in the
forebrain and to participate, via calcium-dependent mechanisms, in the
regulation of monoamine release (Bowery et al., 1980; Allikmets and Rägo,
1983). The enhanced sedative effect of baclofen after 6-OHDA, observed
despite a fall in GABAb receptor density, may be accounted for by a
considerable decrease in the brain content of dopamine for which baclofen
L.H. ALLIKMETS and E.E. VASAR
106
3
e
T able 2 Binding parameters of H-ligands with GABA and benxodiazepine recep­
tors in mouse and rat forebrains after 15 days’ haloperidol treatment (0.25 mg k g '1,
twice daily) or a single intraventricular 6-OHDA injection (60 \lg per mouse, 200jig
per rat).*
G ro u p
GABA a Receptors
(mice)
ä 'd
B m tx
GAHA b Rcccptors
(mice)
Ко
flmax
Benzodiazepine
Rcceplors (rats)
Kd
В шах
Saline
9.8 ± 0.8 805 ± 2 2
52 ± 4 .8
203 ± 1 7 2.5 ± 0 .3
Haloperidol
10.5 ± 0 .9 508 ±32**
49 ± 4 .4
242 ± 2 2 2.6 ± 0 .3 4 482 ±38**
710 ± 4 5
6-OHDA
9.3 ±0.6 708 ± 3 6
56 ± 5 .2
122 ±12* 3.0 ± 0.28
644 ± 4 2
“Values arc the results of three separate experiment s. Binding tests with 3H -muscimol (GABAa
rcceplors) 3I1-GABA (GABAb receptors) and 3H-flunitrazcpam (benzodiazepine receptors)
were performed as described in Nurk e la l (1984), Bowery el al. (1980) and Möhler and Okada
(1978), respectively. Kn - dissociation constant, nmol 1" ; В пал = binding site density, fmol
(mg protein)’ .
In comparison wilh the saline-treated group,/» < : 0.05; 0.01 (Student’s t test).
is a functional antagonist. As for haloperidol, it predominantly blocks dopaminc2 receptors and, as indicated by in vitro evidence, prolonged haloperi­
dol administration significantly increases their density (Seeman, 1980),
which is paralleled by decreases in GABAa and benzodiazepin receptor
densities. A balance therefore appears to exist in the forebrain between
GABAa and benzodiazepine receptors, on the one hand, and dopamine2
rcccptors oti the other, so that the densities o f these receptors are negatively
c orrelated. One result o f these molecular transformations is that the behavio­
ral effects of the GABAa receptor agonist muscimol and the benzodiazepine
antagonist Ro 15-1788 are reversed. However, the reductions in G A BA a and
benzodiazepine receptor numbers cannot fully explain the altered behavioral
effects of muscimol and Ro 15-1788.
Muscimol injected into the raphe nuclei or substantia nigra, but not into
forebrain structures, is known to have a stimulatory effect on animal beha­
vior, the effect being potentiated by benzodiazepine agonists. We have
therefore compared 3H-muscimoI and 3H-flunitrazepam binding in the fore­
brain and afterbrain of rats following their long-term haloperidol treatment.
The density of both G A B A a and benzodiazepine receptors was found to be
increased in the forebrain and decreased in the afterbrain (Table 3). These
changes in receptor density appear relevant to the reversal o f behavioral
LONG-TERM HALOPERIDOL ADMINISTRATION
107
T able 3 Effect of long-term(15 days) haioperdiol treatment (0.25 mg k g '1, twice
daily) on 3H-muscimol and 3H - flunitrazepam binding in rat forebrain and brain stem*
3H-muscimol
G
r o
Saline:
Haloperidol:
u
p
3H-flunitrazepajn
---------------------------------------------------------------------------Forebrain
Brain stem
Forebrain
Kd
9.6 ± 1.6
12.6 ± 1.3
2.1 ± 0 .3
3.2 ± 0 .3
Bmax
908 ± 83
380 ± 3 6
420 ± 43
3.4 ± 0 .4
606 ± 42
Ab
Bmix
10.2 ± 1.8
13.2 ± 1.3
1060 ± 9 2
1.8 ± 0 .4
640 ± 50
508 ± 36
780 ± 76
Brain stem
*See footnote to Table 2. For forebrain binding tests, a frontal incision was made along the
optic chiasm line, and the brain structures anterior to the incision lines were used; for brain
stem tests,a frontal incision was made along the posterior line of the diencephalon, and the
stem structures were then teased off from the ccrebcllum and cortical formations.
In comparison with saline group p < 0.05 (Student’s t test).
effects of muscimol and Ro 15-1788. It would seem that G A B A a and
benzodiazepine receptors in the afterbrain differ functionally from those of
the forebrain in that the ‘stimulatory’ receptors preponderate in the former
and ‘inhibitory’ ones in the latter and that long-term haloperidol administra­
tion increases ‘stimulatory’ receptor numbers, with the result that the mus­
cimol and Ro 15-1788 actions are reversed. Different rats, however, may
Table 4 Effects of diazepam. Ro 15-1788 and naloxone administered twice daily
for 10 days together with apomorphine on apomorphine-induced aggresiveness in
male rats.8
D ru g
Apomorphine + saline
Apomorphine + diazepam
(2.5 mg kg“1)
Apomorphine + Ro 15-1788
(5 mg k g '1)
Apomorphine + naloxone
(0.5 mg kg“1)
Apomorphine + diazepam +
Ro 15-1788
Apomorphine + diazepam +
naloxone
Day 3
Day 7
Day 10
0
1.9 ±0.31
3.0 ± 0.22
0.5 ± 0.28
0.8 ± 0.42
1.3 ±0.41*
0
1.3 ±0.39
2.3 ± 0.34
0.8 ± 0.42
2.9 ±0.18*
3.2 ± 0.25
0
2.3 ±0.17
3.5 ± 0.18
1.9 ±0.38*
3.2 ±0.24*
4.0 ± 0.0*
‘ Values are points scored for intensity of aggressiveness using the scoring system described in
Allikmets et al. (1979). All drugs were given 15 min before apomoiphine (1 mg kg-1).
In comparison with the apomorphine + saline group p < 0.05 (Mann-W hitney U test).
108
L.H. ALLIKMETS and E.E. VASAR
already differ in pretreatment sensitivity of GABA a and benzodiazepine
receptors, as is indicated by the differential effects o f diazepam on apomorphine-induced aggressive behavior. Thus, diazepam at 2.5 mg kg' had a
striking accelerating effect on this behavior in some male rats while blocking
it in others (Table 4). As shown in this table, Ro 15-1788 at 5 mg k g 1 had a
very weak antagonistic effect on apomorphine-induced aggression, while the
opiate antagonist naloxone blocked only the antiaggressive action o f diaze­
pam. These findings indicate that Ro 15-1788, like haloperidol on chronic
administration, is capable o f blocking the functionally predominant subtype
o f benzodiazepine receptors. This may explain the reversal o f the behavioral
effect o f Ro 15-1788, as seen after long-term haloperidol treatment, and the
elimination by this drug of both the anti- and proaggression effects of
diazepam in male rats.
2.2 Effects of Diazepam and Ro 15-1788 on Adaptational Changes in the
GABA-ergic System and Benzodiazepine Receptors During Long-term
Haloperidol Treatment
The benzodiazepine antagonist diazepam has been shown to alleviate
symptoms of tardive dyskinesia. In animal experiments, administration of
diazepam in combination with haloperidol was effective in countering the
development of behavioral supersensitivity in dopamine receptors and the
haloperidol-induced increase in dopamine2 receptor density in the caudate
nucleus (Zharkovsky and Allikmets, 1986). In the studies reported here, we
examined how diazepam or Ro 15-1788, given to mice in combination with
haloperidol, influence the latter’s ability to cause adaptational changes in the
GABA-ergic system and benzodiazepine receptors. Ro 15-1788 plus halo­
peridol administration was found to block the development o f hypersensitiv­
ity to amorphine-induced stereotypy and Quipazine-induced head twitchs,
i.e. o f dopamine2 and s егоton im receptor hypersensitivity. Diazepam (2.5
mg kg’1) plus haloperidol (0.25 mg k g 1) administration failed to prevent
adaptational changes in GABAa receptors, but was effective in countering
the decrease in benzodiazepine receptors in the forebrain (Table 5). Diaze­
pam like haloperidol, reversed the sedative effect of muscimol in mice.
Long-term administration of the benzodiazepine antagonist Ro 15-1788
enhanced the sedative effect of muscimol and eliminated the haloperidol’s
reversal of this effect (see Table 5). In our radioligand binding experiments,
Ro 15-1788 diminished 3H-muscimol and 3H-flunitrazepam binding in
mouse forebrain while significantly diminishing the effect of haloperidol on
GABAa and benzodiazepine receptors on long-term administration.
These findings indicate that the benzodiazepine antagonist Ro 15-1788,
16
9.7 ± 1.6
9.4 ± 1.2
9.4 ± 1.6
500 ± 54**
890 ± 1 0 0
630 ± 50*
9 0 0 ± 120
510 ± 5 0 “
520 ± 60**
9.6 ± 1.6
В m ix
10.2 ± 1.8
9.8 ± 1.4
Ко
d
2.5 ± 0.19
2.8 ± 0.21
2.6 ± 0.20
2.6 ± 0.18
2.7 ± 0.25
2.5 ± 0.22
K
670 ± 5 0
570 ±42**
760 ± 6 0
620 ± 50*
670 ± 6 5
810 ± 48
Втм
3H-muscimol binding in
3H-flunitrazepam binding in
_______ mouse forebrain_________________rat forebrain
‘See footnotes to Tables 1 and 2. The tests were carried out 48 h after withdrawal from haloperidol treatment.
In comparison with control p < : 0.05; 0.01.
320 ± 28**
354 ±36**
128 ± 15
83 ± 1 2
Diazepam (2.5 mg kg-1)
Haloperidol + Ro 15-1788 (5 mg k g 1)
Ro 15-1788 (5 mg kg"1)
144+ 18**
308 ± 32**
(counts/30 min)
Effect of muscimol (0.75 mg
k g '1) on motor activity
Saline (control)
Haloperidol
Haloperidol + diazepam (2.5 mg kg’ * )
T re a tm e n t
Table 5 Effect of 15 day treatment of mice with diazepam or the benzodiazepine antagonist Ro 15-1788 in combination with
haloperidol-induced (0.25 mg k g '1) adaptational changes in GABA a and benzodiazepine receptors.®
ЧС
О
110
L.H. ALLIKMETS and E.E. VASAR
unlike the benzodiazepine agonist diazepam, strongly counteracts adapta­
tional changes in GABAa and benzodiazepine receptors during prolonged
haloperidol treatment.
3. CCK8-ergic M echanisms in Haloperidol Actions
As already noted, dopaminergic and cholecystokinin (CCK8)-ergic mech­
anisms in the forebrain are closely interrelated morphologically and func­
tionally. Convincing evidence exists that CCK8 is a dopamine cotransmitter
in neurons supplying its mesolimbic and mesocortical structures (Hökfelt et
al., 1980). On the other hand, blockade of these dopaminergic mechanisms
explains the antipsychotic action of neuroleptics (Carlsson, 1983), which are
the most potent antipsychotic drugs known today. CCK8 has been reported
to exert a marked influence on dopaminergic processes by altering dopamine
metabolism and release and to affect the affinity of dopamine receptors
(Zetler, 1985).
Dopamine, in turn, has been shown to regulate CCK8 release in the caudate
nucleus. Prolonged blockade of dopamine receptors by a neuroleptic (halo­
peridol, chlorpromazine or clozapine) results in elevated CCK8 levels in
forebrain subcortical structures and appreciably increases CCK8 receptor
density in the forebrain.
We have investigated the effects of cerulein (a CCK8 receptor agonist) on
dopaminergic mechanisms and of long-term haloperidol administration on
CCK8-ergic mechanisms.
3.1 Effect o f Cerulein on Dopaminergic Mechanisms
The decapeptide amide cerulein, which is related to CCK8 in chemical
structure, is a potent agonist o f central CCK8 receptors (Zetler, 1985). We
have found 3H-CCK8 (1 nmol Г1) binding to be 50% inhibited by cerulein
in a concentration of 4 nmol Г1 by sulfated CCK8 at 15 nmol Г1 and by
proglumide, a CCK8 receptor antagonist, at 650 |Япо1 Г1. These findings,
and also behavioral experiments (Zetler, 1985), indicate that cerulein has a
markedly higher affinity for CCK8 receptors than other ligands.
It has been documented by many authors that systemically administered
CCK8 and cerulein eliminate the stereotypic behavior elicited by dopaminomimetics and inhibit the motor excitation caused by phenamine, an indirectly
acting dopaminomimetic (Zetler, 1985).
We thought it worthwhile to examine the mechanism by which the
antidopaminergic action o f cerulein is mediated. To this end, cerulein was
16*
LONG-TERM HALOPERIDOL ADMINISTRATION
111
T abic 6 Effects of cerulein (50-125 |ig kg’1) and proglumide (25-100 mg k g '1) on
phenamine-induced (3 mg k g '1) excitation in mice.*
M otor activity
Drugs
-----------------------------------Counts/15 min
Counts/30 min
Saline
184 ± 2 2
315 ± 3 6
Phenamine
280 ± 42
565 ± 76
Phenamine + cerulein:
Phenamine + proglumide:
50 fig kg 1
244 ± 46
528 ± 62
75 M-g kg-1
lOOfigkg“1
112 ± 20*
237 ±34*
74 ±16**
152 ±26**
125 ng kg" 1
7 0 ± 14**
144 ±22**
25 mg kg-1
328 ± 46
604 ± 66
50 mg kg-1
100 mg kg-1
313 ± 5 0
288 ± 44
575 ± 78
555 ± 69
181 ±28***
332 ±40***
Phenamine + proglumide
(50 mg kg-1) + cerulein (75 ^ g kg-1)
.
Phenamine + proglumide
(100 mg kg'"1) + cerulein (100 |ig kg”1)
162 ± 36
268 ± 65
*
'Phenam ine was administered 15 min before the test, while cerulein and proglumide were given
immediately before it.
In comparison with the phenamine only groupp < : 0.01; 0.001;
0.0001 (Mann-W hitney
U test).
examined for its impact on phenamine (amphetamine)-induced excitation in
male mice and rats and on the behavioral effects o f apomorphine in male rats.
In mice, cerulein injected at 15 min after phenamine (3 mg kg'1) caused a
dose-dependent inhibition of the latter’s excitatory effects on motor behavior
(Table 6), while the CCK8 receptor antagonist proglumide, did not modify
these effects in doses of 25-100 mg kg . Proglumide only weakened the
inhibitory action of cerulein in doses of 50 mg k g 1 and 100 mg kg’1. In rats,
cerulein did not alter the major behavioral effects of phenamine (2.5 mg kg' )
such as stereotypy and motor activity enhancement. Prolonged treatment
wilh cerulein (lO O iig k g 1) plus apomorphine (1 mg kg'1) suppressed ag­
gressive behavior in rats given apomorphine and had an attenuating effect on
apomorphine-induced stereotypy. The animals thus treated were sluggish
and drowsy. However, cerulein (100 or 200 jig k g 1) failed to suppress
112
L.H. ALLIKMETS and E.E. VASAR
Table 7 Effect of single-dose treatment with ccrulein or haloperidol on phenamine
(3 mg kg"1) sensitivity in mice.'
Motor activity
Drugs
Saline + saline
Saline + phenamine
Haloperidol (0.25 mg kg-1) +phenamine
Cerulein (50 pg kg“1) + phenamine
Cerulein (100 pg kg“1) + phenamine
Cerulein (50 pg kg-1) + haloperidol
(0.25 mg kg“1) + phenamine
Cerulein (100 pg kg“1) + haloperidol
(0.25 mg kg-1) + phenamine
Counts/15 min
Counts/30 min
199 ± 24
280 ± 38
314 ± 36
390 ± 42*
549 ± 62
702 ± 64*
325 ± 39
540 ±61
488 ± 68**
439 ± 54*
914 ±82**
844 ±76**
438 ± 48*
845 ± 82**
*Cerulein and haloperidol were given 48 h before phenamine; motor activity was measured 15
min after phenamine injection.
In comparison with saline + phenamine group p < : 0.05; 0.01 (M ann-W hitney U test).
aggressive behavior in rats that had been sensitized to apomorphine aggress­
iveness by 10-day administration of apomorphine (1 mg kg’ twice daily).
Cerulein, therefore, influenced only the development of apomorphine- medi­
ated aggressiveness.
In further experiments, cerulein was found to reinforce phenamine’s
behavioral effects in mice (although only in the dose of 100 p.g kg’1) (Table
7) rather than weakening them as in the previous experiments where it was
given after phenamine (see Table 6); this sensitizing action of cerulein was
stronger than that o f haloperidol (0.25 mg kg'1). When cerulein (50 [ig k g 1)
was combined with haloperidol (0.25 mg kg’1), the sensitizing action of the
latter with respect to phenamine-mediated excitation was markedly en­
hanced, as is evident from Table 7. In rats, on the contrary, the excitatory
effect o f phenamine (2.5 mg kg'1) was attenuated both by cerulein alone (40
f-tg kg' ) and, even more, by cerulein plus haloperidol (0.25 mg kg'1) (Figure
2). Proglumide (50 mg kg' ) did not alter the effect o f cerulein plus haloperi­
dol administration on the excitatory activity of phenamine.
These studies indicate that the effects of cerulein on phenamine-induced
excitation in mice are very different from those in rats: in mice cerulein, like
haloperidol, exerts a strong antiphenamine action if administered after phe­
namine and causes hypersensitivity to the latter if given before it; in rats
cerulein either has no effect on phenamine activity or, when given before
LONG-TERM HALOPERIDOL ADMINISTRATION
113
120
100
80
60
40
20
“ 1—
14
Days
FIGURE 2 Effects of single-dose treatment with cerulein (40 jxg k g '1), haloperidol (0.25 mg
kg’ ), and cerulein plus haloperidol on phenamine-induced (2.5 mg kg’1) motor excitation in
rats. Phenamine was given at 1, 7 or 14 days after haloperidol or cerulein. M otor activity was
measured in an open field (see footnote a to Table 1). Д .Saline + phenamine; □ , haloperidol
+ phenamine;A, cerulein + phenamine; ■ , cerulein + haloperidol + phenamine. * P < 0.05 vs
the saline + phenamine group by Mann-Whitney U test.
phenamine, weakens the phenamine-mediated excitation. The mechanisms
of antiphenamine action by cerulein may therefore be different in these two
species.
In an attempt to identify the mechanisms o f cerulein action in mice and
rats, we studied cerulein for its effects on the binding o f 3H-spiroperidol and
3H-lysergic acid diethylamide (3H-LSD). In vivo, cerulein inhibited in a
dose- dependent manner the binding of these monoaminergic ligands in the
forebrain o f mice (the data for 3H-spiroperidol are shown in Figure 3), but it
114
L.H. ALLIKMETS and E.E. VASAR
Dose (ц-g/kg)
FIGURE 3 Effects of cerulein and N-propylnorapomorphine (NPA) in various doses on
H-spiroperidol (5 (J.g k g '1) binding in mouse forebrain subcortex in vivo. Mean values from
three separate experiments performed as described previously (Vasar et al., 1984b). Cerulein
and NPA were given 15 min before 3H-spiroperidol, and the animals were decapitated 20 min
after being injected with the radioligand.
should be noted that it only interacted with a portion o f dopamine receptors.
Thus, cerulein mainly acted on those 3H-spiroperidol binding sites with
which N-propylnorapomorphine (NPA) interacted when given in the low
dose of 5 pig kg'1. These results suggest that, in mice, cerulein passes across
the blood-brain barrier with relative ease and, moreover, acts primarily on
high-affinity dopamine2 receptors.
LONG-TERM HALOPERIDOL ADMINISTRATION
115
log Molar Cerulein Concentration
FIGURE 4 Effects of cerulein in various concentrations on 3H-spiroperidol and 3H-lysergic acid
diethylamide (3H-LSD) in vitro binding in the association state in rat forebrain. Mean values
from four separate experiments performed as described by Creese el al., (1977). Brain
membranes were preincubated with the indicated cerulein concentrations for 20 min at 25*C,
after which a radioligand was added, followed by further incubation for 8 min at the same
temperaure. Д, 3H-LSD, 0.25 nmol Г1
3H-LSD, 5 nmol l'^ A , 3H-spiroperidol, 0.125 nmol
Г1 ; ■ , 3H-spiroperidol, 1 nmol I'1.
In vitro, cerulein had virtually no effect on the binding o f either Hspiroperidol o f 3H-LSD to dopamine receptors in forebrain samples from rats
if it was in a state o f equilibrium, but did inhibit the binding when in a state
of association (Figure 4). These findings suggest that cerulein in the associ­
ation state is capable o f destabilizing the interaction o f dopaminergic ligands
with dopamine 2 receptors. This mechanism appears to underlie the inhibitory
effect o f cerulein on H-spiroperidol binding in vivo. Similarly, cerulein may
be thought to exert potent antidopamine activity by destabilizing the interac­
tion of endogenous dopamine with dopamine2 receptors. It is the antidopam inergic action o f cerulein that is responsible for the behavioral
hypersensitivity developing to phenamine after a single cerulein injection
(see Table 7). The molecular basis behind this phenamine hypersensitivity
L.H. ALLIKMETS and E.E. VASAR
116
в„.
500
400
300
200
100
*
I
Forebrain
*
1
Brainstem
L
FIGURE 5 Effects of single-dose and multiple dose (10 injections once daily) treatments with
cerulein (100 jig kg ') on in vitro 3H-LSD binding in mouse forebrain and brainstem. Mean
values from three separate experiments. Assays were done as described in Creese et al. (1977)
24 h after the single or last cerulein injection. Bm« = 3H-LSD binding site density in fmol (mg
protein)'1. □ , 10-day treatment with saline; 0 , single dose of cerulein; ■ , cerulein for 10 days.
* P < 0.05; ** P < 0.02 vs the saline-treated group by Student’s t test.
was an increase in dopamine2 receptor numbers in the forebrain and a
reduction in dopamine autoreceptor density in the brainstem (Figure 5). It
should be noted that the increase in dopamine2 receptor numbers in the
forebrain and the decrease in dopamine autoreceptor density in the brainstem
were more marked after multiple cerulein injections.
The inhibition of apomorphine aggressiveness in rats by cerulein may
likewise be accounted for by its destabilizing effect on apomorphine inter­
action with dopamine2 receptors. That cerulein is able to exert antidopaminergic effects in rats (and not only in mice) has been indicated by
Dumbrille-Ross and Seeman (1984) who found that single-dose cerulein
treatment elicited a sustained elevation of dopamine2 receptor density in the
nucleus accumbens and striatum. However, when injected into rats that had
been presensitized to apomorphine-mediated aggressiveness, cerulein was
without effect even in a dose as high as 200 (ig kg'1. Cerulein, therefore, is
capable of blocking the development of hypersensitivity in dopamine2 recep-
I /
LONG-TERM HALOPERIDOL ADMINISTRATION
117
Table 8 Effects of cerulein and haloperidol, 48 h after a single injection, on
3H-etoTphine binding in the accumbcns and caudate nuclei of rats.*
Nucleus accumbens
Nucleus caudatus
Treatment
В m i*
Kd
В m ix
Kd
Saline
Haloperidol (0.25 mg kg-1)
0.53 ±0.04
606 ± 32
0.61 ±0.04
493 ± 30
0.54 ± 0.03
632 ± 36
581 ±31*
Cerulein (40 |ig kg-1)
Haloperidol + cerulein
0.7 ± 0.03
0.60 ± 0.03
0.54 ± 0.04 502 ± 28*
0.51 ±0.03 450 ± 32** 0.60 ± 0.04
489 ± 32
562 ± 3 6
'Results of three separate experiments; the binding tests were performed as described in Owen
et al. (1985). See footnote to Table 2 for definition of Кd and Втлхln comparison with saline-treated groupp < : 0.05; 0.001 (Student’s l test).
tors in response to repeated apomorphine injections but fails to counteract
the effect of apomorphine if these receptors are already hypersensitive.
As shown by Matsubara and Matsushita (1986), cerulein interacts with
phenamine (amphetamine) in rats in a different way than in mice. When given
to rats in a single dose together with haloperidol, cerulein was found to exert
a long-lasting antagonistic effect on the excitatory action o f phenamine.
These authors have clearly demonstrated that this antagonistic effect is
mediated through release of ß-endorphin and its interaction with opioid
receptors in the nucleus accumbens. ß-Endorphin stimulation of opioid
receptors in this nucleus strongly inhibited presynaptic dopaminergic activity
in the mesolimbic system (Matsubara and Matsushita, 1986). These results
have been confirmed by our radioligand binding experiments in which
preinjecting rats with a single cerulein dose (40 fig kg'5) led to a significant
decrease in the. amount of bound 3H-etorphin, an opioid receptor ligand, in
the nucleus accumbens; still less JH-etorphin, was bound when cerulein was
given together with haloperidol (Table 8). Cerulein failed to exert a similar
effect on opioid receptors in the nucleus caudatus.
The findings presented above indicate that cerulein can mediate its antidopaminergic effects not only by enhancing the sensitivity of dopamine
autoreceptors (Zetler, 1985) but also by causing CCK8 receptors to interact
with post-synaptic dopamine2 receptors in forebrain structures such as the
striatum and mesolimbic system, as well as by inducing more ß-endorphin
to be released in the nucleus accumbens. The augmented ß-endorphin release
strongly inhibits the activity of presynaptic dopaminergic mechanisms in the
mesolimbic system. In mice, cerulein does not elicit enhanced ß-endorphin
release and thus fails to exert a sustained antiphenamine effect.
118
L.H. ALLIKMETS and E.E. VASAR
3.2 Adaptational Changes in the CCK8-ergic System on Long-Term
Haloperidol Treatment
The results discussed above indicate that the CCK8 receptor agonist
cerulein acts much in the same way as do neuroleptics. Although these do
not interact with CCK8 receptors, chronic neuroleptic treatment is known to
alter substantially the activity of CCK8-ergic mechanisms in the brain.
Prolonged (two weeks) administration of haloperidol or reserpine increases
the density of binding sites for 125I-CCK-33 in mouse forebrain while 2-week
treatment with haloperidol, clozapine or chlorpromazine raises CCK8 levels
in the forebrain subcortex.
Our previous studies have shown that mice treated long-term with halo­
peridol become tolerant to the sedative action of cerulein, and that this effect
correlated well with the reversal of the effect of cerulein on 3H-spiroperidol
binding (Vasar et al., 1986).
We have now examined in detail the changes that occur in the CCK8-ergic
T able 9 Modification of behavioral effects of cerulein in mice by long-term halo­
peridol treatment (0.25 mg kg' 1 twice daily for 15 days).*
Long-term treatment
Treatment
Saline
Haloperidol
Saline
359 ± 39
276 ± 17
Cerulein (20 (ig kg-1)
198123*
252 ± 2 7
Orienting/exploratory activity (counts/30 min)
Electric pain sensitivity (no. o f aggressive contacts/2 min)
Saline
Cerulein (50 |j.g kg-1)
13.5 ± 0.95
6.8 ± 0.82**
8.6 ± 0.93
21.8 ±2.99**
Picrotoxin-induced convulsions (survival time; min)
Saline
17.4 ± 1.7
18.1 ± 1.5
Cerulein (125 (J.g kg })
26.1 ± 1.6*
21.5 ± 1 .5
*A11 tests were started 48 h after the last haloperidol injection. Orienting/exploratory activity
was measured with a photoelectric actometer, cerulein was given immediately before the
measurements. Aggressiveness was assayed in foot-shock boxes each containing a pair of mice
which received 48 electric shocks (40 V) over a 2 min period, with cerulein being given 20
min before the assay. In the group with picrotoxin-induced (8 mg kg"1) convulsions, it was
given 10 min before picrotoxin.
In comparison with saline-treated group p < : *0.05; **0.01 (Mann-Whitney U test).
17*
2.2 ± 0.38
2.7 ± 0.70
0.8 ±0.26*
3.0 ± 1.39
58.4 ± 7 .5
45.5 ± 10.9
93.0 ± 9 .2
53.0 ± 5 .6
40.4 ±3.1
28.8 ± 4 .4
27.4 ±3.6*
52.0 ± 6.6
C e ru le in (5 ng)
C e ru le in (50 n g )
C e ru le in (500 n g )
Saline
5.9 ± 0 .9 2
4.8 ± 0 .6 9
4.2 ± 1.03
6.2 ± 1.10
Haloperidol
4.2 ± 1.07
4.5 ± 0.87
12.0 ± 2 .86*
3.7 ± 0.55
8.2 ± 1.45*
11.3 ± 1.83**
5.0 ±0.61
4.8 ± 0.77
Haloperidol
No. of head dips
*The assays were started 48 h after the last haloperidol administration using the open field method: 1 min after saline o r cerulein injection, the rats were
placed in the center of an open field, measuring 100 x 100 x 40 cm.
In comparison with control (intraventricular injection erf physiological saline) p < : 0.05; 0.01 (M ann-W hitney U test).
S alin e
Saline
No. of rea rings
Haloperidol
No. of counts
Saline
T re a tm e n t
T able 10 Effects of intraventricular cerulen injection on orienting/exploratory activity in rats pretreated with haloperidol for 15 days
(0.25 mg kg' 1 twice daily).8
120
L.H. ALLIKMETS and E.E. VASAR
160
140'
120'
8. 100.
80
2
60
40-
20
7
14
Days
FIGURE 6 Enhanced antiphenamine action of cerulein after 15-day haloperidol treatment (0.25
mg k g '1 twice daily). Cerulein was given in a dose of 40 (ig kg' 48 h after withdrawal from
haloperidol treatment, followed by phenamine (2.5 mg kg’1) 1,7 or 14 days later. Д , saline +
saline + phenamine;Q, haloperidol + saline + phenamine; ▲, saline + cerulein + phenamine;B.,
haloperidol + cerulein + phenamine. * P < 0.05; ** P <0.01; *** P < 0.001 vs the saline + saline
+ phenamine-treated group by Mann-Whitney U test.
LONG-TERM HALOPERIDOL ADMINISTRATION
121
NPA
16000
14000
Cerulein
10000
Dose (fig/kg)
FIGURE 7 Effect of cerulein and N-propylnorapomorphine (NPA) in various doses on in vivo
3H-spiroperidol (5 fig kg"1) bindmg in mouse forebrain subcortex after 15-day haloperidol
treatment (0.25 mg k g '1 twice daily). Assays were carried out 48 h after withdrawal from
haloperidol treatment using the previously described procedure (V asari/ al., 1984b). Ordinate:
inhibition (-) or stimulation (+) of 3H-spiroperidol binding.O, NPA after 15 days of saline; И ,
cerulein after 15days of saline;B, NPA or cerulein after 15days of haloperidol. (See also legend
to Figure 3 ■)* p < 0.01 vj the saline-treated group by Student’s l test.
system during prolonged blockade of dopamine and serotonin receptors by
haloperidol, a typical ncurolcptic. Fifteen-day haloperidol treatment (0.25
mg kg'1 twice daily) altered all the major behavioral effects of cerulein in
mice (Tabic 9). Thus, the sedative and anticonvulsant actions o f cerulein were
considerably attenuated, and cerulein (50 ng k g 1) enhanced aggressive be­
havior rather than exerting the moderate antiaggrcssion effect observed for
animals not treated with haloperidol. As for rats (Table 10), an intraventricu­
lar cerulein injection in a dose of 5 or 50 mg weakened orienting/exploratory
activity in untreated animals and (after the 50 mg dose) did not affect or
122
L.H. ALLIKMETS and E.E. VASAR
markedly stimulated this activity in those treated with haloperidol for 15
days.
In contrast, the long-lasting antagonistic effect of cerulein on phenaminemcdiated excitation was significantly increased by the prolonged haloperidol
treatment, the rats thus treated being stimulated by phenamine much less than
control animals (Figure 6).
To identify the mechanisms by which the observed changes in cerulcin’s
action arc brought about by long-term haloperidol treatment, we studied
3H-CCK8 binding in rat forebrain in vitro. At 48 h alter discontinuation of
haloperidol treatment, the affinity of 3H-CCK8 binding sites had changed
N u cleu s accum bens
Striatum
FIGURE 8 Effcct of single vs multiple dose (0.25 mg k g '1) haloperidol treatment on 3H-etorphin
bending in rat nucleus accumbens and striatum. Assays were carried out 48 h after haloperidol
ireatment. Ordinate 3H-etorphin binding site density in fmol (mg protein)’1. □ , saline; Ш .
single-dose haloperidol treatment; ■ , 15-day haloperidol treatment. P < 0.05 vs 15-day
treatment with saline by Student's I test.
LONG-TERM HALOPERIDOL ADMINISTRATION
123
but slightly (the dissociation constant being 0.62 ± 0.05 vs 0.78 ±0.05 nmol
Г1 in the control), whereas their density had decreased significantly (21.7 ±
1.8 vs 29.1 ± 1.3 fmol (mg protein)'1 in the control). In mice, long-term
haloperidol treatment reversed the inhibitory effect o f cerulein on 3H-spiro^eridol binding in vivo: the forebrains of mice thus treated bound more
H-spiropcridol after cerulein injection (Figure 7). The reversal of the ccrulein effect was associated with a decrease in the density o f high-affinity
dopamine2 receptors since the long-term haloperidol treatment also attenu­
ated the effect of NPA (5 p.g kg"1).
Prolonged treatment with neuroleptics is known to cause behavioral
supersensitivity to opioid peptides in limbic structures (Stinus el al., 1986).
As shown above, the sustained antagonism of phenamine-induced cxcitaion
by cerulein is associated wilh a decrease in opioid receptor numbers in the
nucleus accumbcns. Our study of how long-term haloperidol treatment
affects 3H-etorphin binding indicates that such treatment results in a signifi­
cant elevation of opioid receptors in this nucleus (Figure 8), suggesting their
heightened sensitivity in mesolimbic structures, which in turn may account
for the enhanced antiphenamine action of cerulein observed under such
haloperidol treatment.
To sum up, long-term haloperidol treatment attenuates or reverses the
inhibitory behavioral effects o f cerulein with one exception: the sustained
antagonist effect o f cerulein on phenamine- induced excitation is enhanced
rather than weakened. Critical to the attenuation of many cerulein effects is
a reduction in the densities of CCK8 receptors and high-affinity dopaminc2
receptors. Long-term haloperidol administration probably changes the latter
receptors to a low- affinity state in the test forebrain structures. The high-af­
finity dopamine2 receptors occur, in the main, on intemeurons of the caudate
nucleus and limbic structures. This indicates that long-term haloperidol
administration most likely reduces the functional activity of interneurons in
subcortical structures of the forebrain.
An antagonistic effect o f cerulein on phenamine excitation occurs only
when the endopioid system is intact, but not when it has been destroyed or
blocked, which implies that ß-endorphins act as intermediaries between
CCK8 and dopaminergic mechanisms in the mesolimbic system. Single- or
multiple-dose cerulein administration elicits cerulein hypersensitivity in
opioid rcceplors of the nucleus accumbens, and this in turn leads to enhanced
antiphenamine action of cerulein.
124
L.H. ALLIKMETS and E.E. VASAR
4. Conclusions
The foregoing discussion clearly demonstrates that the effects resulting
from long-term administration of the typical neuroleptic haloperidol are due
to changes it causes not only in dopaminergic system but also in the G AB Aand CCK8-ergic systems which are closely related morphologically and
functionally with the latter. The studies described above warrant the follow­
ing conclusions:
1. Alterations in the GABA-ergic system under the action of haloperidol are
specific in that they occur on GABA a receptors and the closely related
benzodiazepine receptors.
2. Haloperidol-induced changes in GABA a and benzodiazepine rcceptors
may be of two kinds because o f the existence of two functional types of
these receptors. One type, the so-called inhibitory receptors, occur in
forebrain structures, are closely linked with post synaptic dopamine
receptors and inhibit their activity and thus the activity o f the dopam inergic
system. The other type, ‘stimulatory’ receptors, are found in the brainstem,
are linked with monosynaptic autoreceptors, and inhibit the latter’s
activity to augment the function of monoaminergic systems. Long term
h a lo p e rid o l ad m in istration c a u se s stim u la to ry G A B A A and
benzodiazepine receptors to preponderate with the result that the
behavioral effects of muscimol (a GABA a receptor agonist) or o f Ro
15-1788 (a benzodiazepine receptor antagonist) may be reversed.
3. The benzodiazepine antagonist Ro 15-1788, unlike the agonist diazepam,
is capable of rectifying the changes induced by long-term haloperidol
treatment in GABA a and benzodiazepine receptors.
4. Cerulein, a CCK8 receptor agonist, resembie. haloperidol in its behavioral
(antidopaminergic action) and biochemical (changes in the affinity of
dopamine2) effects, but unlike haloperidol, itacts on dopamiricz receptors
indirectly, via CCK8 receptors, thereby apparently destabilizing the
interaction of dopamine and dopamine agonists with dopam ine: receptors.
Some of the effects seen on long-term haloperidol treatment may therefore
be mediated via CCK8-ergic mechanisms which are closely linked
morphofunctionally with dopaminergic systems
5. Adaptational changes in the CCK8 systems under the action of piolonged
haloperidol treatment arc directly dependent on alterations и other
18
LONG-TERM HALOPERIDOL ADMINISTRATION
125
neurotransmitter (dopaminergic and endopioid) systems with which they
have close morphofunctional links. Such haloperidol treatment decreases
CCK8 receptor density in rat forebrain (in the cerebral cortex, mesolimbic
structures, and nucleus accumbens). Behavioral analysis, however,
indicates that haloperidol lowers sensitivity in only a fraction o f CCK8
receptors. The hypersensitivity of these CCK8 receptors is probably
associated with the haloperidol-induced transition o f a proportion of
high-affinity dopamine2 receptors to a low-affinity state. The other CCK8
receptors, which are closely associated with endopioids, develop
hypersensitivity, as is indicated by the augmented antiphenamine activity
of cerulein after repeated haloperidol injections. It should be noted that
such interactions between CCK8, endopioids, and dopam inergic
mechanisms occur in rats, but not in mice.
6. The adaptational alterations in dopam incrgic, G A B A -ergic, and
CCK8-ergic systems described above underlie those side-effects and
antipsychotic actions of haloperidol seen on its long-term administration.
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caerulein and A/-propy!norapomorphine in mice. Pharmacol. Biochem. Behav , 24,469-478
Wang, R.Y., White, F.J., Voight, M.M. (1984) Cholecystokinin, dopamine and schizophrenia.
Trends Pharmacol. Sei, 9, 436-438
Wise, S.P., (1985) Co-localization of GABA and neuropeptides in the cerebral cortex. Trends
Neurosci.,3,92-95
Zetler, G. (1985) Neuropharmacological profile of cholecystokinin-like peptides Ann. N.Y.
Acad. Sei., 448, 448-469
Zharkovsky, A.M. and Allikmets, L.H. (1986) Analysis of dopamine receptor supcrsensitivity
after chronic neuroleptic treatment. In Valdman, A. V. (Ed.) Drug Dependence and Emotional
Behaviour New York: Plenum, pp.286-302
18 *
A H.hm«». Vol. ?5. |>p 8S5-KSV ® Pergamon Press pk. I W
U091-305 7/90 S3 .UO f .00
Printed in Uh. U S.Ä
Similar Behavioral and Biochemical
Effects of Long-Term Haloperidol and
Caerulein Treatment in Albino Mice
EERO VASAR, LEM BIT ALLIKM ETS, ANDRES SOOSAAR AND AVO LANG
Laboratory o f Psychopharmacology. Institute o f General & Molecular Pathology
Tartu University, 34 Veski Street, 202400 Tartu, Estonia, USSR
Received 6 June 1988
VASAR, E., L. ALLIKMETS, A. SOOSAAR AND A. LANG. Similar behavioral and biochemical effects of long-term haloperidol
and caerulein treatment in albino mice. PHARMACOL BIOCHEM BEHAV 35(4) 855-859, 1990. —Behavioral and biochemical
experiments on male albino mice have revealed similar effects after the cessation of repeated (15 days) haloperidol (0.5 mg/kg daily
IP) and cacrulcin (0.1 mg/kg daily SC) treatment. Tolerance developed to the action of muscimol (a GABA-A agonist, 1 mg/kg IP),
caerulein (a CCK-8 agonist, 15 p-g-'kg SC) and flumazenil (a benzodiazepine antagonist, 10 mg/kg IP). Muscimol and caemlein were
not able to suppress (he motor activity of mice after !5 days treatment with haloperidol and caerulein. Flumazenil, which increased
motor activity in saline-treated animals, also failed ю affect activity after extended haloperidol or caerulein treatment. In contrast, the
motor excitation induced by amphetamine (an indirect dopamine agonist. 3 mg/kg IP) was increased after haloperidol or caerulein
administration. In radioligand binding studies the density of dopamine-2-receptors in .striatum, opioid receptors in mesojimbic
structures, and benzodiazepine and GABA-A receptors in brainstem was significantly elevated after long-term haloperidol or caemlein
treatment. Simultaneously, the number of CCK-8, benzodiazepine and GABA-A receptors in cerebral cortex was deceased. It is
probable that CCK-8-crgic mechanisms are involved closely in the action of repeated haloperidol treatment. CCK-8 seems to modulate
the action of haloperidol through altering the sensitivity of dopamine, opioid. GABA-A and benzodiazepine receptors.
Long-term treatment
Haloperidol
Caerulein
Exploratory activity
IT is generally accepted that the antipsychotic potency of neuro­
leptic drugs is correlated with their affinity to dopamine-2reccptors in striatum (21,22). However, long-term treatment with
neuroleptic drugs causes significant changes not only in dopamine
receptors, but also in receptors for the other ncurotransmitters. For
example, the administration of haloperidol for 15 days increases
the number of glutamate receptors in striatum (26), but decreases
the density of GABA-A and benzodiazepine receptors in forebrain
structures of rat (1,2) Recently, the involvement of cholecystokinir. octHpeptide (CCK-8) in the action of neuroleptic drugs has
been established. Repeated administration, but not acute treat­
ment. of different neuroleptic drugs (clozapine, chlorpromazine
and haloperidol) evidently increases the amount of CCK-8 in
.'•ria'.um and mcsolimbic structures (II) Chauj- et al. (6) have
shown that long-term treatment with halopcr-dol increases the
density Of CCK-8 receptors in cortical and iimbic structures of
mice. In addition. Bunney <•/ al. (3, 4. 7) have demonstrated that
repeated, hut not iicute. administtaiiou of different neuroleptic
drugs (haloperidol. chlorproma/ine. clozapine, etc ) induces de*
polarization and subsequent inactivation of dopamine neurons in
midbrain. Acute treatment with CCK 8 causes the same cffect and
proglumide. an antagonist of CCK-8. reverses completely the
effect of neuroleptic drugs (4) The above studies support the idea
that CCK-8 is playing an obvious role in the mediation of
biochemical and behavioral effects of neuroleptic treatment. The
:-!i.t of the present study was a farther clarification of the
iiuolvcmuit oi ( t ’K-S mi the action. i neuroleptic drugs To stud;»
Radioligand binding
this problem the behavioral and biochemical effects of long-term
haloperidol and caerulein, an agonist of CCK-8 receptors, treat­
ment were compared. The changes in dopamine-2-, opioid,
GABA-A, benzodiazepine and CCK-8 receptors were studied in
both behavioral and radioligand experiments. Carlsson (5) has
suggested that neuroleptic drugs cause their antipsychotic effect by
blocking mesolimbic dopamine receptors. Other investigators
have reported that G ABA- and CCK-8-ergic systems have dense
morphofunctional connections with dopaminergic system in me­
solimbic structures (12,14). Because the normal functioning of the
mesolimbic dopamine system appears to be critical for the regu­
lation of locomotor activity in rodents (8) we used the exploratory
locomotor activity of mice to determine the long-term behavioural
effects of haloperidol and caerulein. Haloperidol was chosen as the
neuroleptic for investigation in ‘his study, because it is a potent
and widely used antipsychotic drug. It is also noteworthy that in
drug discrimination experiments there is substantial generalization
between haloperidol and CCK-8 (9). Caerulein was selected for
investigation because it is most effective among the available
CCK-8 analogs.
MbTHOD
Animals
Male albino mice unknown >train. weighing 25 . t } g. were
useii. Mice wen maintained at 20 1 2°С am! on I2 hr light.
.cn К a m and S p.m wiiSi U-od and water ad lib.
betA
VAS M t
Determination o f Exploratory Activity
Exploia’ory activity was measured in individual cages. The
cage for registration of exploratory activity was a cylinder with an
inner diameter 40 cm and 2 photocells (located in walls) for
detection of motor activity. Exploratory activity was' counted
between 15 and 45 min after intraperitoneal administration of
amphetamine <an indirect dopamine agonist. 3 mg/kg j, muscimol
(a GABA-A agonist, I mg/kg) and flumazenil (a benzodiazepine
antagonist Ro 15-1788. 10 mg^kg). or between 0 and 30 min in the
case of subcutaneous treatment of caerulein (a CCK 8 agonist. 15
Th* doses of amphetamine, caerulein. muscimol and
flumazenil were chosen according to results i»t our j^evious
studies. These doses cause only moderate, but statistically evident,
changes in exploratory activity. Thus, an increase or decrease in
activity due to the action of these drugs can be detected alter
repeated treatment with haloperidol or caerulein
Preparation of Brain Membranes for Radioligand Studies
Following decapitation (between 10 and 12 a.m.) the whole
brain was rapidly removed from skull. The different brain regions
(cerebral cortex, striata, mesolimbic structures/nucleus accumbens
and tuberculum olfactorium/and brainstem) were dissected on ice.
Freehand method was used for dissection of brainstem, whereas
the other structures were dissected according to the method of
Glowinski and Iversen (13). Brain regions from ten mice were
pooled and homogenized in 10 volumes of ice-cold 50 mM Tris
HC1, pH 7.4 at 4°C, using motor-driven Teflon-glass homogenizer
for 12 strokes. The homogenate was centrifuged at 40000 x g for
15 min, resuspended in the same volume of buffet and again
centrifuged for 15 min. The membrane preparation for all radio­
ligand studies was the same, except for [H]-etorphine binding. In
this case the homogenate of the mesolimbic structures was
incubated for 45 min at 37°C between two centrifugations (for
elimination of endogenous opioid peptides). In the case of {'Hjmuscimol binding the membranes were washed (centrifuged) 7
times at 40000 x g for 15 min.
Radioligand Binding Studies
Different incubation mixtures were used for the radioligand
binding experiments. The binding of [3H]-etorphine (36 Ci/
mmole, Amersham International. U.K.). [3H|-flunitrazepam (81
Ci/mmole, Amersham International, U.K.) and iJH)-nuiscimol
(19Ci/mmole. Amersham International, U.K.) were performed in
50 mM Tris HC1 (pH 7.4 at 4°C). (JH)-Spiroperidol (77 Ci/
mmole. Amersham International, U.K.) binding was determined
in an incubation buffer consisting of the following: 50 mM Tris
HC1 (pH 7.4 at 4°C), 120 mM NaCl. 5 mM KCI* 2 mM CaCU, 1
mM MgCl?, 1 mM EDTANa2. 50 p.M pargyline and 0.1*31'
ascorbic acid. l3H]-Pentagastrin (81 Ci/mmole. NEN-Dupont.
USA) binding was studied in the following incubation medium: 10
mM HEPES-KOH (pH 6.8 at 4°C). 5 mM MgCU. 1 mM
FL)TANa; , 0.2% bovine serum albumin.
For the binding experiments each polypropylene tube (1.5 ml)
received 50 jju of j ‘H|-ligand. 50 p.1 of incubation medium or
displacing compound and 400 ц! of brain membrane homogenate
1 1-4 mg ol‘ original tissue wet weight ). {'Hj-Flunitrazepam was
.idded in concentrations *rom 0.6 ю 16 nM The nonspecific
binding was determined by using 1 ц-M llunitiazepam. The
membranes of cerebral cottex and brainstem were incubated at0cC
tor 60 min. 1*H|-Muscimul was used in concentrations from 1 to
SO nM. The nonspecific binding w as measured by 100 цМ
muscimol. Tbc membranes ofcerebr.il cortex and brainstem were
mcubated for 10 min at !) ('. J ’Щ-ГлогрЫпе was added m
I I Al
concentration?, from 0.05 to 3 nM, the uonsprcifi« bimiui*» was
detected by adding naloxone (10 jtM). 11k- incubation uf n.r.olm?
bic membranes was performed at 25°C for 45 nun. (‘HI-Spiro
peridot was used in concentrations from 0 .1 to 2 nM and the
nonspecific binding was measured by adding I jiM spiroperidol.
The membranes oi murine sttnt!a weie incubated for 30 min at
17X i 'H j-Pentagastim v as addrd to the incubation medium in
concentrations Iron! 0 ! to 20 iiM, nonspecific binding was
detected with I jxM cam iltin. Inenbation of | ’H(-pentagastrin was
performed for 75 nun at 25°C.
In all cares the binding experiment was stopped by rapid
centrifugation {Beckman microfuge mode! !2) for 3 min at
11000 v g. The supernatant was carefully discardeu and remaining
pellft was washed with ice-cold incubation buffer and the lips of
polypropylene tubes were *ut into counting vials Radioactivity wf
samples ^as counted after stabilization in scintillatioii locktati
within 24 hours using a Becl'nutn LS 6300 'counting efficacy
50-54** l. The binding experiments were repeated at least three
mves and the data analy sed usine the Scatch>»rd method (19).
JM'gs and Their Administration
The a/ugs usea in the present investigation are caerulein
(Cerule;idc Farm italic O m o Erba, Italy), haloperidol fGedeon
Richter. Hungary), spiroperidol (Janssen Pharmaceutica, Bel­
gium), naloxone (Dupont. USA), flunitrazepam and flumazenil
(Ro 15-1788 \ (Hoffmann-La Roche. Switzerland), muscimol (Serva,
FRG», pargyPne (Sigma, USA), amphetamine (USSR). Caer­
ulein, muscimol, amphetamine and commercial solution of halo­
peridol were prepared in saline. The injection solution of flumazenil
was made soluble in saline by adding some drops of Tween-80.
Each injection was done in a volume of 0.1 ml/10 g body weight.
Haloperidol (0.5 mg/kg IP) and caerulein (0.1 mg/kg SC) were
injected once daily for 15 days. The doses of haloperidol and
caerulein were chosen according to our previous studies. Acute
administration of haloperidol (0.5 mg/lcg) and caerulein (0.1
mg/kg) caused significant neuroleptic effects in mice (catalepsy
and the reversal of the behavioral effects of dopamine agonists).
The behavioral and radioligand experiments were performed 72
hours after the cessation of haloperidol and caemlein treatment.
Statistics
The results of the binding studies were evaluated by the
Student's Mest. The results of the behavioral experiments were
analyzed by the Mann-Whitney U-test
RESULTS
According to our preliminary experiments the cessation oi
long-term administration of haloperidol and caerulein did not
cause significant signs of withdrawal. The basal motor activity of
mice was unaltered 72 hours after the last injection of repeated
treatment with saline and haloperidol as well as caerulein (Table
1). In addition, we found that quinolinic acid- and picrotoxininduced seizures were identical after the withdrawal of long tetin
saline, haloperidol or caerulein administiation. The behavioral
effects of ketamine (motor excitation, stereotyped behavior) re­
mained unchanged after the withdrawal of haloperidol and caer­
ulein (data no; presented). In addition, there were no significant
differences in the binding values of ('HJ-spiroperidol, I3H)tlunitrazepam and [ ’HJ-pemagastrin if the tissues were obtained 2
or 72 hours after the last injection of haloperidol and caerulein.
Consequently, the changes in mice behavior and radioligand
binding described below were not caused by the withdrawal of
haloperidol and caerulein. but rather were induced by the repeated
administration of both drugs.
SIM ILAR EFFECTS O F HALO PERID OL AND CAERULEIN
857
THE EFFECT OF CAERULEIN. AMPHETAMINE. MUSCIMOL AND FLUMAZENIL ON
EXPLORATORY ACTIVITY AFTER 15-DAY HALOPERIDOL OR
CAERULEIN TREATMENT IN MICE
Long-Term Treatment
Saline
Haloperidol
Caerulein
Motor Activity Counts During 30 Min
%
Drug/dose
Saline
Caerulein 15 tig/kg
Amphetamine 3 mg/kg
Muscimol I mg/kg
Flumazenil 10 mg/kg
171
104
409
89
261
±
±
±
±
r
15
10
30
10
17
100
100
100
100
100
%
188
172
598
203
162
±
±
±
±
±
14
15*
45*
36*
15*
no
165
146
228
62
%
184
190
704
170
193
±
*
±
±
±
18
15*
62*
28*
16*
108
183
172
191
74
The study was performed 72 hours after ihe cessation of haloperidol, caerulein or saline
treatment. The mean values * S.E M. are shown. *p<0.05 (U-test Mann-Whitney, compared
to mice, receiving saline injections for 15 days).
Seventy-two hours after the cessation of 15 days o f haloperidol
(0.5 mg/kg daily) and caerulein (0.1 mg/kg daily) treatment the
effects of different drugs on mice motor activity were changed.
The motor excitation induced by amphetamine (3 mg/kg) was
evidently increased after haloperidol or caerulein treatment (Table
1). However, tolerance developed to the action of muscimol (I
mg/kg), caerulein (15 p-g/kg) and flumazenil (10 mg/kg). Musci­
mol and caerulein were not able to suppress the motor activity of
mice after haloperidol or caerulein administration (Table 1).
Flumazenil, which increased the motor activity in saline-treated
animals, failed to affect activity after 15 days of haloperidol or
caerulein treatment.
The prolonged haloperidol and caerulein treatment also af­
fected the binding of different radioligands to washed brain
membranes in a similar way. They changed mainly the number of
binding sites of different radioligands, but failed to affect the
affinity of the radioligands for their sites. The density of (3H]spiroperidol binding sites in striatum (mainly dopamine-2-receptors) was significantly increased after the administration of
both drugs (Table 2). Similar increase of [3H]-etorphine (labelling
mu-f delta- and kappa-opioid receptors) binding sites was detected
in mesolimbic structures. Differently from [3H]-spiroperidol and
[3H]-etorphine binding the number of [3H]-pentagastrin (a ligand
interacting with central CCK-8 receptors) binding sites was
evidently decreased in cerebral cortex. The changes in [3HJflunitrazepam and [3H]-muscimol binding were dependent on the
brain region studied, in cerebral cortex their number was reduced,
whereas in brainstem the density of [3H]-flunitrazepam and
[3HJ-muscimol binding sites was increased after 15-day treatment
of haloperidol and caerulein (Table 2).
DISCUSSION
Zetler (27) has shown in his experiments on mice that caerulein
causes haloperidol-like behavioral effects, but the further pharma­
cological analysis revealed marked differences in the action of
haloperidol and caerulein. Haloperidol, the potent antipsychotic
drug, preferentially blocks dopamine-2-receptors (18), whereas
, (pmoles/'g tissue)
Ku (nM)
Haloperidol
Radioligand. Brain Structure
f3HJ-spiroperidol, striatum
[JH J-flunitrazepam . cerebral
cortex
(JH J-flunitrazepatn. brainstem
p H l-m uscm iol. cerebral
cortex
[3HJ-rnusvurn il, brainstem
(}B ) - e ty p h m ^ mesoiinibic
| JH j-pentagastr:n. cciebral
Haloperidol
Caerulein
45 .0 ± 2.5'*
144 ± 15*
49.2 ± 3.2*
138 £ 14*
0 .4 7 ± 0 .0 5
1.70 ± 0.25
0 .6 2 i 0.05
1.60 ± 0.25
0 63 - 0.05
1.50 ± 0 .18
34 .8 ♦ 3 0
198 ± 12
2.42 ± 0 20
У.6 ± 1 6
1.92 г 0.18
10.2 .r I 8
2.62 = 0 .17
11.0 r 1.2
103 £ 8
91 ± 8
12.0 i 1.3
0 .6 2 i 0.05
13.2 t 1 3
0.61 ± 0 .05
14.3 z l . \
0 .7 7 r 0,05
38 £ 4
33 = 2.4
51 r 4
42 г 2 .5 '"
50 г 5
46 л 3.2*
J 50 * 0 .4 0
* 20 r 0 .30
1.20 i 0.32
5 ± 0.4
3 5 i 0.3-'
3.2 i 0.3*
125 г 12
64 ♦ 5*
142 £ 16*
63 2: 6*
The vtudy was perform ed
1hours after the e<ovation o f haloperidol. caerulein or saline treatment. T he mca и values o f three
independent e x p erim e n t are sf»»** и Statistically ev idem difference;&from salme-trcakrd mice: */)'" 0.05 (Student's м test). K„. constant
Of dissocial И*11 «nM-. И
Itpp.ircnt number »4 binding sites (pmoles/g * e t weight tissue)
VASAR £ T AL.
caerulein stimulates CCK-8 receptors (27). Despite the significant
differences in the molecular action of the two drugs, long-term
treatment with haloperidol and caerulein has a similar effect on
behavior and causes similar changes in radioligand binding to
washed brain membranes. Our data suggest that both compounds
increase the number of dopamine-2-receptors in striatum and
opioid receptors in mesolimbic structures. The increased sensitiv­
ity of mice to moior stimulating effecl of amphetamine, a
compound that increases the release of dopamine, probably
reflects the enhancement of dopamine-2-receptors density after
haloperidol or caerulein treatment. Some authors have demon­
strated С17.23) that opioid receptors in limbic structures play an
important role in the regulation of dopamine receptors’ sensitivity.
The prolonged administration of different neuroleptic drugs (halo­
peridol. sulpiride. flupenthixol. etc.) leads to the hypersensitivity
not onl> of dopamine receptors, but also of opioid receptors in
mesolimbic structures 120.23). It seems probable that the in­
creased sensitivity of opioid receptors is obligatory for the devel­
opment of -hypersensitivity in dopamine receptors in mesolimbic
area.
After 15 days of haloperidol and caerulein administration a
marked decrease in CCK-8 receptors density in cerebral cortex is
found. The significant reduction of motor depressant effect of
caerulein after haloperidol or caerulein treatment is probably
related to the decrease of CCK-8 receptor number in brain.
Consequently, haloperidol and caerulein treatment cause a sub­
sensitivity of CCK-8 receptors. A similar subsensitjvity (decrease
of [3H}-CCK-8 binding sites in mouse and rat brain, tolerance or
inversion of caerulein’s behavioral effects) of CCK-8 receptors
was found after long-term haloperidol treatment in our previous
experiments (25). Many behavioral studies now support the idea
that CCK-8 acts as a functional antagonist of dopamine and
endogenous opioid peptides in brain (10, 16, 27). Accordingly,
the subsensitivity of CCK-8 receptors seems to be necessary for
the development of hypersensitivity of dopamine and opioid
receptors. However. Chang et al. (6) have shown the opposite
effect, the increase of the number of CCK-8 receptors, after
repeated haloperidol treatment in mice. They have used [,a5I]CCK-33 for labelling of CCK-8 receptors and they have admin­
istered significantly higher dose of haloperidol (2-3 mg/kg) to
mice. These factors may explain the differences between our study
and that of Chang* al. (6). Despite the discrepancy the above­
mentioned results would support the idea that CCK-8-ergic mech­
anisms play a crucial role in the mediation of the effects of
prolonged neuroleptic treatment.
The changes in benzodiazepine and GABA-A receptors differ
from those of the other neurotransmitter receptors after long-term
haloperidol or caerulein administration. In frontal cortex the
density of benzodiazepine and GABA-A receptors is reduced and
it is parallel to the reduction of CCK-8 receptors. The number of
benzodiazepine and GABA-A receptors in brainstem, on the
contrary, is increased after haloperidol or caerulein treatment. The
similar alteration of CCK-8 and benzodiazepine-GABA-A recep­
tors in cerebral cortex may be linked to the finding that CCK-8 and
GABA are co-mediators in the same neurons of cerebral cortex
and hippocampus (15). The molecular changes in benzodiazepine
and GABA-A receptors are probably associated with tolerance of
behavioral effects of GABA-A agonist muscimol and benzodiaz­
epine antagonist flumazenil. Muscimol did not suppress and
flumazenil did not increase the motor activity of mice after
long-term treatment of haloperidol and caerulein. The possible
explanation for these changes may consist of the existence of
functionally different benzodiazepine and GABA-A receptors in
forebrain and brainstem structures (24). The results of present
study show that CCK-8 may have, through CCK-8 receptors
indeed, a modulating action on the sensitivity of GABA-Abenzodiazepine, opioid and dopamine receptors. This opinion is
supported not only by present study, but also by other investiga­
tors. CCK-8 and caerulein inhibit not only the action of amphet­
amine and methylphenidate, interacting with presynaptic dopaminergic
mechanisms, but also the effects of apomorphine, a direct agonist
of dopamine receptors (27).
In conclusion, the similar actions of haloperidol and caerulein
after long-term treatment seem to be related to the fact that the
effects of haloperidol are effected not only through dopaminergic,
but also via CCK-8-ergic mechanisms. The effecl of CCK-8 seems
to be related to the modulation (through CCK-8 receptors) of the
sensitivity of different neurotransmitter receptors (dopamine, en­
dogenous opioid peptides and GABA).
ACKNOWLEDGEMENTS
We would like to thank Faimitalia Carlo Erba. Janssen Pharmaceutica,
Hoffmann-La Roche and Dupont for the generous gifts of drugs.
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25 Vasar. K. E .; Soosaar, A. H .; Maimets, M. C .; A llikm eis. L H
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1991, 891: 215-233
T H E C H AN G ES A T CH O L ECY ST O K IN IN R E C E PT O R S A FT E R LO N G -T ER M
TR E A T M E N T W IT H D IAZEPAM
E.Vasar, J.Harro, A.Soosaar, A.Lang
L aboratory o f P sychopharm acology, Tartu U niversity
2 0 2 4 0 0 TARTU, Estonia
SUM M ARY
The effect of benzodiazepine withdrawal was studied on CCK-8 receptors in experiments on male
rodents. Benzodiazepine anxiolytic diazepam (5 mg/kg i.p.) was injected for two-weeks. The
behavioural and radioligand binding studies were performed 24-72 hours after the last injection of
diazepam. The significantly suppressed exploratory activity of mice in elevated plus-maze was
reflecting the obvious anxiety after benzodiazepine withdrawal. CCK-8 agonist caerulein (500 ng/kg),
which induced anxiogenic-like effect in control animals, was not able to change the exploratory
activity of mice pretreated with diazepam for two-weeks. The sedative effect of caerulein (15 ng/kg)
was reduced after withdrawal of benzodiazepine treatment. The mild antiaggressive effect of
caerulein (40 (ig/kg) was replaced by proaggressive action in male mice pretreated with diazepam.
The
anticonvulsant effect of caerulein (125 ng/kg) against picrotoxin-induced seizures was
completely reversed after withdrawal of diazepam treatment. These results suggest that the
subsensitivity is developing at CCK-8 receptors after long-term diazepam treatment. However,
according to radioligand studies the number of ^HpCCK-8 binding sites was increased in frontal
cortex, piriform cortex and hippocampus of rat after benzodiazepine withdrawal. It seems possible
that repeated treatment with diazepam is causing the opposite changes at different subtypes of CCK-8
receptors. The sensitivity of one subtype is reduced, whereas the affinity of others is increased to
CCK-8. Probably the described changes at CCK-8 receptors are related to increased anxiety after
withdrawal of long-term diazepam treatment.
K E Y WORDS: DIAZEPAM; BENZODIAZEPINE WITHDRAWAL; CCK-8
RECEPTORS; CAERULEIN; ANXIOGENIC-LIKE EFFECT; SEDATIVE
EFFECT; AGGRESSIVENESS; ANTICONVULSIVE EFFECT; MOUSE; RAT
IS
INTRODUCTION
Cholecystokinin octapeptide (CCK-8), neuropeptide widely distributed in CNS, is shown to
colocalize with major inhibitory transmitter GABA in cerebral cortex and hippocampus (Kosaka et
al., 1985). Interaction between CCK-8 and GABA seems to be antagonistic, because several
behavioural effects of CCK-8 and caerulein, its structural analogue, can be antagonized by the
administration of benzodiazepine anxiolytics (Kubota et al., 1985; 1986). It is widely accepted that
benzodiazepine anxiolytics exert their action through the facilitation of GABAergic neurotransmission
in the brain (Haefely et al., 1985). Two benzodiazepines, diazepam and lorazepam, depress at very
low doses selectively the CCK-8-induced excitation of rat hippocampal pyramidal cells (Bradwejn,
De Montigny, 1984). It lias been suggested that this effect of benzodiazepine tranquillizers might be
related to their anxiolytic properties (Bradwejn, De Montigny, 1985). The blockade of CCK-8
receptors by CCK antagonist lorglumide is shown to potentiate several behavioural effects of
iliazepam (Panerai et al., 1987). In our previous studies the potent amuogenic-like effect of CCK-8
agonists (caerulein, pentagastrin) is established (Harro et al., 1989b). Subchronic, but not acute,
treatment with diazepam is able to antagonize the anxiogenic-like action of caerulein (Harro et al.,
.989a). Recent evidence suggests that repeated treatment with benzodiazepine anxiolytics reduces
rfectrophysiologically measured neuronal responsiveness to CCK-8 in hippocampus (Bouthillier, De
Montigny, 1988). The above described studies probably reflect very significant role of CCK-8 in the
action of benzodiazepine tranquillizers. The aim of present study was to reveal the role of CCK-8
receptors in benzodiazepine withdrawal. The different behavioural effects of caerulein (anxiogenic,
sedative, antiaggressive and anticonvulsant) and -%pCCK-8 binding were studied after withdrawal of
two-weeks diazepam administration in rodents.
MATERIALS AND METHODS
Male unstrained albino laboratory mice (20-25 g) and male Wistar rats (220-250 g) were used in
this study. Every experimental group consisted of 8-10 animals. Diazepam (5.0 mg/kg i.p. daily,
Seduxen, commercial solution, G edeon R ichter, Hungary) or diazepam vehicle (40% propylene
glycol, 10% ethyl alcohol, 5% sodium benzoate, 1,5% benzyl alcohol) were injected for 14 days. The
behavioural and radioligand binding experiments were performed 24-72 hours after the last injection
of drag or vehicle. The anxiogenic-like effect of CCK-8 agonist caerulein (F arm italia-C arlo Erba,
Italy) was studied according to the original method of Fellow et al. (1985) (so-called 'elevated plusmaze ) in our slight modification (Harro et al., 1989a). Exploratory activity of mice in elevated plusmaze was detected 15 min after the injection of caerulein (500 ng/kg i.p.). The locomotor activity of
animals was measured in individual cages. The cage for registration of motor activity was a cylinder
with an inner diameter 40 cm and 2 photocells (located in walls) for detection of motor activity.
Locomotor activity was counted between 0 and 30 min after subcutaneous treatment with caemlein
(IS |ig/kg). The action of caerulein on aggressive behaviour was determine according to the method
of foot-shock-elicited aggressiveness. Each pair of mice received during 2 min 48 foot-shocks (2 mA)
and number of aggressive contacts (bitings, boxings etc.) was counted. Caerulein 40 ng/kg was
injected 15 min before the experiment. In the case of study of anticonvulsive effect of caerulein the
mice were placed in individual observation boxes 15 min before the start of the experiment After this
habituation period each animal was injected with caerulein (125 ng/kg) or saline 10 min prior to
picrotoxin (10 mg/kg. Sigma, USA). Mice were observed for 30 min and the latencies to onset of
clonic seizures, tonic extension and death were registered. ^HpCCK-8 (60 Ci/mmole, Am ersham
International p ic , UK) binding was measured by the method of Praissman et al. (1983) with slight
modifications. The rats were killed by cervical dislocation respectively 30 min and 24 h after the last
injection of diazepam and die brains immediately removed from the skull. The different brain regions
- frontal cortex, piriform cortex and hippocampus - were dissected on ice. Brain regions from 5 rats
were pooled. Saturation curves of ^HpCCK-8 binding (0.05-2 nM) were analyzed using the
ENZFOTER program on IBM microcomputers (Leatherbarrow, 1987).
RESULTS
The exploratory activity of mice in elevated plus-maze was significantly suppressed after
withdrawal of repeated diazepam administration in comparison with animals receiving vehicle (table
1). The administration of caerulein (500 ng/kg) caused the anxiogenic-like effect (reduction of
exploratory activity) in vehicle pretreated mice, whereas after repeated treatments with diazepam it
did not change the behaviour of mice. After withdrawal of diazepam injections the motor activity of
mice was somewhat reduced in comparison with vehicle treated group (table 2). 15 Mg/kg caerulein
suppressed significantly the motor activity in control group, whereas in benzodiazepine pretreated
mice the sedative effect of caerulein was reduced. In control mice 40 ng/kg caerulein slightly reduced
the number of aggressive contacts between animals. However, after diazepam withdrawal caerulein
increased markedly the aggressiveness (table 3). 125 jig/kg caemlein evidendy antagonized seizures
induced by picrotoxin in control mice, but not in animals after diazepam withdrawal (table 4). Long­
term treatment with diazepam or withdrawal of diazepam administration did not alter the affinity (Kj)
of 3HpCCK-8 binding sites in different forebrain structures (table 5). Two-weeks treatment with
diazepam elevated the number of CCK-8 binding sites in piriform cortex. After the withdrawal of
diazepam treatment die density of CCK-8 binding sites was increased in all three brain structures
Table 1
Effect of caerulein (500 ng/kg) on exploratory activity of mice after repeated (14 days)
treatment with diazepam in elevated plus-maze.
Latency of first
No of sectors
Total time
Fretreatment/
open part entry
crossed in
spent in
drug
(s) in plus-maze
open part of
open part of
plus-maze
plus-maze(s)
31±2.8
16±4.1**
16±3.7**
17±4.3
96±7
61±12*
64±12*
49±11
Vehicle+saline
Vehicle+caeru'.ein
Diazepam+saline
Diazepam+caerulein
10±2
53±29
16±3
41±15
The last treatment was given 72h prior to the experiment Saline and caerulein were
administered 15 min prior to experiment. * - p < 0.05 significantly different from
vehicle+saline; **- p < 0.01 significantly different from vehicle+saline, Duncan's test
following significant ANOVA.
Table 2
Effect of long-term treatment with diazepam on motor depressant effect of caerulein (15
Hg/kg) in mice
Number o f motor activity counts
Long-term treatment+
drug
Vehicle + saline
Vehicle + caerulein
during
15 min
%
30min
184±17
100
325±39
100
115±19*
63
198±35*
61
%
Diazepam + saline
177±19
100
263±22
100
Diazepam + caerulein
116±16**
66
214dt32
81
The experiment was performed 48 hours after the last injection of diazepam. Caerulein (15
pg/kg) was injected immediately before the experim ent The number of mice in each group
was 10-12. * - p < 0.05 (Student's t-test, in comparison with vehicle+saline treated group); ** p < 0.05 (Student's t-test, as compared with diazepam+saline).
Table 3
Effect of caerulein (40 ng/kg) on foot-shock induced aggressiveness after long-term treatment
with diazepam.
Number of aggressive contacts
Long-term treatment +
during
2 min
drug
Vehicle + saline
14±1.0
Vehicle + caerulein
9±1.6
Diazepam + saline
13±2.5
Diazepam + caerulein
28+3.2* **
The experiment was performed 48 hours after the last
Caerulein (40 ng/kg) was injected 20
each group
injection of diazepam.
min before the experiment. The number of mice in
was 10-12. * - p < 0.002 (Newman-Keuls test following significant ANOVA
as compared with diazepam + saline).** - p < 0.0001 (Newman-Keuls test following
significant
ANOVA, in comparison with vehicle + caerulein).
Table 4
Anticonvulsant effect of caerulein (125 |ig/kg) against picrotoxin (10 mg/kg) induced seizures
after repeated treatment with diazepam in mice.
Latency
Long-term treatment+
Clonic
Tonic
drug
seizures
seizures
(sec.)
(min.)
Death
Number of
(min.)
vived seizures
mice sur­
Vehicle + picrotoxin
442±25
16.1+1.5
17.4±1.7
0/9
Vehicle + caerulein +
775±141*
23.0±2.0*
24.3±2.5*
5/9
Diazepam + picrotoxin
615± 151
21.0±2.1
2L2±2.0
1/10
Diazepam + caerulein +
564±61
19.5±2.6
19.8±2.5
1/10
picrotoxin
picrotoxin
The experiment was performed 48 hours after the last injection of diazepam. Caerulein (125
pg/kg) was injected 10
min prior to picrotoxin (10 mg/kg). * - p< 0.05 (Newman-Keuls test
following significant ANOVA, as compared
with vehicle + picrotoxin).
Table 5
3H-CCK-8 binding with rat brain homogenates after repeated treatment and after withdrawal
of diazepam (5 mg/kg per day
i.p. for two weeks) or vehicle.
Repeated treatment
Brain region/group
Withdrawal
K/d
B/max
K/d
B/max
VEHICLE
0.27±0.01
23.4±0.7
0.19±0.02
20.6±1.3
DIAZEPAM
0.22±0.03
24.2±1.3
0.22±0.04
27.2±2.1*
VEHICLE
0.29±0.04
30.4±2.4
0.27±0.03
31.5±1.7
DIAZEPAM
0.33±0.04
47.0±4.2*
0.39±0.07
38.7±3.4
VEHICLE
0.46±0.09
9.5±1.5
0.66±0.16
10.5±1.7
DIAZEPAM
0.31±0.14
9.8±2.6
0.70±0.12
15.6±1.9*
Frontal cortex
Piriform cortex
Hippocampus
Rats were decapitated 30 min or 24 h after last pretreatment injection respectively. Results
are from pooled tissue of 5 animals. B/max expressed as fmol/mg protein; K/d expressed as
nM. * - p < 0.05, Student's t-test, as compared to corresponding vehicle group.
DISCUSSION
The present results show the significance o f CCK-8 receptors in the development of withdrawal
signs after long-term diazepam treatment. All the studied behavioural effects o f CCK-8 agonist
caerulein are changed after withdrawal o f long-term diazepam treatment. The significantly suppressed
exploratory activity o f mice in elevated plus-maze is reflecting obvious 3nxiety in these animals after
benzodiazepine withdrawal. It seems to be the main reason why diazepam pretreated mice did not
react to anxiogenic-like effect of caerulein in elevated pius-maze. The reduction o f sedative effect of
caerulein is probably related to decreased basal motor activity of mice after diazepam withdrawal
The mild antiaggressive effect of caerulein was replaced by proaggressive action after 14 days
diazepam medication The anticonvulsant action of caerulein against picrotoxin-induced seizures was
reversed by repeated diazepam pretreatments t o two weeks These results suggest that subsensitivity
is developing at CCK-8 receptors after withdrawal of long-ierm diazepam treatment. This opinion is
supported by the study of Bouthillier and De Montigny (1988). They have shown that 14-day
treatment with either diazepam or flurazepam reduced the responsiveness of rat dorsal hippocampus
pyramidal neurons to CCK-8.
However, according to ^HpCCK-8 binding studies the number of
CCK-8 receptors is elevated in different structures of rat forebrain after 14-day treatment with
benzodiazepine anxiolytic. Probably two opposite processes are taking place at CCK-8 receptors
during repeated benzodiazepine treatment. The sensitivity of one subtype of CCK-8 receptors is
reduced, whereas the other subtype is becoming more sensitive to CCK-8 after diazepam withdrawal.
The existence of different subtypes of CCK-8 receptors is described in radioligand (Moran et al.,
1986, Wennogle et aL, 1988) and electrophysiological experiments (Mac Vicar et al., 1987). Mac
Vicar et al. (1987)
have shown that low concentrations o f CCK-8 are inhibiting the activity of
hippocampal pyramidal neurons, whereas only very high concentrations of CCK-8 are causing the
excitation of these
cells (Dodd, Kelly, 1981) From radioligand binding studies the existence of
CCK-A ("visceral") and CCK-B ("brain'1) receptors is described in different brain regions (Moran et
al., 1986; Hill et al., 1987; Barrett et al., 1989). But the relation of CCK-A and CCK-B receptors to
subtypes o f CCK-8 receptors, in which long-term diazepam treatment is evoking respectively sub- and
hypersensitivity, is still unclear.
In conclusion, the present study is supporting the idea of Bouthillier and De Montigny (1988) that
long-term diazepam treatment is affecting the sensitivity of CCK-8 receptors. But differently from
this study it is possible that repeated treatment with diazepam is causing the opposite changes at
different subtypes o f CCK-8 receptors. Two-weeks administration o f diazepam is reducing the
sensitivity of one subtype, but increasing the affinity of others to CCK-8. It is probable that described
changes at CCK-8 receptors are related to increased anxiety after benzodiazepine withdrawal.
20
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20*
In: Psychopharmacology, 1991,105: 393-399
DIFFERENTIAL INVOLVEMENT OF CCK-A AND CCK-B RECEPTORS
IN THE REGULATION OF LOCOMOTOR ACTIVITY IN THE MOUSE
Eero Vasar*, Jaauus Harro, Aavo Lang, Anu Põld,
and Andres Soosaar
Psychopharmacology Lab, Institute o f General
and M olecular Pathology, Tartu University,
34 Veski Street, 202400 TARTU, Estonia.
Telefax: (USSR code) 01434 30365
* - To whom all correspondence should be addressed
ABSTRACT
The influence o f the CCK-A antagonist devazepide and the CCK-B/gastrin antagonist L365,260 on the locomotor activity o f m ice was studied. Devazepide and L -365,260 had opposite
effects on spontaneous locomotor activity, and on caerulein- and apomorphine-induced
hypomotility in the mouse. Devazepide in high doses (0.1-1
mg/kg IP) reduced
spontaneous
motor activity, whereas L-365,260 at a high dose (1 mg/kg IP) increased the activity o f m ice.
Devazepide (0.1-10
ng/kg) moderately antagonized the sedative effect o f apomorphine (0.1
mg/kg SC) and caerulein
(25 |ig/kg SC), whereas L-365,260 (1-10 pg/kg) significantly
potentiated the actions o f dopamine and CCK agonists. Concomitant administration
of
caerulein (15 ng/kg SC) and apomorphine (0.1 mg/kg SC) caused an almost com plete loss of
locomotor activity in the mouse. Devazepide and L-365,260 (0.1-10 ng/kg) were com pletely
ineffective against caerulein-induced potentiation of apomorphine's hypomotility. Devazepide in
high doses (0.1-1 m g/kg), reducing the spontaneous motor activity o f m ice, counteracted the
motor excitation induced by d-amphetamine (5 mg/kg IP). The CCK agonist caerulein (100
pg/kg SC) had a similar antiamphetamine effect. Devazepide (1-100 ng/kg) and L -365,260 (1
pg/kg) reversed com pletely the antiamphetamine effect of caerulein
The results o f present study reflect apparently distinct role o f CCK-A and CCK-B receptors in
the regulation o f motor activity. The opposite effect o f devazepide and L -365,260 on caeruleinand apomorphine-induced hypolocom otion is probably related to the antagonistic role o f CCK-A
and CCK-B receptor subtypes in the regulation o f m esencephalic dopaminergic neurons. Ihe
antiamphetamine effect o f caerulein is possibly linked to the stimulation o f CCK-A receptors in
the mouse brain, whereas the blockade o f both subtypes o f the CCK-8 receptor is involved in the
antiamphetamine effect ol devazepide.
Key words: Caerulein - CCK-A receptors - CCK-B receptors - Devazepide - L -365,260 -
Locomotor activity - Apomorphine -Amphetamine
INTRODUCTION
Dopamine coexists with cholecystokinin octapeptide (CCK-8) in som e m esencephalic
neurons, innervating m esolim bic and cortical regions (H ökfelt et al.,
1980). M esolim bic
dopamine is known to have a significant role in the regulation o f motor activity in rodents
(Bradbury et al., 1983; Costall et al., 1985). System ic treatment with CCK agonists (CCK-8 and
caerulein) in low doses significantly suppresses locom otor activity in rodents (Zetler, 1985) and
in higher doses the compounds are able to block stereotyped behaviour and hyperlocom otion
induced by dopamine agonists (Zetler, 1985; Matsubara and Matsushita, 1986; Vasar et al.,
1988). It has been suggested that several behavioural effects o f CCK-8 and caerulein are
generated through
peripheral m echanism s (M orley,
1987). It is thought that the motor
depressant effect o f CCK-8 and the suppression o f dopam inergic activity by large d oses o f CCK
agonists are peripheral origin since they could be abolished by abdominal vagotom y in rats
(Crawley and Kiss, 1985; Hamamura et al., 1989). The highly selective antagonist at peripheral
CCK (CCK A subtype) receptors devazepide com pletely reversed the motor depression induced
by CCK-8 in m ice (Khosla and Crawley, 1988) and in rats (Soar et al., 1989). N evertheless, not
all authors have been able to reproduce the finding that vagotom y can reverse the behavioural
effects o f CCK agonists in rodents. Moroji and Hagino (1987) have demonstrated that bilateral
subdiaphragmatic
vagotomy
does
not
prevent
the
behavioural
effects
of
system ically
administered caerulein in m ice. The suppression o f electrical self-stimulation by caerulein is
com pletely insensitive to vagotom y in rats (D e W itte et al., 1986). Altar and Boyar (1989) have
shown that peripherally injected CCK-8 interacts through CCK-B receptors
(brain or central
subtype) with central dopaminergic mechanisms. R ecently two different subtypes o f the CCK
receptor (CCK-A and CCK-B) have been shown to occur in the brain o f rodents (Moran et al.,
1986; Dourish and Hill, 1987). The C CK -В subtype is ubiquitous in the brain, whereas CCK -A
receptors were shown to be localized in certain discrete regions o f brain, including the area
postrema, nucleus o f the soli<ary tract and the interpeduncular nucleus (Moran et al., 1986; Hill
et al., 1987). However, recent behavioural, electrophysiological and hom ogenate radioligand
binding studies (Crawley et al., 1985, Rovati, 1988; Barrett et al., 1989; Gerhardt et al., 1989;
Vickroy and Bianchi, 1989) show CCK-A receptors to have a more widespread distribution in
the mammalian brain than suggested by above CCK autoradiographic studies.
The aim o f present study was to analyze further the role o f CCK-A and CCK-B receptors in
the regulation o f motor activity o f mice. Therefore, two highly selective CCK antagonists
deva/epide (CCK-A
antagonist)
(Chang
and Lotti, 1986) and L -365,260 (CCK-B/gastrin
antagonist) (Lotti and Chang, 1989) were used to exam ine the role o f CCK receptor subtypes in
the regulation o f motor activity and in the action o f peripherally injected caerulein, an agonist at
( ( К receptors. 1 he action ot deva/epide and L-365,260 was
studied on spontaneous motor
activity, apomorphine-induced hypolocomotion and amphetamine-induced hyperlocomotion, and
on the behavioural effects of caerulein (caerulein-induced hypolocomotion, potentiation of
apomorphine-induced hypomotility by caerulein, antiamphetamine effect of caerulein) in mice.
METHODS
Animals. Male albino mice, weighing 20-25 g, were used throughout the study. Mice were
maintained at 20±VC and on 12- hr light, between 9 a.m. and 9 p.m., with food and water ad lib.
All the experiments were performed between 3 and 9 p.m.
Procedure. Spontaneous locomotor activity and hypolocomotion induced by apomorphine and
caerulein were studied in an open-field Animals were placed singly into the centre of the openfield area (30x30x18 cm, divided by lines into 16 equal squares) and observed during 3 min. The
number of line crossings, rearings and head-dippings into holes was counted. Apomorphine (a
dopamine agonist, 0.1 mg/kg ) and caerulein (a potent CCK-8 agonist, 15 and 25 Mg/kg) were
given subcutaneously 15 min before the experiment. CCK antagonists (devazepide and L365,260)
were
administered
intraperitoneally
30
min
prior
to
open-field
test.
Amphetamine-induced hyperlocomotion and antiamphetamine effect of caerulein were measured
in individual photocell cages. The cage for registration of mote» activity was a cylinder with an
inner diameter 40 cm and 2 photocells (located in walls) for detection of motor activity. Motor
activity was counted between 15 and 45 min after intraperitoneal administration of damphetamine (an indirect dopamine agonist, 5 mg/kg). CCK antagonists were given
intraperitoneally 15 min before the injection of d-ampbetamine. Caerulein (100 Mg/kg) was given
subcutaneously 5 min after the administration of amphetamine.
Drugs. The following drugs were used in the present
study: caerulein (Bachem), d-
amphetamine (Sigma), apomorphine (Sigma), devazepide and L-365,260 (M erck Sharp &
Dohme). Caerulein, d-amphetamine and apomorphine were prepared in saline. Some drops of
0.001 N H O was added for stabilizing the injection solution of apomorphine. Devazepide (1methyl- 3-(2- indoloyl)amino-5-phenyl-3H-l,4-benzodiazepin-2-one) and L-365,260 (3R(+)-N(2,3-dihydro-l-methyl-2-oxo-5-phenyl-lH-
l,4-beazodiazepin-3-yl)-N'-(3methyl-pheny()urea)
were made soluble in saline by adding 1-2 drops of Tween-85. The same vehicle, 1-2 drops of
Tween-85 in saline, was the control injection for CCK antagonists. Each treatment was given in
a volume of 0.1 ml/10 g body weight.
Statistical analysis. Results are expressed in the tables and figures as means ± S.E.M. The
behavioural data were analyzed using one-way analysis of variance (ANOVA). Post hoc
comparisons between individual groups were made by using Newman-Keuls test.
RESULTS
Io tbe behavioural studies CCK antagonists (devazepide and L -365,260) in low doses failed to
affect the locom otor activity o f m ice in an open-field (figure 1), only in high d oses were they
able to change the behaviour o f animals.
D evazepide (0.1-1 m g/kg) decreased the number o f
line crossings in an open-field [F(5,54)=2.88, p<0.05] (figure 1), whereas L -365,260 (1 m g/kg)
had the opposite effect [F(5,54)=2.52, p<0.05) The system ic administration o f caerulein in a
moderate dose (25 ng/kg) reduced the number o f line crossings and head-dips in the open-field
test (table
1). The pretreatment o f anim als with devazepide (0.1-10 ng/kg) only partially
antagonized the effect o f the CCK agonist, particularly on head dips. However, a high dose o f
devazepide (100 ng/kg) reduced the spontaneous locom otor activity o f animals and enhanced the
eflect o f caerulein [F(5,54>= 2.62, p<0.05 for crossings; F(5,54)=4.08, p <0.005 lor head-dips].
L -365,260 (0.1-1000 ng/kg) enhanced the sedative effect o f caerulein [F(6,63)= 3.86, p< 0.01 for
crossings; F(6,63)= 3.72, p<0.01 for rears; F(6,63)= 6.86. pcO.OOOl for head-dips] (table 1). The
dopamine agonist apomorphine in low dose (0.1 m g/kg) reduced the motor activity o f m ice
(table 2). L -365,260 (1-10 ng/kg) significantly enhanced the sedative effect o f apomorphine in
the mouse
{F(6,63)=4.06,
p<0.005
for crossed lines; F(6,63)= 2.36,
p<0 05
for rears;
F (6.63)= 8.15, pcO.OOOOl for head-dips]. Sm all d oses o f devazepide (1-10 ng/kg) only partially
attenuated the effect o f apomorphine, whereas high d oses (100 and 1000 pg/kg) enhanced the
effect o f the dopamine
for rears]
(table
2).
agonist [F(5,54)=4.68, pcO.OOl for line crossings; F(5,54)= 2.83. p<0.05
Pretreatment
apomorphine-induced hypolocom otion
with
caem lein
in the m ouse
(15
ng/kg)
significantly
[F(3,36)=38.4
potentiated
pcO.OOOOOl
for
line
crossings, F(3,36)= 20.7, pcO.OOOOl for rears, F (3,367)= 5 01, p<0.01 for head-dips] (figure 2).
The coadministration o f apomorphine and caem lein caused nearly com plete loss o f motor
activity in
m ice. Several animals lay m otionless in the centre o f open-field area. Neither
devazepide, nor L -365,260 could antagonize the effect o f concom itant treatment with
apomorphine and caem lein (data not shown)
Ал indirect dopamine agonist d-amphetamine (5 m g/kg) caused a three fold increase in the
number o f motor activity counts (figure 3). L -365,260 had no effect on d-amphetamine induced
hyperactivity, whereas devazepide in high dose (1 m g/kg) suppressed spontaneous motor activity
and com pletely antagonized the motor
stim ulation induced by d-amphetamine [F(S,86)=3.1,
p<0.005 for 30 min period] Caerulein (100 ng/kg) also potently reversed the motor excitation
induced by d- amphetamine (figure 4). The pretreatment o f m ice with devazepide over a w ide
dose range (1-100 ng/kg) com pletely blocked the antiamphetamine effect o f caerulein
[F(7,104)=9.56, pcO.OOOOOl for 30 m in period]. The administration o f L -365,260 at low dose
(1 ng/kg) also counteracted the antiamphetamine effect o f CCK agonist PR 7,104)=4.48,
p<0 0001 for 30 min period].
LEGENDS TO THE FIGURES
Figure 1. THE EFFECT OF CCK-8 ANTAGONISTS ON THE SPONTANEOUS MOTOR
ACTIVITY OF MICE IN AN OPEN-FIELD. L-365,260 (0.0001-1 mg/kg, i.p.) and devazepide
(0.0001-1 mg/kg, i.p ) were administered 30 min before the experiment. The number of crossed
lines during 3 min is presented in the figure Each bar represents the mean ± S.E.M. for 10
animals. Data subjected to one-way analysis of variance and Newman-Keuls test. * - p<0.05
(significantly different from vehicle treated animals).
- Vehicle;
- Devazepide;
- L-
365,260
Figure
2.
THE
EFFECT
OF
CAERULEIN
ON
APOMORPHINE-INDUCED
HYPOLOCOMOTION. Apomorphine (0.1 mg/kg, s.c.) was given 15 min and caerulein (15
ng/kg, i.p.) 10 min prior to the open-field test. The number o f crossings, rears and head-dips
during 3 min is presented here. Each bar represents the mean ± S.E.M. for 10 animals. Data were
subjected to one-way analysis o f variance and follow ed by Newm an-Keuls test.
p<0.01
- V ehicle;
(statistically
evident
difference
from
- Apomorphine (0.1 mg/kg);
vehicle
* - p< 0.05; **
treated
m ice).
- Caerulein (15 ng/kg);
Apomorphine + caerulein
Figure 3. THE INTERACTION OF CCK ANTAGONISTS W ITH AMPHETAMINEINDUCED HYPERLOCOMOTION. CCK antagonists (0.001-1 mg/kg, i.p.) were given 15 min
before d-amphetamine (5 m g/kg, i.p ), whereas d-amphetamine was injected 15 min prior to the
experiment. The locomotor activity o f m ice was measured in the individual cages. The number
o f counts was registered during 30 min. Each bar represents the mean ± S.E.M. for 10 animals.
Data were subjected to one-way analysis o f variance
and Newman-Keuls test. * - p<0.05
(significantly different from vehicle treated group); ** - p<0.01 (if compared to vehicle+damphetamine).
amphelamine;
- V ehicle;
- V ehicle + d-amphetamine;
- Devazepide + d-
- L-365,260 + d-amphetamine
Figure 4 THE INTERACTION OF CCK ANTAGONISTS WITH ANTI AMPHETAMINE
EFFECT OF CAERULEIN. CCK antagonists (0.0001-1 mg/kg, i.p.) were injected 30 min, damphetamine
(5 mg/kg, s.c.) 15 min and caerulein (0.1 mg/kg, s.c.) 10 min before the
experiment. The number o f motor activity counts was registered in the individual cages during
30 min. Each bar represents the mean ± S.E.M. for 10 animals. Data were subjected to one-way
analysis o f variance and followed by
from
Newman-Keuls test. * - p<0.05 (significanUy different
vehicle+saline); ** - p<0.05 (if compared to vehicle + d-amphetamine); *** - p<0.05;
**** - p<0.01 (if compared to d-amphetamine+caerulein).
amphetamine;
amphetamine
- D evazepide + caerulein + d-amphetamine;
- V ehicle;
- V ehicle + d-
- L -365,260 + caerulein + d-
iz
Number o f cro sse d lines
VEHICLE
0.0001
(mg/kg)
0 .0 1
FIGURE
0.001
DOSE
О
O
O
O
O
O
O
O
O
O
O
8 0
70 60 -
C rossings
FIGURE 2
R earings
Head
Ч-
rfNj
О
ö)
го
с
v.
Т>
со
D
с
4 -'
О
и
1800
1440 h
1080 -
720 -
360 Ь
0 .0 0 1
FIGURE 3
0 .0 1
DOSE (mg/kg)
0.1
1
m otor a c tiv ity counts during 3 0 min
-J L
ы
О
0)
о
Nl
ю
о
00
О
00
о
о
T--------------1--------------1--------------г
vehicle
+ caeriiein
0.0001
DOSE
0.001
(mg/kg)
0.01
о
о
о
Table I
The interaction of L-365,260 and devazepide with caeruleininduced hypolocomotion in the mouse.
Line crossings
Drug / dose
Rears
(during
3
Head-dips
min)
Mean values ± S.F.M
29+6.3
22-t2.9
56±5.6*
16±4.3
9±1.4*
Deva/epide 0.1 ng/kg + 54±5.4*
13±4.8
16±3.3#
16+5 7
18±3.4#
19+3.2
14±l.5#
Vehicle
77±6.2
Vehicle +
caerulein 25 jjg/kg
caerulein 25 ng/kg
Deva/epide 1 це/kg +
58±12.4
caerulein 25 ng/kg
Deva/epide 10 ng/kg + 66±7.5
caerulein 25 ng/kg
Deva/epide 100 ng/kg + 38±8.0*
13+^7
10±1.8*
caerulein 25 ng/kg
L-365.260 0.1 ng/kg + 52±6.0*
15±3.6
11±2.2*
caerulein 25 ng/kg
L-365,260 1 ng/kg +
35±6 8*’#
5±1.8*’# 10±1.8*
41±8.5*
8+4.6*
caemlein 25 ng/kg
L-365.260 10 ng/kg +
13±2.5*
caerulein 25 ng/kg
L-365,260 100 ng/kg + 40±10 5*
№±5.2*
8±1 6*
caerulein 25 ng/kg
L-365,260 1000 ng/kg + 56±9 4
14±4 8
14±1.4#
caerulein 25 ng/kg
CCK antagonists were administered 30 min and caerulein 15 min
before the experiment
* - jx 0 05 (Newman-Keuls test alter
significant one-way ANOVA, if compared to vehicle treated mice).
^ - p<0 05 (Newman-Keuls test, in comparison to vehicle +
caerolein treatment)
Table 2
The effect of devazepide and I -365,260 on apomorphinemduced hypolocomotion in micc.
I ine crossings Rears
D rug/dose
(during
Head-dips
3 min)
values -c S H M
Vehicle
60*X X
14.2±3 0
7.4±0.9
Vehicle +
40 ±', V
I0.3±3.0
7 5± 1 2
3X±3 6*
:>.2±2.6
5.2±2.0
аротоф Ьш е 0 I
I -3hS,260 0 I m-/kg +
apomorphine 0 .1 ;ng/kg
I.-3n5.2oO 1 ng/kg f
24±8 5*
5.4±l,6*
2.f>±0.7*-#
.ipoinurphinc 0 1 mg/kg
I -K.S.260 l()ug/kg+
5 .7 ± |У
2 3±0 7*’#
apuinorpiiine 0 I mg/kfi
I .-’ 6S.1N) 1(Юцй/к » +
7'
7.5±2 4
?.8±0.X*’#
I (MO pg/kg -г 36±5 6 *
9.6±2.5
4.0±0.6*
apomorphine 0 I mg/kg
! - '6 S y . i j
apo;n< «pinne 0 1 mg/kg
v.-hicle
7<)ii0.2
22±4,X
7.9±2.6
V'-hidc +
4S±S.6*
11+2.9
5.5±l .8
55±6.6
13±2.6
5.1±1.6
54±5.X
12±2.6
6.1±2.0
ар; |ШОф1г1пе 0.1 in^/kg
Devazepide ‘ j><i/'kg +
upninorphiiic U. 1 mg/kg
D c v a , ■, idt lOpg/ke +
a*4>u!*iT»hine 0.1 nig,-leg
i Vv.i/epuic 100 до/kg
’
1<) o*
6±2 f
2 8±0 9
S+2.3 "
3(>±1.0
л р о т о ф п т с 0.1 mp.kp
D< va/epidc 1000 ug/kg+ i4±7л*
apoiiioipliine ■' S mg/kg
< i ‘K af!*:s
!ii:,i- were given ! 5 mil: pnor to аротоф Ы пе, whereas
•ipoiii.■!'. !. ii-.* v :-s injected !
jv о 05 • Newman- F
ANOV/V n romparis
iur. bc^.ie (tie experiment.
• u*si. !(.!i’ov^ng significant one-way
v;-t,icL- -x.aitd micc): * - p<0.05 (if
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DISCUSSION
In these behavioural studies the CCK-B/gastrin antagonist 1,-365,260 and Ihe CCK-A
antagonist devazepide had opposite effects on spontaneous locomotor activity, and on
apomorphine- and caerulein-mduced hypolocomotion in mice The spontaneous motor activity
was affected only by high doses of CCK antagonists, but the hypolocomotion induced by
caerulein and apomoiphine was changed by low doses of devazepide and L-365,260. Devazepide
(0.1-1 mg/kg) reduced the spontaneous motor activity of mice, whereas L-365,260 (1 mg/kg)
increased this behaviour. It is very puzzling that CCK antagonists affect in a similar way
apomorphine- and caerulein-induced hypolocomotion. Devazepide partially antagonized the
sedative effect of low doses of apomorphine and caerulein. whereas L-365,260 significantly
potentiates the action of caerulein and apomorphine. According to the existing data the motor
suppressant effect of apomorphine and caerulein are thought to lie related to Ihe decreased
activity of dopaminergic cells in the
mesencephalon (Strömbom. 1977; Zetler, 1985). The
behavioural effects of CCK antagonists probably leflect the distinct role of CCK-A and CCK-B
receptors in the regulation of presynaptic dopaminergic activity in the mouse brain. 1Ъе
blockade of CCK-B receptors by L-365,260 decreases the dopaminergic activity, whereas the
interaction of devazepide with CCK-A receptors
increases it in the mouse brain. It seems
probable that die CCK-A receptors at which caerulein and CCK-8 act to reduce locomotor
activity are in the periphery and associated in some way through the vagal afferent pathway with
dopaminergic neurons (Crawley and Schwaber, 1984, Crawley and Kiss, 1985; Homraer et al.,
ll>85). However, it is important to siress that in the present study devazepide, in contrast to the
investigation of Khosia and Crawley (1988), only moderately antagonized the motor depressant
effect of caerulein Hamilton et al. (1990) have shown that devazepide only partially antagonizes
the suppression of self-stimulation induced by caerulein in the rat. All these experiments support
the idea that not only the CCK-A receptor subtype is involved in Mediating the effect of
caerulein. ГЬс concomitant treatment with a low dose of apomorphine ar.d caeruiein causes
nearly complete loss of motor activity in the mice. Several animals lav motionless in the middle
of the open field Deva/epide and L-365.260 in low doses, which do not affect locomotor
activity of animals per se, were completely ineffective against the moior depression induced by
the simultaneous administration of caerulein and apomorphine. According to the studies of
Homrner
et al. (1986) and Crawley (1989) the CCK-8 receptors that mediate the potentiation of
dopamine-mduced hypolocomotion and suppression >ü die electrical activity of dopamine
■icurons in the rat mesencephalon by CCK-8 belong to die CCK-B subtype. Altar and Bov.*;
<1984» have found that the antagonistic effect of centrally oi peripherally administered (X'A-8
.‘gonisi'. [C< К-4. devulfated CCK-8 and CCK-4) on amphetamine evoked dopamine release in
the mouse striatum is also related to the CCK-B receptor subtype. Nevertheless, it is not clear
whether CCK 11 receptors are involved in the potentiation of apomorphine induced
hypolocomotion by caerulein in the mouse and it remains to be elucidated.
The interaction of CCK antagonists with amphetamine-induced hyperlocomotion and the
antiamphetamine effect of caerulein is somewhat different from their action on caerulein and
apomorphine elicited hypolocomotion. It is suggested that the different pharmacology of CCK-8
against dopamine-induced hypolocomotion and hyperlocomotion is related to the involvement of
distinct brain regions in tbe development of two opposite behavioural effects of dopamine in the
rat (Crawley. 1989). The potentiation of dopamine-induced hypolocomotion is linked to the
interaction of CCK-8 with dopamine "autoreceptors” in the ventral tegmental area, whereas the
potentiation of dopamine-induced hyperlocomotion is related to an interaction with post-synaptic
dopaminergic receptors in tbe posteromedial part of the nucleus accumbens (Crawley et al.,
1985; Crawley, 1989). The CCK-B/gastrin antagonist L-365,260 did not significanüy change
amphetamine-induced hyperlocomotion, but paradoxically it reversed (at a low dose) the
antiamphetamine effect of caerulein
Devazepide antagonized tbe antiamphetamine effect of
caerulein at low doses, where it probably interacts selectively with CCK-A receptors. However,
at a high dose (1 mg/kg), which also interacts widi CCK-B receptors (see Dourish et al., 1989;
1990), devazepide per se
reverses the motor excitation induced by d-amphetamine. The
antiamphetamine effect of devazepide is in good agreement with our previous studies in which
the unselective CCK antagonist proglumide (5-15 mg/kg) also blocked the effect of damphetamiiie (Vasar ei al., unpublished data). According to the studies of Moroji and Hagino
(1987) the antiamphetamine effect of caerulein in mice is completely resistant to the vagotomy.
It is worthy to note that nearly 10 times higher doses of caerulein are required for blocking
amphctamine-induced hyperlocomotion in comparison to the sedative effect of caerulein.
Accordingly, it seems very probable that the CCK-A receptors involved in the antiamphetamine
effect of caerulein are distinct from the CCK-A receptors related to caerulein and apomorphineinduced hypolocomotion
Hie above mentioned study (Moroji, Hagino, 1987) suggest the
possibility that these CCK-A receptors arc located in the mouse brain. This idea is supported also
by the study of Hagino et aJ. (1989) in which the intracerebroventricular administration of CCK8 and caerulein, but not desulfated CCK-8 and CCK-4, antagonizes amphetamine induced motor
excitation in the mouse. The recent behavioural, electrophysiological and radioligand binding
studies (Rovati, 1988, Crawley, 1989; Vickroy and Biancbi, 1989, Gerhardt et al., 1989) have
established that there is a wider distribution of CCK-A receptors in the rat brain than was
previously thought (Moran et a l. I98f>; Hill
et al., 1987). The possible mediation of the
antiamphetamine effect ot caerulein through the CCK-A receptors in the mouse brain may
reflect the substantial difference between CCK-A receptors in the mouse and rat brain. Crawley
and colleagues (19XS; Crawley. 1989) have shown that CCK-8 by interacting with CCK-A
receptors laciiitates dopamine-induced hyperlocomotion in the posteromedial part of the nucleus
accumbens ot the rat. The different pharmacology of CCK-A receptors in the mouse and the rat
brain seems to account for the interspecies differences in the behavioural effects of caerulein in
lu n y x a n Journal of Р !ш пт иЫ т . 202 (1991) 385-390
|9 9 | Mlscvier Science Publishers B.V. All rights reserved 0014-2999/91/503.50
ADONIS 0014294991006298
385
E JP 52043
Changes in motor activity and forebrain [propionyl-'H]propionylated-CCK-8
binding in mice after repeated administration of drugs affecting
cholecystokinin receptors
Eero Vasar
John D. Stephenson and Brian S. Meldrum
1 Psychophurmacology Laboratory, Tartu University, Tartu, Estonia and Departments o f Neuroscience and Neurofogy, Institute o f Psychiatry .
London SE5 8AF, U.K.
Received 25 June 1991. accepted 9 July 1991
The cffects of acute or repeated treatment of male albino BKW mice with caerulein, a cholecystokinin octapeptide (CCK-8)
agonist, and wilh devazepide (MK-329) and L-365,260, antagonists at C CKA ( peripheral’) and C CKB (‘central') receptors
respectively, on motor activity and [propionyl- 'H]propionylated-CCK 8 ([’H]pCCK-8) binding were studied. Acute treatm ent
with a large dose of caerulein (100 iig/V .g s.c.) suppressed motor activity (line crossings and rearings) whereas devazepide (2
m g/kg i.p.) had the opposite action. L-365,260 (2 m g /k g i.p.) increased only the number of rearings. Tolerance developed to the
locomotor cffects of caerulein and devazepide when these same doses were administered once daily (caerulein) or twice daily
(devazepide) for 10 days. Twice daily administration of L-365,260 (2 m g/kg) for 10 days did not significantly alter the locomotor
activity of mice. The sedative effect of caerulein (20 д g / kg s.c.) was markedly reduced in mice receiving repeated injections of
either a larger amount of caerulein (100 Mg/kg) or devazepide but not after L-365.260. The stimulant effect of (+ )-amphetamine
(2 m g/kg s.c.) on motor activity was increased by subchronic administration of either devazepide iir caerulein, but not by
L-365.260. All three compounds (caerulein. devazepide and L-365,260) increased the number of ([ 'HlpCCK-S binding site;, in
mouse forebrain but the increase was only significant after L-365,260. The effects of long-term treatment with caerulein are
probably related to the stimulation of CCKA receptors, whereas the paradoxically similar action of devazepide may be linked to
the blockade of both subtypes of the CCK-8 receptor. The results of the present study can therefore be explained by postulating
different roles for ССКЛ and C CK B receptors in the regulation of motor activity, the C CKA receptors being the more important.
Caerulein; Devazepide; L-365,260; CCK receptors; Amphetamine; Motor activity
1. Introduction
D opam ine coexists w ith cholecystokinin o cta p ep tid e
(C C K -8) in th e n e u ro n e s o f th e v en tra l teg m en ta l area,
innervating m esolim bic stru c tu re s o f ra ts (H okfelt et
al., 198Ш. M esolim bic d o p am in e is know n to have a
crucial role in the re g u la tio n o f m o to r activity in ro ­
d e n ts (B radbury et al., 1983; C ostall e t al.. 1485).
S ystem ic tre a tm e n t w ith low doses of c ith e r C C K -8 or
ca eru le in significantly sup p re sse d locom otor activity o f
ro d e n ts (Z e tle r, 1985) and at hig h er doses hyperlocom otion induced by d o p am in e agonists w as also blocked
(Z e tle r, 1985; M atsu b ara and M atsushita, 1986; V a sar
el al.. I‘>88a). A ccording to som e re p o rts, p erip h e ra l
CC K -8 re cep to rs linked w ith th e vagus nerve have
( \> m -4poinlcinv in: H.S. M cU huni. IK 'p aitm c n t o f N etm »lop\. lu sh
tu te u l l \ u h u l r > . l) e C resp ig n y P a rk . D en m ark Mill. 1 o m lo n S I *5
SAI II k I d . 14 71.7113 >411. c \ l »398.
significance fo r m o to r d ep ressan t an d a n tid o p a m in e rgic activity o f C C K -8 (C raw ley an d S ch w a b cr. 1983;
K aw asaki e t ul.. 1983: H a m a m u ra et al.. 1989). H ow ­
ever, A lta r a n d B oyar (1989) show ed th at p erip h e ra lly
injected C C K -8 affec ted ce n tra l d o p am in erg ic m e c h a ­
nism s directly. T w o d iffe ren t su b ty p es o f th e C CK -8
re c e p to r have now b ee n d esc rib ed in th e b ra in s o f
ro d e n ts (M o ran e t al.. 1986; D ourish a n d H ill, 1987).
С С К Л (‘p e rip h e ra l' type) re c e p to rs w ere show n to be
localized in c e rtain d iscre te re g io n s o f b ra in , including
th e a re a p o strem a . th e nuclcus o f th e solitary tra c t and
th e in te rp e d u n c u la r nuclcus (M o ran e t al., 1986; H ill et
al.. 1987). H ow ever, clcctrophysiological an d hnm o g en a lc ra d io lig an d bin d in g stu d ie s (B a rre n e t at..
1989; t ic r lia r d t et al.. 1989; Vickroy an d liia n ch i. 198°)
have show n th e C C K A rc cep lo rs to be m o re widely
d is trib u te d in th e b ra in th an suggested by th e above
au to ra d io g ra p h ic stu d ie s. T h e aim o f th e p re s e n t study
was lo exam ine th e role o f C C K 4 an d C C k „ re cep to rs
m (lie re.uulation o f m o to r activity ol m ice. M otor
48ft
activity was su p p re sse d by ca eru le in , a C C K -8 agonist,
an d s tim u late d by a m p h e ta m in e , an indirectly acting
d o p am in e agonist. Sensitivity o f th e C C K re cep to rs was
ch a n g ed by 10 days daily tre a tm e n t w ith the highly
selective C C K A a n d C C K B an ta g o n ists, dev a ze p id e
a n d L-365,260 respectively, a n d w ith c a eru le in . T h e
binding o f (p ro p io n y l-1H ]propionylated-C C K -8 < ['H ]
pC C K -8) to fo reb ra in nuclei w as d e te rm in e d co n c u r­
re n tly w ith th e beh a v io u ral studies.
2. M aterials and m ethods
M ale BKW m ice (25-30 g) o b ta in e d from B antin and
K ingm an (H u ll) w ere used th ro u g h o u t th e experim ents.
C a eru lein (100 f ig /k g o n ce daily), dev a ze p id e (2 m g /k g
tw ice daily) an d L-365,260 (2 m g /k g twice daily) w ere
a d m in iste re d for 10 days. T h e effects o f ca eru lc in and
am p h e ta m in e on m o to r activity w ere stu d ie d 24 h a fte r
th e last injection o f C C K -8 an ta g o n ists a n d c a eru le in .
[ ’ H ]pC C K -8 binding e x p e rim en ts w ere c a rrie d o u t 2 h
a fte r th e last injection o f C C K -8 a n ta g o n ists an d 24 h
a fte r th e last injection o f ca eru le in . T h e s h o rte r tim e
interval for th e C C K -8 an ta g o n ists w as selec ted b e­
ca u se [ ’H ]pC C K -8 b inding h ad re tu rn e d to norm al by
24 h a fte r the last o f the twice daily injections o f
devazepide.
2 . 1. Measurement o f motor activity in mice
M o to r activity o f m ice w as q u a n tified in an ‘o p en field ’ (35 x 25 x 20 cm ) divided by lines into six equal
rectangles. T h e to tal n u m b er o f line-crossings an d re a r­
ings w ere c o u n te d for e ith e r 3 o r 5 m in. In th e case o f
re p e a te d tre a tm e n ts , th e m o to r activity o f m icc was
re g iste red 20 m in a fte r th e first and last injection of
ca eru le in , dev a ze p id e , L-365,260 o r vehicle. A fte r KIday tre a tm e n t w ith C C K -8-ergic d ru g s th e effect of
c a eru lc in (20 M g /k g ) an d ( + (a m p h e tam in e (2 m g /k g )
on m o to r activity w as stu d ie d 15 m in a fte r injection of
c a eru lc in a n d 30 m in a fte r a d m in istratio n o f ( + )am p h e ta m in e . V eh ic le-in jec te d c o n tro l anim als were
always p a ire d w ith d ru g -tre a te d anim als to control for
d iu rn al flu ctu a tio n s in m o to r activity. T he re su lts w ere
analyzed using a one-w ay A N O V A follow ed by Newm an-K culs test for significance.
2.2. I 'H lpC ( K K hint/inn experiments
l'H lp C C K -8 binding was m ea su red by th e m ethod
o f P raissm an el al. (1483). Ih e m ice w ere killed by
cervical dislocation p rio r to d e c a p ita tio n a n d the brains
im m ediately rem oved a n d bisecte d across the optic
chiasm a. Ih e s tru c tu re s a n te rio r to this cut (cortical
regions, s tria ta , m esolim bic stru c tu re s , sep tu m , etc.)
w ere used fo r th e binding stu d ie s. T h e bra in m aterial
from th re e micc was p ooled and ho m o g en a te s p re ­
p a re d by h o m ogcnization in 75 volum es o f 50 m M
T ris-H C I (S igm a, pH 7.4 at 2 0 “ C l using a polytron
h o m ogcnizer (S ilverson) for 15 s. T h e ho m o g en a te s
w ere c e n trifu g ed a t 37 000 x g fo r 2(1 m in, re su sp en d ed
in th e sam e volum e o f b u ffer and re cen trifu g ed as
d esc rib ed above. T h e final pellet w as hom ogenized in a
s ta n d a rd incubation buffer: H E P E S (Sigm a, 10 m M ),
N aC l (Sigm a, 130 m M ), KCI (Sigm a, 5 m M ), M gC I,
(Sigm a 5 m M ), sodium E D T A (Sigm a, 1 m M ), bovine
serum album in (Sigm a, 0.4 m g p e r ml, pH 6.7 at
20 °C ). T en m illilitres of incu b atio n b uffer w as ro u ­
tinely used for ea ch 100 mg o f o riginal tissue wet
w eight. T h e binding e x p e rim en ts w ere p erfo rm ed in
polypropylene tu b es (1.5 m l), ea ch co n ta in in g 50 I o f
[ 3H ]pC C K -8 (73.2 C i/m m o l. A m ersh am In tern atio n al
pic, 0.03-2.0 nM ), 50 д1 o f in cu b a tio n b u ffer o r 200 nM
C C K -8 (B achem ) a n d 400 д1 freshly p re p a re d brain
m em branes. T h e b rain m em b ra n es w ere p re in c u b a te d
fo r 25 m in a t 2 3 ° С w ith o r w ithout 200 nM CCK -8,
a fte r which th e ra d io la b elled ligand w as ad d e d and the
s am ples w ere carefully m ixed. T he m em b ra n es w ere
in cu b a te d in th e p re sen ce o f rad io lig an d for 2 h at
2 3 ° С an d incu b atio n w as te rm in a te d by rapid c e n trifu ­
gation in a B eckm an m icrofuge (11 000 x x ) for 3 min
at room te m p e ra tu re . T h e s u p e rn a ta n ts w e re carefully
asp ira ted a n d the p ellets w ashed th re e tim es w ith 250
/il o f ice-cold incubation b u ffer b efo re tra n sfe r to
scintillation vials. A tissue solubilizer (S oluene-350,
C a n b e rra -P a c k a rd ) w as ad d e d (300 p.1) to the vials
w hich w ere th en in cu b a te d at 6 0 ° С fo r 1 h. S cintilla­
tion cocktail (O p tip h ase , H isafe II, LK B -W allac) was
th en ad d e d a n d radioactivity was c o u n le d by liquid
scintillation sp ec tro m etry a fte r a 12 h stabilization p e ­
riod. T he specific binding o f ['H ]p C C K -8 was defined
as th e diffe ren c e b etw een th e deg ree o f binding in the
a b sence and p re sen ce o f 200 nM o f CCK -8. T h e sp e ­
cific binding o f [ 'H ]p C C K -8 was 7()-8(lr > o f to tal b in d ­
ing in the case o f low er ligand co n c en tra tio n s (0.03-0.25
n M ) a n d approxim ately 5 Ш with h ig h er c o n c e n tra ­
tions (above I nM ). S atu ratio n curves w ere analyzed
using a n o n -lin ea r regression p ro g ram (E N Z F IT T E R ,
L c a th erb a rro w , 1487).
2..1. I)rug\
C aeru lein (B ac h cm ) an d ( 4 (-am p h etam in e (Sigm a)
w ere dissolved in saline (0 W r N aCl solution w /’v).
D evazepide (M K -3291, l-m cthyl-3-(2-indolovl)am ino5-p h c n y l-3 H -I.4 -b en z o d ia ze p in -2 -o n e) an d I .-365.260
(3K( t )-N-( 2.3 -d ih y d ro -1-m cthyl-2-oxo-5-phciiyl-1H l,4 -b en z o d iaze p in -3 -y l)-N '-(3-nicth y l-p h en y llu rca were
su sp en d e d in saline with 1-2 d ro p s o f T w cen-80. T h e
sam e vehicle w as used as th e in jeeta te for th e co n tro l
e x p e rim e n ts . D c v a /c p id c anil I.-365.2M I (M e rck
3K7
S h arp & D o h m e) w ere given i p w h e rea s ( + )am p h e ta m in e and caeru lein w ere injected s.c.
S p o n ta n eo u s m otor activity a n d m otor activity in­
duced hy ca eru le in . apo m o rp h in e and ( + (-am p h eta­
m ine in the albino m ouse w ere affected d ifferently by
doses o f devazepide and L-365,260 in the range O.IKHII-I
m g /k g (V asar e t al., 1991). F or the p re sen t, chronic
study, larger doses o f the two an tag onists w ere used (2
m g /k g ) to en su re com plete blockade o f C C K A and
C C K B re cep to rs th ro u g h o u t th e interval betw een in­
jections.
IA B I 1 2
T h e effect of c ae ru le in (20 / ig / k g ) on m ouse m otor activity given 24
h a fte r th e last o f a scries o f in jections o f d e vaz epide (2 m g /k g . twice
daily). t.-3h5.2W) (2 m g /k g . Iwice daily) and c ae ru le in (100 ^ g / k g ,
once daily). R earin g s a n d line-crossings w ere rec o rd e d over a 3-min
p e rio d 15 min a fte r c ae ru le in injection.
Treatment
n
Rearings
Line-crossings
Vehicle + saline
Vehicle + caerulein
Devazepide + caerulein
L-365.260 + caerulein
Caerulein + caerulein
12
10
10
10
10
41 ±3.3
24 ±5.0
35 ±5.5
28 ±6.4
53 ±3.8 h
132 ±
85±
104±
84 ±
125 ±
7.8
11.2 е
11.1 a
11.7 е
6.0 a
1 P < 0.05: ь P < 0.01 (in co m p arison w ith vehicle + c ae ru le in group);
L P < 0.01 (in c o m p ariso n w ith vehicle + saline g roup) (N ew m anK euls analysis follow ing significant A N O V A ). n = nu m b e r o f m ice in
th e group.
3. Results
A single injection o f a large dose o f caerulein (100
fj-g/kg) significantly sup p re sse d th e m otor activity of
m icc (F 0 .2 4 ) = 5.10, P < 0.05, N ew m an-K euls P < 0.05
for rearings; FC 1,24) = 8.80, P < 0 .0 1 , N ew m an-K euls
P < 0.01 for Iine-crossings, as c o m p a red to vehicle
trea tm e n t; table 1). T h e С С К Л antagonist, devazepide
(2 m g /k g ) increased th e frequencies o f rearings and
line-crossings (F (l,2 4 ) = 7.43, P < 0.05, N ew m an-K euls
P < 0 .0 5 for rearings; F( 1,24) = 6.18, P < 0.05, N ew­
m an-K euls P < 0.05 for line-crossings), w hereas the
C C K B an ta g o n ist, L-365,260 (2 m g /k g ) only increased
th e nu m b er o f rearings (F (l,2 4 ) = 5.36, P < 0.05, Newm an-K euls P < 0.05). T o leran ce developed to the loco­
m otor effects o f ca eru le in and th e antagonists a fte r
th e ir re p e a te d adm inistration (table 1). H ow ever to le r­
ance to devazepide was not seen in all m ice, about
20% becom ing aggressive w ith re p eate d trea tm e n t.
T h ese m ice atte m p te d to bite th e backs o f o th e r m ice
in th e cage.
A dm inistration o f a m o d era te dose o f ca eru le in (20
/u g /k g ) 24 h a fte r th e last injection o f C CK -8-ergic
drugs re duced m otor activity only in control anim als
p re tre a te d w ith e ith e r vehicle (F( 1,20) = 9.08, P < 0.01,
N ew m an-K euls ns. for rearings, F( 1,20) — 13.02, P <
0,01, N ew m an-K euls P < 0.01 for line-crossings, as
co m p a red to vehicle + saline tre a tm e n t) o r w ith L365,260 (F( 1,20) = 4.59, P < 0.05, N ew m an-K euls ns.
for rearings; F ( l,2 0 ) = 12.86, P < 0 .0 1 , N ew m an-K euls
P < 0.01 for line-crossings; tab le 2). T h e sedative effect
o f ca eru le in (20 /x g /k g ) was significantly re d u ced in
mice p re tre a te d w ith d ev azepide ( F( 1,20) = 4.50, P <
0.05; N ew m an-K euls ns. for line-crossings co m p a red
w ith vehicle + saline tre a tm e n t; table 2) a n d w as re ­
versed in m ice p re tre a te d w ith a larg e r dose of
ca eru le in , 100 j i g /k g . T h u s, ca eru le in , 20 ^ g / k g , in­
crease d m otor activity bo th w hen co m p a red w ith a
g ro u p receiving c a eru le in a fte r ch ro n ic ad m in istratio n
o f vehicle ( F ( I ,I 8 ) = 19.88, P < 0.001, N ew m an-K euls
P c O .O l for rearings: F (l,1 8 ) = 8.57, P < 0 .0 1 . Newm an-K euls P < 0.05 for line-crossings, as c o m p a red to
vehicle + ca eru le in tre a tm e n t) a n d w hen co m p a red
w ith a g roup receiving saline a fte r chronic ad m in is tra ­
tion o f vehicle (F( 1,20) = 4.59, P < 0.05, N ew m anK euls ns. for rearings, as c o m p a red to vehicle + saline
group; table 2).
A d m in istratio n o f ( + (-am p h etam in e p ro d u c ed a
54 '/r increase in th e nu m b er o f line-crossings (F (l,2 0 )
= 6.04, P < 0.05, N ew m an-K euls P < 0,05 to r linecrossings, as co m p a red to vehicle + saline tre a tm e n t.
T A R I.E I
T he effect
rep e n te d trea tm e n t (fo r 10 days) w ith cae ru le in (1(H)
MK/'kg. once daily), d ev azep id e (2 m g /k g . twice daily) a n d l.-3(i5.2Ml
12 m g /k g . twice daily) on m o to r activity o f mice. T h e n u m b er of
rearings a nd line-crossings w as c o u n ted over a 3-m in p e rio d 21) mm
a fte r the first and last injection o f d ru g o r vehicle.
1 rea tm e n t
n
R earin g s
Firsl
V ehicle
C aeru le in
D evazepide
L-3(,5.2NI
14
12
12
12
lin e -cro ssin g s
Last
First
l.ast
4 1 + .1 3
37 + 4.2 1 3 2 + 7.X
П ч+ТЙ
3 0 + 3.4 '4 0 + 4 .2
KO + fc.K1'
44 + 11 7
54 + 3 .3 '1 4 6 + 5 .7 I?» + 7.4 ■■ 1 1 0 + IH .h
5 3 + 3 .0 - '
4 4 + 4.4 1 3 1 + 7 .4
117 1 10.0
" P <0.1)5, h 1* • 1)111 co m p ared with v eh icle -tre a ted anim als. In
N ew m an-K euls analysis follow ing significant A N O V A ). n n u m b e r
of anim als in the g io u p
IM il 1 . 3
T h e effec t o f ( + 1 a m p h eta m in e (2 m g /k g ) on m ouse m otor activity
given 24 h a lte r the last of a scries o f injections o f cae ru le in .
d e v az ep id e and I -3(,5.2M). R earings and line-crossings w ere m e a ­
su red o v e r a 5-111(11 p erio d 30 min a fte r am p h eta m in e .
T rea tm en t
V ehicle * Šaline
V eh icle t a m p h i'tan u m
D eva/ipH lii* t ain p h cla m ine
C a eru le in ‘ a m p h ela m ine
L -365.2M 1am p h ela m me
i:
10
10
10
10
R earings
L ine-crossings
58 + 5
77+ 14
I I I + 14 h
110+ X 1,
КЧ f 17 -
184 f
242+
372 t
365+
2‘>N+
12
26 1
36 "
3 1 11
24 1
1 Г 11.05; h 1* • 0.IH (co m p a red wiith vehicle - ••aline g roup. New
m au-K culs anal\4is lo Mowing sigm l icant A N O V Л ) и
inim tH i ol
an im als in Hie group.
1.4N
1/Mil 1 1
1In- i-Hi t i ol Iic|H-.ilt-i! tic a lin c u t w ilh e .itiiili'in . ile v a/ep iide a iitl
! Ib V ’MI on 1 ' 11К I K s b in d in g 1in m ouse lo itltia in П н­ stu d ies
m i r iii mk* .» It a t lei Ihe Iasi injcrliiин 1 III days lie .it Dll' n l ) ol с е к к
ailta g o n isls and1 J i l t alle« Ih r Iasi a<Innnisiialion ol eae in le in I h e
values iiic llu- 1i.e an s 1 S I- M o l six; n u le p em len l stu d ies.
T re a tm e n t
В.....
( p in o l/V Ossllt •)
V ehicle
C a eru le in
D ev a /e pule
1 -.4i5.2f)0
S.(i7 ♦ 0.П0
7.AI +0.42
7.41 +0.4.1
I0.3N + 0.4S
k,i
(i)M>
O.hK t 0 \h
0.53 + o.o: *
0.47 4 0.02 **
0.45 i 0.04
1 V 0.05; b 1’ < 0.01 (statistically significant A N O V A ) c P < 0 ,0 0 1
tin c om p ariso n w ilh v e h ic le -tre a ted an im als. S tu d e n t's t-test).
table 3) and this effect was not altered by 10 days
pretreatm ent with L-365,260. Injection o f ( + )amphetamine in animals pretreated with devazepide
increased motor activity more than in animals pretreated with vehicle (F(l,18) = 2.83, P = 0.10 for rear­
ings; F( 1.18) = 3.60, P = 0.075 for line-crossings). This
increase was more marked when compared with the
saline + vehicle group (1X1.20) = 13.58, P < 0.01, New­
man-Keuls P < 0.01 lor rearings; F (l,2 0 )= 18.63. P <
0.001, Newman-Keuls P < 0.01 for line-crossings). The
effects of repeated treatm ent with caerulein were simi­
lar to those of repeated treatm ent with devazepide
inasmuch as ( + (-amphetamine produced a motor stim­
ulation which was more pronounced when compared
with the vehicle + saline-treated animals (F(1.20) =
17.06, P <0.001. Newman-Keuls P < 0.01 for rearings;
R 1 .2 0 )» 1910. P < 0.001, Newman-Keuls P < 0.01 for
linc-crossings) than with the vehicle + amphetamine
treated animals (F(I,I8) 3.60. P = 0.075 for rearings;
F(I.I8) = 2.95. P - 0.10 lor line-crossings. table 3).
Repeated treatm ent with each of the three CCK-8ergie compounds altered ['H lpCCK -8 binding in mice
lorchrain (tabic 4). Treatment with devazepide and
caerulein slightly increased the affinity of [ 'H]pCCK-8
binding sites (F d .lO )-7 .6 ti. P < 0.05. for caerulein;
F(1.I0 )= 12.05. P < 0 .0 l. for devazepide) whereas af­
ter L-365.260 there was no significant change. All the
chronic drug treatm ents tended to increase the number
of [ ’HJpCCK-X binding sites, but only the 83', in­
crease after I -365.260 was significant (table 4).
4. D iscussion
The p re sen t s tu d y d e m o n s lia tc ' significant d iffe r­
en c es b etw e en t h e effects o f s c lc d iv c ( V K л an d C< k „
an ta g o n ists o n th e re g u la tio n ol C C K re c e p to r density
suggesting that th e two re c e p to r types differen tly a i l e d
I lie re g u la tio n o f locom nini activity in m ice. The C C K N
re cep to r an ta g o n ist, d e v a /e p id e a d m in iste re d daily lot
III days, increased loconiotoi re sp o n ses in ( i >-
amphetamine but icduccd sensitivity I n eaciulcin with
out significantly affecting tile number of lorchrain ( ( k
receptors. In contrast, repealed injections of the C C k „
antagonist. 1.-365.260 markedly increased receptor
density but did not affect motor responses to caerulein
and < t ( amphetamine. The effects of devazepide were
similar, surprisingly, to those produced by chronic ad­
m inistration of the non-selective CCK agonist,
caerulcin. However the present study used large doses
of devazepide (2 m g/k g twice daily) and it cannot be
assumed that its effects were solely due to prolonged
CCK л icceptor blockade. In rats for example, large
doses of devazepide (I m g/kg) enhanced morphine
analgesia in the paw pressure and tail flick tests
(Dourish et al., 1988; O'Neill et al., 1989) effects which
are mediated by CC K B receptors (Dourish et al., 1990).
Administration of a single high dose of caerulein
(100 M g/kg) suppressed the motor activity of mice,
whereas devazepide (2 m g/kg) had the opposite effect.
Tolerance to the motor effects of both compounds
developed after their repeated administration, al­
though approximately 20% of animals receiving de­
vazepide did not develop tolerance, but became aggres­
sive with repeated injections. These mice frequently
attacked the other animals in the cage usually biting
the victim’s back.
The sedative effect of a m oderate dose of the CCK-8
agonist, caerulein (20 щ / kg) was prevented by chronic
caerulein pretreatm ent suggesting that the CCK-8 re­
ceptors became desensitized to caerulein although this
was not reflected in changes to the binding of
[ ’H]pCCK-8. The sedative effect of caerulein was also
reduced after repeated injections of the CC K A antago­
nist, devazepide. This was unexpected, because chronic
administration ot an antagonist usually leads to recep­
tor up-rcgulation and an enhanced sensitivity to ago­
nists. There arc no obvious reasons why chronic de­
vazepide treatm ent did not render the animals more
sensitive to caerulein but the sedative effect of caerulcin
was also reduced after administration of proglumide
(10 m g /k g twice daily), a non-selective antagonist of
CCK-8 (Vasar et al.. unpublished data). Devazepide
has previously been reported to antagonize the loco­
motor effects produced by CCK-8 (Khosla and Craw­
ley. 1988). One explanation for these findings is antag­
onism of Ixith receptor sub-types by the dose-schedulc
of deva/epide used in this study (see Dourish et al.,
1490). и has been shown that the motor depressant
actions of caerulcin and apomorphine arc reduced by
low doses of deva/epide (0.1-10 p.g/kg) whereas low
doses of L-365.260 (1-10 M g / k g ) have the opposite
effect (Vasar el al.. in press). Since CCKA and CCK„
receptors appear to affect the regulation of motor
activity differently, an interaction of devazepide with
both CCK-8 ’ecoptor subtypes might explain the o th er­
wise paradoxical reduction of the motor depressant
effect of caerulcin after chronic treatm ent with de­
vazepide. An alternative explanation for this finding is
that С С К Л rcceptors located on either side of the
blood-brain barrier may have opposite effects on mouse
motor activity. The receptors responsible for the seda­
tive effects of CCK are located in the nucleus of the
solitary tract and vagus nerve (outside the blood-brain
barrier). Stimulation of these, reccptors suppresses
dopamine release in basal ganglia induced by peripher­
ally injected CCK-8 agonists (Crawley and Schwaber,
1983; Hamamura et al., 1989). In contrast, С С К Л re­
ceptors in the striatum and mesolimbic structures pro­
mote dopamine release and potentiate dopamine-in­
duced hyperlocomotion (Crawley et al., 1985; Vickroy
and Bianchi, 1989). Chronic treatm ent with proglumide
increased the activity of dopaminergic cells in the
mesolimbic system (Chiodo et al., 1987). A similar
effect occurring after repeated treatm ent with de­
vazepide might explain the reduced sedative effect of
caerulein. The motor stimulant effect of ( + )-amphetamine was increased after repeated administration
of caerulein and devazepide, but not after L-365,260.
This was most likely due to increased sensitivity of
striatal and mesolimbic dopamine D2 rcceptors be­
cause long-term administration of CCK antagonists
(proglumide, devazepide) and agonists (CCK-8 and
cacrulein), increased the number of dopamine D j re­
ceptors in the basal ganglia of rodents (Dumbrille-Ross
and Seeman, 1984; Csernansky et al., 1987; Vasar et
al.. 1988b; Vasar et al.. unpublished data) but probably
by different mechanisms. Increased dopamine D , rcceptor sensitivity would also account for the increased
aggressiveness seen in some mice during repeated
treatm ent with devazepide.
It has been shown that the two subtypes of the CCK
receptor have opposite effects on mesolimbic dopamin­
ergic activity. Stimulation of С С К Л receptors potenti­
ates dopamine-induced hyperlocomotion from the me­
dial nucleus accumbens (Crawley et al.. 1985) and
increases the release of dopamine from the same re­
gion (Vickroy and Bianchi, 1989). Injection of CCK-8
into the dorsomcdial accumbens also increased the
frequency of apomorphine-induced jaw movements, an
effect prevented by the selective L'( KA antagonist,
lorglumidc (Koshikawa el al., 1990), In contrast, C C K B
receptors potentiated apomorphine-induced inhibition
of dopaminergic ventral tegmental cells and the reduc­
tion in motor activity produced by dopamine applied to
the same area (Honimer et al.. 1986; Crawley, 1989).
Injections of CC'K-8 into the ventrolateral accumbens
did nol affect Ihe frequency of apomorphine-induced
jaw movements, in contrast to the facilitation seen
after its injection into the medial part of the nucleus
accumbens (Koshikawa el al.. IWO). These opposite
effects of the two receptor sub-types on the activity of
dopaminergic systems could explain why repeated ad­
23
ministration of the agonist, caerulein and the antago­
nist, devazepide affected behaviour similarly if it is
assumed that high doses of caerulein also affected
C C K B receptors. In recent acute studies, low doses of
devazepide (1-100 ^ g /k g ) have been shown not to
affect amphetamine-induced hyperlocomotion but to
completely prevent (Vasar et al., in press) the antago­
nistic effect of caerulein (Zetler, 1985; Allikmets and
Vasar, 1990). However, large doses of devazepide alone
antagonized the effects of amphetamine (Vasar et al.,
in press), effects similar to those of caerulein and
which have been shown to be due to selective С С К Л
receptor stimulation. This suggests that caerulein and
large doses of devazepide would also be expected to
have similar effects on sensitivity to ( + Vamphetamine
after chronic administration. The mechanism of the
anti-am phetam ine effect of large doses of devazepide
is not clear because in contrast to selective C C K B
receptor agonists, devazepide (10 m g/kg) did not de­
crease amphetamine-induced dopamine release, m ea­
sured as 3-methoxytyramine (Altar and Boyar, 1989).
In conclusion, repeated treatm ent with caerulein
and devazepide significantly affected the locomotor
activity of mice and their behavioural responses to
caerulein and ( + )-amphetamine. The results are inter­
preted in the light of recent suggestions that CC K A
and C C K B receptors have opposite effects on
dopamine-m ediated behaviours (Crawley. 1989; Koshi­
kawa et al., 1990; Vasar et al.. in press). It is difficult to
explain the discrepancy between the increase in the
density of CCK-8 receptors and Ihe lack o f any changes
in behaviour after 10 day treatm ent with L-365,260. It
may be that the behaviours studied are more depen­
dent on CC K A receptors and that L-365,260 does not
affect these receptors even at high doses.
Acknowledgement
R.V. w as su p p o rte d by a B ritish C ouncil Fellow ship.
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IN: Molecular Pharmacoloj»y of Receptors. IV. (I.. Allikmets, ed.). Л»(и tl ( nmmcnlatlonts
Dnivendtatis TartueiwlN,
929: 34-41.
PILOCARPINE-INDUCEI) LIMBIC SEIZURES - AN INVOLVEMENT OF CCK
RECEFIORS.
E. Vasar, J. Ilarro, A. Lang, A Soosaar
Psychopharmacology Lab, lartu University
SUMMARY
A muscarinic agonist pilocarpine (380 mg/kg) induced in all injected male mice the fatal seizures.
The pretreatment of mice with CCK-8 (25-200 pg/kg) antagonized
significantly the effect of
pilocarpine, whereas the CCK-B/gastrin agonist pentagastrin (CCK-5, 2500 pg/kg) only moderately
inhibited the action of muscarinic agonist. Devazepide (10-1000 pg/kg), a selective antagonist at
CCK-A receptors, and L-365,260 (10- HXM) pg/kg), a selective antagonist at CCK-B receptors,
antagonized the anticonvulsant effect of CCK-8. However, only a high dose (1 mg/kg) of
devazepide and L-365,260 reversed significantly the action of CCK-8.
In rats the administration of pilocarpine (380 mg/kg) decreased significantly the number of [3H]pCCK-8 binding
sites in the several forebrain structures (the frontoparietal cortex, striatum and
hippocampus). The comparison of [3H]-pCCK-8 binding in the brain strucfures of rats with and
without seizures revealed evidently higher decrease of CCK-8 receptors' density in animals
experiencing seizures. In the hippocampus the difference between the values of responders and non­
responders was statistically evident. Ihe significant reduction of [3H]-pCCK-8 binding density in
the rat brain during pilocarpine-induced seizures probably reflects the involvement of CCK-B
receptors. However, the weak reversal of pilocarpine-induced seizures by CCK-5, and nearly similar
action of L-365,260 and devazepide against the anticonvulsant effect of CCK-8 in the mouse seems
to support the involvement of both subtypes of the CCK-8 (CCK-A and CCK-B) receptor in the
modulation ol pilocarpine-induced limbic seizures in rodents.
KEY WORDS: LIMBIC SEIZURES; CCK-8 RECEPTORS; PIUX’ARPINE: DEVAZEPIDE;
L-365,260; CCK-8 ; MOUSE, RAT.
23*
Ли involvement of cholecystokinin octapeptide (CCK-8) in the regulation of seizure activity
has been suggested by
numerous pharmacological studies. Ihus, systemic or
intracerebral
administration of CCK-8 and its analogue caerulein inhibits seizures with different genesis [6, 20,
21 J. On the other hand, the unspecific CCK-8 antagonist proglumide reverses the anticonvulsant
effect of caerulein against picrotoxin and quinolinate-induced seizures, and potentiates seizures
induced by quinolinate, an agonist at N-methyl-D-aspartate receptors [19, 20]. The highest levels of
CCK-8 immunoreactivity and receptors are found in the different limbic and cortical structures
(piriform cortex, amygdala, hippocampus etc.) [5, 12, 17], which are known to be involved in the
regulation of seizure activity [4, 14]. Limbic seizures with varied genesis have been demonstrated
to cause nearly complete loss of CCK-8 immunoreactivity from hippocampal mossy fiber system
|3], The potent convulsant picrotoxin is shown to reduce CCK-8 immunoreactivity in the several
limbic regions |7]. The systemic treatment with muscarinic agonist pilocarpine is shown to cause
very typical limbic seizures in rodents [18]. Magnani et al. [10, 11] have shown that the systemic
treatment with CCK-8 and caerulein significantly affects the release of acetylcholine from the
cerebral cortex of the rat "in vivo". Therefore, the aim of present work was to establish the role of
CCK-8 receptors in the regulation of limbic seizures induced by pilocarpine in mice and rats. CCK8, CCK-B/gastrin agonist pentagastrin (CCK- 5) and two selective antagonists at CCK-8 receptors
L-365,260 (antagonist of "brain" or CCK- B receptors) and devazepide (antagonist of "visceral" or
CCK-A receptors) [1, У| were used for clarifying of this problem. Simultaneously with the
behavioural experiments, the effect of pilocarpine-induced seizures was studied on the parameters of
CCK-8 receptors in the different brain structures of the rat.
MA rERI ALS AND METHODS
Male albino mice (25-30 g) and male Wistar rats (250-300 g) were used throughout the
experiment. The mice were placed into individual observation boxes 15 min before the start of
experiment. After this habituation period CCK antagonists - L-365,260 (3R(+)-N-(2,3-dihydro-lmethyl-2-oxo-5-phenyl-lH-l,4-benzodiazepin-3-yi)-N'-(3-methyl-phenyl)urea. CCK-B antagonist,
0.01-1 mg/kg) and deva/epide (formerly MK-329, CCK-A antagonist, 0.01-1 mg/kg) - were injected
15 min. and CCK-8 (25-200 ng/kg) and pentagastrin (CCK-5. 2.5 mg/kg) 10 min prior to muscarinic
agonist pilocarpine (380 mg/kg). Mice were observed for 60 min and the latencies of onset of
tremor, and tonic seizures and death were registered. In the radioligand binding experiments with
[propionyl-3Il]propionylated CCK-8 (3[H]-pCCK-8) scopolamine methylnitrate (an antagonist at
peripheral muscarinic receptors) was injectcd 30 min prior to saline or pilocarpine (380 mg/kg).
1 wo subgroups of rats - responders and non-responders to pilocarpine-induced limbic seizures -
were selected for radioligand binding studies Animals, respectively with and without seizures, were
killed by decapitation 60 min after the administration of pilocarpine The brains were removed
rapidly from the skulls and the frontoparietal cortex, mesolimbic structures (nucleus accumbens,
tuberculum olfactorium), piriform cortex, striata and hippocampus were dissected [ 15|. T he method
of Praissman et al. [ 16] in our slight modification was used for 3[1Г|-рССК-8 binding studies.
Saturation curves were analyzed using ENZHT1ER program for IBM microcomputers (8].
RESULTS.
Systemic treatment with muscarinic agonist pilocarpine (380 mg/kg) evoked in all injected
male mice (n=39) the fatal seizures. The pretreatment of mice with CCK-8 (25-200 ng/kg)
significantly antagonized the effect of 380 mg/kg pilocarpine (figure 1). 50 pg/kg CCK-8 obviously
reversed the effect of muscarinic agonist, the further increase ol CCK-8 dose did not enhance the
effect of neuropeptide 13 mice from 39 tested survived pilocarpine- induced seizures after
administration of 200 pg/kg CCK-8. CCK- 8 antagonist devazepide in the high dose (1 mg/kg)
evidently antagonized the anticonvulsant effect of CCK-8 (figure 2) CCK-B antagonist L-365,260
also after the administration of high dose (1 mg/kg) reversed the anticonvulsant action of CCK- 8
(figure 3). However, L-365,260 (10-1000 pg/kg), differently from dcva/epide, completely blocked
the antagonism of CCK-8 against the pilocarpine-induced lethality
Pilocarpine up to 1 mM did not interact with
3[H]-pCCK-8 binding in the radioligand studies
"in vitro” The administration of high dose of pilocarpine (380 mg/kg) changed the parameters of
3H-pCCK-8 binding sites in the several forebrain structures (table). Ihlocarpine reduced
significantly the number of 3|H|-pCCK-8 binding sites in the striatum, frontoparietal cortex and
hippocampus (table). In ihe hippocampus affinity of 3H-pCCK-8 binding sites was also increased
alter administration of pilocarpine The comparison of [3H|-pCCK-8 binding parameters in Ihe
animals, responding and non-responding to pilocarpine induced seizures, revealed more significant
changes in the brain
structures <>1 rats, experiencing seizures (table). In Ihe hippocampus the
difference between the values ol |3H|-pCCK-8 binding in responders and non-responders was
statistically evident (table)
I >ISt I ISSH )M
Ih e results ol present study are rellecting a significant role ol ( ’( 'К X receptois in th e
m odulation ot epileptogenic ellect of a m uscarinic agonist piiocaipine ( ' ( 'K-X potently antagonizes
the seizures induced by the lethal dose o l pilocarpine. <)ni’ fluid ol m ice survive pilocarpine
induced seizures aller prelrcalineiil with 200 pg/kg t '( К X CCK l./gastnn agonist |H-nlngaslrui only
LEGEND TO THE FIGURES
Figure 1. The effect o f CCK -8 (25-200 pg/kg) and pentagastrin (CCK-5,
2500 |ig/kg) on pilocarpine-induced seizures in mice. CCK -8 and CCK-5 were
given 10 min prior to pilocarpine (380 m g/kg). The animals were observed for
60 min after the administration o f pilocarpine. Significant differences between
vehicle/pilocarpine and CCK -8 or CCK- 5/pilocarpine treated groups were
determined by Newm an-Keuls test after significant AN O V A . F 5,l 16= 8.71,
pcO.OOOl (for tremor); F 5,116= 10.46, pcO.OOOOOl (for tonic seizures and
death). * - p< 0.05; ** - p< 0.01; *** - p<0.005 (if compared to pilocarpine
treated m ice).
Figure 2. The effect o f devazepide (10-1000 ng/kg) on the anticonvulsant
action o f CCK -8 (200 ng/kg) against pilocarpine-induced seizures in mice.
Devazepide was injected 15 min and CCK -8 10 min prior to pilocarpine.
Significant
differences
between
pilocarpine,
CCK- 8/pilocarpine
and
devazepide/CCK- 8/pilocarpine treated groups were determined by NewmanKeuls test after significant A N O V A.
F4,77=2.4, p<0.05
(for tremor),
F4,77=2.5 (for tonic seizures and death). * - p<0.05 (if compared to
pilocarpine treated animals); ** - p<0.05 (if compared to CCK- 8/pilocarpine
treated m ice).
Figure 3. The effect o f L-365,260 (10-1000 (ig/kg) on the anticonvulsant
effect o f CCK -8 (200 ng/kg) against pilocarpine-induced seizures in mice. L365,260 was injected 15 min and
Significant differences between
CCK -8 10 min prior to pilocarpine.
pilocarpine,
CCK- 8/pilocarpine
and L-
365,260/CCK-8/pilocarpine treated groups were determined by NewmanKeuls test after significant AN OVA. F4,77= 2.86, p<0.05 (for tremor); F4,77=
3,69, p<0.01 (for tonic seizures and death). * - p<0.05 (if compared to
pilocarpine treated moce); ** - p<0.05 (if compared to CCK- 8/pilocarpine
treated animals).
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о
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Table
The binding parameters of [3H]-pCCK-8 in tbe brain
responding and non-responding rats to pilocarpine (380 mg/kg) seizures.
Brain structures
structures
Saline
Non-responders
Responders
0.42±0.02
0.40*0.03
0.50*0.15
Bmax 4.51±0.32
4.13±0.22
3.55*0.53
Kd
0.43±0.02
0.63*0.06
0.68*0.08
Bmax 6.30±0.20
5.22*0.29
5.58±0.75
Frontoparietal
Kd
0.33*0.02
0.41*0.03
0.26*0.02
cortex
Bmax 5.15±0.30
3.76*0.76
2.71*0.23a
Striatum
Kd
0.45±0.07
0.36*0.04
Bmax 5.23*0.28
4.25*0.19a
3.74±0.24b
Dorsal hippo­
Kd
0.63*0.04
0.37±0.05a
0.15*0.02b,c
campus
Bmax 1.87±0.17
1.18±0.28a
0.56±0.05b,c
Mesolimbic area
Piriform cortex
Kd
0.27±0.01
of
The brain structures of 4-5 rats have been pooled. The mean values * S.E.M. of 4
independent experiments are presented in table. Kd - dissociation constant in nM;
Bmax - apparent number of binding sites in pmoles per gram original tissue wet
weight a-p < 0.05; b -p < 0 .0 1 (compared to saline treated rats, Student's t-test); c p< 0.05 (compared to non-responders, Student's t-test).
moderately reduces (he convulsant action of pilocarpine Accordingly, it seems probable that the
peripherally injected CCK-8 alleds (lie cholinergic neurotiansmiNsioii in the brain. It is suggested
that several behavioural ellects of CCK-8 and caerulein are generated through primarily peripheral
uicchanisms [I3| It is thought tliat the sedative effect of large doses of CCK-8 is of peripheral
origin and could be abolished by abdominal vagotomy |2|. Magnani et al 110. 111 have shown that
CCK-8, iu the doses 10 pg/kg and higher, potently inhibits Hie release of acetylcholine from the rat
cerebral cortex This effect of CCK-8 is not affected by bilateral vagotomy or by the lesion of
dopaminergic cclls in the substantia nigra Ihe selective CCK antagonists devazepide and L365.260 reverse the anticonvulsant effect of CCK X. However, it liappens only alter the
administration of very high dose {1 mg/kg) of CCK antagonists. It is noteworthy that the effect of I 365.260 is somewhat stronger.
L-365,260, in wide dose range ( 10-ИХХ) pg/kg), antagonize also the effect of CCK-8 on pilocarpineinduced lethality. Nevertheless, the both subtypes of CCK-8 (CCK-A and CCK-B) seems to be
involved in the anticonvulsant effect of CCK-8. According to the radioligand binding studies "in
vitro", pilocarpine (up to 1 mM) does not interact directly with CCK-8 receptors in the brain
However, the systemic administration of very high dose of pilocarpine (380 mg/kg) is reducing ihe
density of CCK-B receptors in the frontoparietal cortex, striatum and hippocampus of the rat brain
The comparison of 3[H]-pCCK-8 binding parameters in rats, responding and non- responding to the
administration of pilocarpine with seizures, reveals markedly higher reduction of CCK-B receptors
in animals with seizures. It supports the idea that CCK-B receptors are involved in the modulation
of seizures induced by muscarinic agonist.
In conclusion, it is very likely that the both subtypes of CCK-B receptor are involved in the
modulation of limbic seizures induced by the muscarinic agonist pilocarpine. This idea is supported
by the findings that CCK-B/gastrin agonist pentagastrin only moderately antagonized the effect of
pilocarpine, the selective CCK-8 antagonists devazepide and L- 365,260 have nearly similar effect
on the anticonvulsant effect of CCK-8, and during pilocarpine-induced seizures the density of CCK8 receptors is significantly reduced in the several brain regions.
Acknowledgment. The authors are grateful to Merck, Sharp and Dohme Ltd. for the generous
gift of
24*
devazepide and L-365,260.
RM-lšRKNCbS
1 ('hang R.S.J.. and I otti V.J. (19X6) Biochemical and pharmacological characterization of an
extremely potent and selective nonpeptide cholecystokinin antagonist Proc Natn.Acad.Sci.il.S.A.
XI: 4923-4926.
2 Crawley J N and Kiss J.Z. (19X5) Tracing the sensory pathway from gut to hrain regions
mediating the actions of cholecystokinin on feeding and exploration Ann.N Y.Acad.Sci 44X: 586-
3 Hall (Л (19XX) Seizure induce dramatic and distinctly different change in enkephalin,
dynorphin and CCK immunoreactivities in mouse hippocampal mossy fibers. J.Neurosci. X: 18521862.
4 llandforth A. and Ackermann R.F. (1988) Electrically induced limbic status and kindled
seizures. In Anatomy of epileptogenesis. (eds. Meldrum B.S., Ferrendelli J.A. and Wieser H.G.).
John Libbey and Co Ltd., London, pp. 71-88.
5 Harris Q.L.G., Lewis S.J., Shulkes A. and Vajda F.J.E. (1988) Regional concentrations of
cholecystokinin in the rat: the effects of kindled and non-kindled seizures. Neuropharmacol. 27:
547-549.
6. Kadar Т., Pesti A., Репке В. and Telegdy G. (1984) Inhibition of seizures induced by
picrotoxin and electroshock by cholecystokinin octapeptides and their fragments in rats after
intracerebroventricular administration. Neuropharmacol. 23: 955-961.
7. Kato Т., Emi Y., Takita M. and Kushima Y. (1988) Time dependent changes in the
concentrations of immunoreactive cholecystokinin octapeptide sulfate in rat brain regions after
systemic injection of picrotoxin. Neurochem.Int 12: 493-497.
8. Leatherbarrow R.J. (1987) Enzfitter, a non-linear regression
data analysis program for the
IBM PC'. Elsevier Science Publisher, Amsterdam.
9. Lotti V.J. and Chang R.S.L. (1989) A new potent and selective non-peptide gastrin
antagonist and brain cholecystokinin receptor (C('K-B) ligand: L-365,260 Eur.J Pharmacol. 162:
273-280.
10 Magnani M., Mantovani P and Pepeu G. (1984) Effect of cholecystokini octapeptide and
ceruletide on release of acetylcholine from cerebral cortex of the rat in vivo. Neuropharmacology
23: 1305-1309.
11. Magnani M., Florian A., Casamenti F. and Pepeu G. (1987) An analysis of cholecystokinininduced increase in acetylcholine output from cerbral cortex of the rat. Neuropharmacology 26:
1207-1210.
12. Marley P.I)., Rehfeld J.F. and Emson P.C. (1984) Distribution
and chromatographic
characterisation of gastrin and cholecystokinin in the rat central nervous system. J.Neurochem. 42:
1523-1530.
13. Morley J.E. (1987) Behavioural effects of administered cholecystokinin. ISI Atlas
Pharmacol. 1: 49-51.
14. Patel S. (1988) Chemically induced limbic seizures in rodents. In: Anatomy of
epileptogenesis. (eds. Meldrum B.S., Ferrendelli J.A. and Wieser G.H.), John Libbey and Co. Ltd.,
London, pp 89-106.
15. Paxinos G. and Watson C. (1982) The rat brain in stereotaxic coordinates. Academic Press
Australia, North Ryde.
16. Praissman M„ Martinez P.A., Saldino C.F., Beikowitz J.M., Steggles A.W. and Finkelstein
J.A. (1983) Characterization of cholecystokinin binding sites in rat cerebral cortex using a [1251]CCK-8 probe resistant to degradation. J.Neurochem. 40:1406-1413.
17. Saito A., Sankaran K., Goldfine D. and Williams J.A. (1980) Cholecystokinin receptors in
the rat brain: characterization and distribution. 208: 1155-1156.
18. Turski W.A., Cavalheiro E.A., Schwarz M., Czuczwar S.J., Kleinrok Z. and Turski L.
(1983) Limbic seizures produced by pilocarpine in rats: behavioural, electroencephalic and
neuropathological study. Behav.Brain Res. 9: 315-322.
19. Vasar E., Rägo L., Soosaar A., Nurk A. and Maimets M. (1985) Modulatory effect of
caerulein on benzodiazepine receptors. Bull.Exp Biol.Med. 102: 711-713.
20. Vasar E., Allikmets L„ Ryzhov I„ Prakhye I., Soosaar A. and Mirzayev S. (1988)
Intraspecies differences in the behavioral effects of caerulein, an agonist of CCK-8 receptors, in
mice and rats. Bull.Exp.Biol.Med. 105: 168-170.
21. Zetler G. (1985) Caerulein and its analogues: neoropharmacological properties. Peptides 6:
33-46.
In: Multiple Cholecystokinin Receptors in the CNS (S.T. CWper ami C.D. D inrbh, eds.),
Oxford University Press, 1991 (In press)
CCK-8 RECEPTORS AND ANXIETY IN RATS
Eero Vasar, Jaanus Harro, Anu Põld and Aavo I^ang
Psychopharmacology Lab, Tartu University,
34 Veski Str., 202400 TARTU, Estonia
The agonists at CCK-8 receptors are shown to induce or potentiate fear-related behaviours in rats.
Caerulein and peatagastrin (CCK-5) have at very low doses an anxiogenic-like effect on rats in an
elevated plus-maze (Harro,
Pöid,
Vasar and Allikmets, 1989). CCK-8 antagonist proglumide
completely reversed the anxiogenic-like action of caerulein and CCK-5 in rodents (Harro et al.,
1989; Harro, Põld and Vasar, 1990). Intraventricular administration of CCK-4 significantly
increases the intensity of foot-shock elicited aggressiveness in male rats (Vasar, Maimets and
Allikmets, 1984). Very recently, the intravenous administration of CCK-4 is shown to cause very
severe anxiety and panic-like attacks in healthy volunteers (De Montigny, 1989). However, it is not
clear where the primary target of anxiogenic-like action of CCK-8 related peptides lies: in the brain
or periphery. Therefore, the present study was dedicated to reveal the primary target of anxiogeniclike effect of CCK-8 agonists on the rat.
The male and female rats (weighing 180-220 grams) were used throughout the study. The
animals were used only once. The anxiogenic-like effect of CCK-8 agonists (caerulein, CCK-5 and
CCK-4) was studied according to the method of Pellow, Chopin, File and Briley (1985) in an
elevated plus-maze. The lowest dose of caerulein to cause the anxiogenic-like effect on the rat was
100 ng/kg. CCK-5 had the similar effect after administration of 500 ng/kg. The subcutaneous
treatment with 10 ng/kg CCK-4 in some experiments also significantly decreased the exploratory
activity of rats. The maximal reduction of animals behaviour was seen after injection of 25 and 50
jig/kg of CCK-4 (table 1). In higher closes (100 ng/kg and 1 mg/kg) CCK-4 failed to affect the rats'
behaviour. The anxiogenic-like effect of CCK agonists was in good accordance with their potency to
inhibit |^Hj-propionylaled-CCK-8 (|%J-pCCK-8, 0.3 nM) binding in the cerebral cortex, bul not in
pancreas (table 2). According to these results it is very probable tliat CCK-B (central subtype)
receptors have a significance in the anxiogeuic-like action of CCK-8 agonists on the rat.
J urther, the interaction of different CCK antagonists (lorglumide. proglumide, devazepide and L365,260) with anxiogenic-like effect of CCK-4 (50 ng/kg) was studied. The pretreatment with 0.01
mg/kg L-365,260, the selective antagonist at CCK-B/gastrin receptors, caused statistically evident
antagonism with the effect of CCK-4 (figure 1). L-365,260 antagonized the action of CCK-4 in 10
limes smaller dose than lorglumide (the effective dose 0.1 mg/kg). CCK-B/gastrin antagonist was
KK) limes more effective than the selective CCK-A (peripheral subtype) antagonist devazepide (1
mg/kg) and proglumide (1 mg/kg). It is worthy noting that the antagonism of glutaramic acid
derivatives (proglumide, lorglumide) against CCK-4 was more pronounced if compared to the
effect of 1,4- benzodiazepines (devazepide, L-365,260).
CCK-8 is shown to localize in some brain regions (cerebral cortex, hippocampus) mostly in
GABA-ergic neurons (Kosaka, Kosaka, Tateishi. Hamaoka, Yanaihara, Wu and Hama, 1985). There
is existing the clear antagonistic interaction between benzodiazepine tranquillizers and CCK-8 m
the electrophysiological experiments (Bradwejn and De Montigny, 1984). Picrotoxin, the potent
anatgonist al chloride channel, at anxiogenic dose (0.5 mg/kg) increased the density of CCK-8
receptors m frontal cortex and hippocampus (figure 2). In higher doses (1 and 2.5 mg/kg) picrotoxin
induced seizures and apparently decreased the density of CCK-8 receptors (figure 2). Picrotoxin
failed in the acute experiments to affect the parameters of benzodiazepine receptors. On the other
hand, ihe anxiogenic doses of CCK-8 agonists caerulein (100 ng/kg) and CCK-5 (500 ng/kg) did not
change the affinity and density of CCK-8 receptors, but decreased (respectively 34 % and 38 %) the
apparent number of benzodiazepine receptors in frontal cortex. It is quite probable that at least in the
frontal cortex the negative interaction is existing between CCK-8- and GABA-ergic systems in the
regulation of anxiety. This opinion is supported also by the experiments, where the rats were
selected according to their behaviour in the elevated plus-maze. There was possible to find the
animals from the population of rats, which exploratory activity differed very significantly. The
radioligand binding experiments with [3H]-pCCK-8 and [^Hj-flunitrazepam revealed very evident
differences between the high ("non- anxious") and low activity ("anxious") rats in the density of
CCK-8 and benzodiazepine receptors in the frontal cortex (figure 3). "Non-anxious" rats had
obviously lower density of CCK-8 and higher density of benzodiazepine receptors in the frontal
cortex if compared with "anxious" animals.
In conclusion, the anxiogenic-like effect of peripherally administered CCK-8 agonists is probably
related to the central subtype of CCK receptor in the rat. At least in the frontal cortex CCK-B
rei cptors seem to have a strong negative interaction with GABA-benzodiazepine receptor complex.
Thus, ihe balance between CCK-8- and (iABA-ergic systems has the significance in the genesis of
anxiety.
Devazepide and 1.-365,260 are the generous gifts from Merck, Sharp and Dohme. Proglumide
and lorglumide were donated by Rotta Pharmaceutici.
IF XI К ) lill I l( il IUI iS
Figure
I
TIIE INTERACTION Ol CCK
ANTAGONISTS Wi l l i ANXHXiFNIC-l IKIi
1.111(4 (>1 l'CK-4 ON RAIS IN AN ELEVATED 1*1 I IS- MAZE.
Data presented in the figure are % of control values of each separate experiment. The scores are
mean (±Sli.M ) percentage of crossed lines in the open part. In the control group the mean value of
crossed Imes during 4 min was between 10 and 14 in the different experiments. l*togluinide (0.1-10
uig/kg, i p.) and lorgluniide (0.01-1 mg/kg, i.p ) were administered 10 min before the CCK-4 (50
Hg/kg, s.c.) treatment, whereas devazepide (0.01-1 mg/kg, i.p.) and 1.-365,260 (0.001-0 I mg/kg,
i.p.) were given 15 prior to the
CCK-4
dose. Lorglumide, devazepide and L-365,260 were
suspended with the help ot some drops of Tween-85 in distelled water The same vehicle (some
drops of Tween-85 in distilled water) was given to control rats. * - p < 0.05; ** - p<0.01, as
compared to respective CCK-4 group values.
Figure 2. THE EFFECT OI PICROTOXIN ON f3H]-pCCK-8 BINDING IN THE RAT BRAIN
Data are expressed as the apparent number of binding sites (В/max) in pmol/g original tissue wel
weight of the frontal cortex and hippocampus of the rat. The mean values of three independent
studies (±S.E.M ) are presented in the table. Picrotoxin (0.1-2.5 mg/kg. s.c.) was injected 30 min
before the decapitation. * - p< 0.05; ** - p<0.01, as compared to control group (Student's t-test)
Figure
3 [3H]-pCCK-8 AND [3H]-FLlINITRAZEPAM BINDING IN THE FRON1AI.
CORTEX OF THE RATS, SELECTED ACCORDING TO THEIR RESPONSE IN THE
F.LLVATED PLUS-MAZE.
In the low activity group the number of crossed lines in the open part was 0 (>±i) 6 In the
intermediate group it was 8.5±0.5 and in the high activity group 18.2±1.5. T he differences between
three selected groups are statistically evident (p< 0.01, Mann- Whitney IJ-test). T he mean values of
three independent selection experiments are presented in the figure. Data are % of home-cage
control values in the frontal cortex The apparent number of [31Ц-рССК-8 binding sites in the
frontal cortex of home-cage controls was 3.66±0.34 pmol/g original tissue, and for | 3H|flunitrazcpam it was I37±12 pmol/g original tissue.
* - p< 0.05, significant difference from home-cage controls;
** - P< 0.05, significant difference from high activity group (Student s t-test)
25
°o
of
c o n tro ls
tis s u e )
original
(p rr, ci/g
PIC R O T O X IN (mg/kg)
I— ... I F rontal
c o rte x
25*
Ю
H ippocam pus
Table 1
Effect of CCK-4 on the exploratory activity of the rat in an elevated plus-maze
Test dose
Latency of
No of lines
Tolal time spent
first open part
crossed in
in open part (s)
entry (s)
open part
Vehicle
42±8
10.3±1.1
57±6
CCK-4 1 Mg/kg
36±24
11.3±2.6
69+12
10 Mg/kg
57+10
7.4±2.6
45+6
25 Mg/kg
81±8*
6.2± 1.2 *
39+5
50 Mg/kg
86± 12*
5.5+1.0*
33+5
100 (ig/kg
69± 14
9.0±2.2
63±10
1000 |ig/kg
39±24
10.9+2.1
58+10
All values are means ± S.E.M. The test time was 4 min. CCK-4 was administered
s.c. 15 min prior to the experiment. *- p<0.05 (significantly different from vehicle
treated group, Newman-Keuls test after ANOVA, one-way ANOVA for entries
F(1.153)=2.18, p<0.05; for crossings F(l,153)=2.36, p<0.05; for total time
F( 1,153)=1.92, p=0.08).
Table 2
Correlation between anxiogenic-like e ffe c t of CCK-8 agonists and their affinity at
CCK-8 receptors in cerebral cortex and pancreas o f the rat.
Suppression of
IC50 values against
exploratory activity
f 3H]-pCCK-8
CCK-8 agonists in elevated plus-maze
cerebral cortex
(pmol/kg)
pancreas
(nM)
Caerulein
0.074
1.1
0.6
CCK-5
0.670
10
6200
CCK-4
43.3
411
>10000
0.99998
0.797
p= 0.004
p= 0.41
Pearson's у
The doses of CCK agonists presented in the table are inducing the statistically
evident anxiogenic-like effect in the elevated plus-maze. The radioligand binding
studies were performed according to the method of Praissman, Martinez, Saldino,
Berkowitz, Steggles and Finkelstein, 1983.
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