석 사 학 위 논 문 만성적 니코틴 투여와 투여 중단이 중추신경계

석 사 학 위 논 문
만성적 니코틴 투여와 투여 중단이 중추신경계
니코틴성 아세틸콜린 수용체 밀도와 Fos 발현에 미치는 효과
Effect of Chronic Nicotine Administration and its Withdrawal on
Neuronal Nicotinic Acetylcholine Receptor Density and Fos
Expression
정 아 정
한 양 대 학 교
2000년
6월
대 학 원
일
석 사 학 위 논 문
만성적 니코틴 투여와 투여 중단이 중추신경계
니코틴성 아세틸콜린 수용체 밀도와 F o s 발현에 미치는 효과
Effect of Chronic Nicotine Administration and its Withdrawal on
Neuronal Nicotinic Acetylcholine Receptor Density and Fos
Expression
지도교수: 채 영 규
이 논문을 이학석사 학위 논문으로 제출합니다.
2000년
일
6월
한 양 대 학 교
대 학 원
생 화 학 과
정 아 정
이 논문을 정아정의 이학석사 학위 논문으로 인준함
2000년
6월
일
심사위원장
이 영 식
(인)
심사위원
김 상 은
(인)
심사위원
채 영 규
(인)
한 양 대 학 교
대 학 원
석 사 학 위 논 문
개 요
만성적 니코틴 투여와 투여 중단이 중추신경계
니코틴성 아세틸콜린 수용체 밀도와 F o s 발현에 미치는 효과
Effect of Chronic Nicotine Administration and its Withdrawal on
Neuronal Nicotinic Acetylcholine Receptor Density and Fos
Expression
지도교수: 채 영 규
한 양 대 학 교
대 학 원
생 화 학 과
정 아 정
Contents
Contents ...............................................................................................................i
Figure contents...................................................................................................ii
Table contents ...................................................................................................iii
Abbreviation table ............................................................................................iv
Abstract (Korean)..............................................................................................v
I. Introduction....................................................................................................1
II. Materials and Methods ................................................................................3
II-1. In vitro receptor binding assay ........................................................ 3
1-1. Nicotine treatment .......................................................................3
1-2. Tissue preparation .......................................................................4
1-3. Equilibrium [3 H]nicotine binding ...............................................4
1-4. Data analysis.................................................................................5
II-2. Fos immunohistochemistry...............................................................6
2-1. Tissue preparation .......................................................................6
2-2. Immunohistochemistry ................................................................6
2-3. Data analysis.................................................................................7
III. Results ..........................................................................................................8
III-1. Effect of chronic nicotine treatment and its withdrawal on number
of [3 H]nicotine binding site in rat brain............................................. 8
III-2. Fos expression after nicotine withdrawal in striatum and nucleus
accumbens...........................................................................................18
IV. Discussion ...................................................................................................23
V. Reference......................................................................................................28
Abstract (English) ............................................................................................33
Acknowledgement ............................................................................................35
i
Figure contents
Figure 1. Effect of chronic nicotine treatment (once-daily injection) and its
withdrawal on [3 H]nicotine binding to rat striatal membranes....11
Figure 2. Effect of chronic nicotine treatment (twice-daily injection) and
its withdrawal on [3 H]nicotine binding to rat striatal membranes
.............................................................................................................13
Figure 3. Effect of chronic nicotine treatment (once-daily injection (A) and
twice-daily injection (B)) and its withdrawal on Bmax of
[3 H]nicotine binding in rat striatum ................................................15
Figure 4. Effect of nicotine administration protocol on nAChR density....17
Figure 5. Photomicrographs illustrating Fos-like immunoreactive cells in
the striatum........................................................................................19
Figure 6. Photomicrographs illustrating Fos-like immunoreactive cells in
the nucleus accumbens ......................................................................20
Figure 7. Effect of a challenge injection of nicotine (0.4 mg/kg, s.c.) on Foslike immunoreactivity in the striatum(A) and nucleus
accumbens (B) of rats during chronic nicotine treatment and its
withdrawal .........................................................................................21
ii
Table contents
Table 1. Nicotine (4 mg/kg, once a day) treatment effects on [3 H]nicotine
binding ..................................................................................................9
Table 2. Nicotine (2.4 mg/kg, twice a day) treatment effects on [3 H]nicotine
binding ................................................................................................10
iii
Abbreviation table
DA
Dopamine
nAChR
Nicotinic acetylcholine receptor
KRH
Krebs-Ringer’s HEPES
HEPES
N-2-Hydroxyethylpiperazine-N'-2-ethane sulfonic acid
DOPAC
3,4-Dihydroxyphenylacetic acid
HVA
Homovanillic acid
PKC
Protein kinase C
cAMP
Cyclic adenosine 5’-monophospate
iv
국문요지
만성 니코틴 투여는 니코틴성 아세틸콜린 수용체의 상향조절과 도파
민성 신경의 작용부위에서의 Fos 발현 지연을 일으킨다. 그러나 만성 니코
틴에 노출이 중지된 동안의 니코틴성 아세틸콜린 수용체 변화와 Fos 발현
에 대한 연구는 미흡하다. 이 연구의 목적은 니코틴 만성 투여와 투여 중단
기간 동안의 니코틴성 아세틸콜린 수용체와 니코틴에 의해 유도된 Fos 발현
의 수를 관찰하는 것이었다. 또한 우리는 서로 다른 니코틴 투여 방법이 니
코틴성 아세틸콜린 수용체 밀도에 미치는 효과에 대해서도 실험하였다.
흰쥐에 니코틴(4 mg/kg, 하루 한번 또는 2.4 mg/kg, 하루 두번) 또는 생
리식염수를 매일 한번씩 14 일 동안 연속적으로 피하주사 하였다. 마지막
약물 투여 후 1 일, 2 일, 7 일에 단두하여 선조체를 분리하여 [3H]nicotine
(0.625-20 nM) 니코틴성 아세틸콜린 수용체 결합을 측정하였다. 또한 Fos 면
역반응을 측정 하기 위하여 측핵과 선조체를 1 일, 2 일, 7 일에 분리하여 면
역화학적 조직염색방법을 사용하였다.
2.4 mg/kg, 니코틴을 하루 두번 처리한 쥐에서 니코틴성 아세틸콜린
수용체의 Bmax 값은 마지막 약물투여 후 1 일째에서 309.4% 증가되었다. 2 일
후 Bmax 는 대조군 수준으로 감소하였고 7 일 후에는 증감의 변화가 없었다.
4 mg/kg 니코틴을 하루 한번 처리한 동물에서도 유사한 결과를 얻었다. 그
러나 2.4 mg/kg 니코틴 하루 두번 처리한 군이 4 mg/kg 하루 한번 처리군에
비하여 1 일 후 Bmax 값의 증가가 더 크게 나타났다.
Fos 발현은 선조체와 측핵에서 1 일째에 대조군과 비교하여 46.2%,
42.1% 감소 하였다. 2 일째에 선조체에서는 대조군에 비해 감소하였지만 측
핵에서는 증가하였다.
7 일째에서는 선조체와 측핵 모두에서 대조군과 처
리군의 차이가 없었다.
결과는 만성 니코틴 노출에 의한 니코틴성 아세틸콜린 수용체의 증가
v
는 투여중지 후 급격히 회복됨을 나타낸다. 투여 중지 후 1 일째에 선조체
와 측핵에서 니코틴이 Fos 발현을 유도하지 못한 것은 만성 니코틴 투여기
간 동안 니코틴성 아세틸콜린 수용체의 불활성화에 의한 것으로 생각된다.
이러한 결과는 만성적 니코틴 투여에 의한 중추신경계의 니코틴성 아세틸
콜린 수용체의 탈감작성에 의해 니코틴성 아세틸콜린 수용체의 증가가 일
어났다는 가설을 지지한다. 또한 그 결과는 니코틴 하루 투여량보다는 그
투여 방법이 니코틴성 아세틸콜린 수용체의 상향조절에 더 중요한 요소임
을 알 수 있다.
vi
I. INTRODUCTION
Nicotine (3-(1-Methyl-2-pyrrolidinyl)-pyridine) has pharmacological properties
leading to a progressive and long-lasting dependence, well known by cigarette
smokers. This alkaloid from the tobacco plant exerts its action dopamine (DA) release
in the central nervous system through ligand-gated ion channel receptors that belong
to the large nicotinic acetylcholine receptor family.
When Schwartz and Kellar (1983) and Marks et al. (1983) discovered
independently that the chronic administration of nicotine to animals resulted in an
increase in nAChR density in brain tissue, this was considered an unusual effect for a
receptor agonist. Many agonists had been shown to cause receptor down-regulation
after chronic administration, whereas receptor up-regulation had generally been
produced by the administration of receptor antagonists (Creese and Sibley, 1980). It is
hypothesized that desensitization of neuronal nAChRs induced by chronic exposure to
nicotine initiate up-regulation of nAChR number.
One of the ways to evaluate the neuronal receptor activity is to study the
regulation of the expression of immediate early genes, such as c-fos and the protein
they encode, as they can be activated by various stimuli including physiological and
pharmacological treatments (Curran and Morgan, 1985; Hunt et al., 1987; Sagar et al.,
1988; Robertson et al., 1989; Morgan and Curran, 1991; Fu and Beckstead, 1992). Fos
expression has been measured to investigate the receptor activity stimulated by drugs
such as cocaine, amphetamine, morphine and phencyclidine. For example, acute
1
exposure to cocaine transiently induces several Fos family transcription factors in the
nucleus accumbens, a region of the brain that is important for addiction. In contrast
chronic exposure to cocaine does not induce these proteins, but instead causes the
persistent expression of highly stable isoforms of deltaFosB (Kelz et al., 1999).
Drug treatments affecting dopaminergic transmission modulates the expression
of Fos protein in the striatum (Robertson et al., 1990; Herrera and Robertson, 1996).
Acute nicotine is known to increase the expression of the Fos protein in neurons of
dopaminergic target areas (Ren and Sagar, 1992; Pang et al., 1993; Valentine et al.,
1996; Salminen et al., 1999). In contrast to the marked c-fos induction produced by a
single injection of nicotine, its repeated administration diminished c-fos indelibility
(Hope et al., 1994; Rosen et al., 1995; Nye and Nestler, 1996; Turgeon et al., 1997;
Merlo et al., 1999; Salminen et al., 2000). However, little is known about the changes
in nAChRs and Fos expression during withdrawal from chronic nicotine exposure.
The purpose of the present study was to investigate the effect of chronic
nicotine administration and its withdrawal on number of nAChRs and nicotineinduced Fos expression. We also examined the effects of different nicotine
administration schedules on the nAChR density.
2
II. MATERIALS AND METHODS
Materials
(–)-[3H]nicotine (specific activity 84.5 Ci/mmol) was obtained from NEN Life
Science (Boston MA.). (–)-Nicotine, polyethyleneimine, bovine serum albumin,
HEPES, Tris-HCl were purchased from Sigma Chemical Co. (St. Louis, MO.).
Whatman GF/B glass fiber filters were obtained from Brandel Harvester Apparatus
(Gaithersburg, MD.) and Aquassure scintillation fluid was purchased from Packard
Bioscience (Groningen, The Netherlands).
Animals
Male Sprague-Dawley rats, weighing 280-320 g, obtained from Daehan
Experimental Animal Center, were used throughout this study. Rats were housed two
per cage and were allowed free access to food and water. The animal room was
maintained on 12 hr light/dark cycle (lights on 8:00-20:00), temperature 22 ± 2℃, and
relative humidity 50 ± 5%.
Methods
1. In vitro binding assay
1-1. Nicotine treatment
Animals received subcutaneous injections of nicotine for 14 consecutive days at
the dose of 4 mg/kg (8:00 AM), once a day or 2.4 mg/kg, twice a day (8:00 AM, 5:30
3
PM). The nicotine was administered in 0.5 ml saline. Control animals were injected
with saline by the same administration protocols.
1-2. Tissue preparation
One day, 2 days and 7 days after the last nicotine or saline administration, the
rats were killed by decapitation. The brains were taken out and the striata were
dissected on a dry ice plate. The tissues were stored at –70℃ until binding assays
were performed. Tissue preparation method was determined using a modification of
the method of Bhat et al. 1994. On the day of the assay, tissue was placed in 10
volumes of ice-cold Krebs-Ringer’s HEPES (KRH) buffer with the followin g
composition: NaCl 118 mM, KCl 4.8 mM, CaCl2 2.5 mM, MgSO 4 1.2 mM, HEPES 20
mM, pH adjusted to 7.5 with NaOH. The tissue was thawed, homogenized in a
Polytron PT-3000 homogenizer (Brinkmann Instrument Inc. Westbury NY.), and the
resulting homogenate was incubated for 5 min at 37℃ to promote the hydrolysis of
endogenous nicotine. After incubation, the homogenate was centrifuged at 18,000 × g
for 20 min. The resulting pellet was resuspended in 20 volumes of distilled water,
incubated for 1 hr at 4℃, and recentrifuged at 18,000 × g for 20 min. The resulting
pellet was resuspended in 10 volumes of KRH buffer and centrifuged at 18,000 × g for
20 min. For the final assays, the pellet was resuspended (10 mg wet weight tissue/ml)
in KRH buffer.
1-3. Equilibrium [3 H]nicotine binding
4
Equilibrium binding of [3 H]nicotine was determined using a modification of the
method of Marks et al. 1986. In brief, incubations used ～500 µg of protein and were
conducted in 12 × 75 mm polypropylene test tubes at 4℃ in a final volume of 250 µl
of KRH buffer containing 200 mM Tris (pH 7.5). Six concentrations of [3 H]nicotine
were used ranging between 0.625 and 20.0 nM. After the 2 hr incubation, the binding
reaction was terminated by dilution of the tissue with 2.5 ml of ice-cold KRH
followed by filtration of the samples onto GF/B glass-fiber filters that had been soaked
in buffer containing 0.05% polyethyleneimine. After the filters were washed four
times with 2.5 ml of KRH, they were transferred to 20-ml scintillation vials for the
determination of radioactivity. After adding 5 ml of Aquassure scintillation fluid
(Packard Bioscience) to each vial, the radioactivity was determined using a Packard
Tri-Carb 2500 liquid scintillation counter. Nonspecific binding was estimated as that
present in samples containing 10 µM nonradioactive (–)-nicotine. Protein content was
determined according to the method of Lowry et al. (1951).
1-4. Data analysis
Binding data derived from the saturation experiments were analyzed using
radioligand data analysis software, KELL (BIOSOFT, U.K.). The Bmax values were
statistically evaluated by using a two-way (drug treatment × time after drug
withdrawal) analysis of variance (ANOVA). A significant interaction effect was
followed by Student’s t-test to compare the Bmax values from the two treatments (saline
and nicotine) at each time after drug withdrawal.
5
2. Fos Immunohistochemistry
2-1. Tissue preparation
Rats were treated with nicotine (2.4 mg/kg s.c., twice a day) or saline for 14
consecutive days. On 1, 2 and 7 days after the last administration of nicotine or saline,
animals received a challenge injection of nicotine (0.4 mg/kg, s.c.) 2 hr before
anesthesia (ketamine 80 mg/kg i.p. plus xylazine 10 mg/kg i.p.). Rats were perfused
intraaortically with ice-cold 0.1 M phosphate buffered saline (PBS), pH 7.4 followed
by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and
post-fixed with the same fixative for 2 hr at room temperature and immersed in a 20%
sucrose solution overnight at 4℃.
2-2. Immunohistochemistry
Immunohistochemistry was performed as described by Robertson and Fibiger
(1992) and Chergui et al. (1996). Brain sections (bregma 0.20 for the striatum; bregma
1.70 for the core of nucleus accumbens) were cut at –20℃ and mounted on silane
(DAKO Carpinteria, CA.) coated slides. The sections were washed in a PBS (2 × 5
min) and placed in 0.3% H2 O2 in methanol for 15 min and then washed in PBS (3 × 5
min). The sections were first incubated in a 2% normal goat serum (DAKO) in PBS-T
(0.1% Triton X-100 in PBS) for 40 min to block nonspecific staining.
The sections were then incubated in polyclonal rabbit Fos antibody (Oncogene
research, Cambridge, MA.) diluted in 1:500 for 24 hr at 4℃. The sections were
washed in PBS-T (10 min) and PBS (3 × 5 min) and incubated for 30 min with
6
biotinlyated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame,
CA.). After washing in PBS (3 × 5 min) the sections were incubated for 30 min in
PBS-T containing avidin-biotinlyated horseradish peroxidase complex (1:100, Vector
Laboratories). The immunoreactive complex was revealed with 3,3’-diaminobenzidine
(Liquid DAB substrate-chromogen-system, DAKO). Sections were washed with PBS
twice and dehydrated with graded ethanol and transferred into xylene and
coverslipped. The Fos-like immunoreactivity was localized by brown-stained cell
nuclei. No Fos-like immunoreactivity has been observed when the primary antibody
was omitted.
2-3. Data analysis
The number of Fos-like immunoreactive nuclei were counted by using IBAS
image analysis system (Zeiss, Germany) within a squared field area of 210 × 210 µm
for nucleus accumbens and 420 × 420 µm for striatum using 200X magnifications.
The mean value of the three to six sections was calculated from each selected region.
The data were statistically evaluated by using a two-way (drug treatment × time after
drug withdrawal) ANOVA. A significant interaction effect was followed by Student’s
t-test to compare the number of Fos positive nuclei from the two treatments (saline
and nicotine) at each time after drug withdrawal.
7
III. RESULTS
1. Effect of chronic nicotine treatment and its withdrawal on number of
[3 H]nicotine binding sites in rat striatum
Figures 1 and 2 show the effect of chronic nicotine treatment and its withdrawal
on saturation of [3 H]nicotine binding to rat striatal membranes. They are calculated
from Table 1 and 2 by software KELL. Also, Figure 3 shows the effect of chronic
nicotine treatment and its withdrawal on B max of [3 H]nicotine binding. In rats treated
with 4 mg/kg nicotine once a day, the Bmax was increased by 70.8% compared to that
of controls after 1 day withdrawal (24.6 ± 0.65 vs. 14.7 ± 0.68 fmol/mg protein, p <
0.001). After 2 day withdrawal the Bmax was decreased to control levels (14.7 ± 1.25 vs.
12.4 ± 0.32 fmol/mg protein) , and remained unchanged after 7 day withdrawal (13.7 ±
0.81 vs. 11.6 ± 1.26 fmol/mg protein). Similarly, in animals treated with 2.4 mg/kg
nicotine twice a day, the Bmax was increased by 309.4% compared to that of controls
after 1 day withdrawal (86.7 ± 27.9 vs. 21.2 ± 0.28 fmol/mg protein, p < 0.0005).
After 2 day withdrawal the Bmax was decreased to control levels (23.9 ± 0.61 vs. 19.4 ±
0.65 fmol/mg protein, p < 0.005), and remained unchanged after 7 day withdrawal
(22.5 ± 0.37 vs. 19.4 ± 1.20 fmol/mg protein). However, after 1 day withdrawal, the
magnitude of B max increase was significantly (p < 0.0005) higher in animals treated
with 2.4 mg/kg nicotine twice a day than in those treated with 4 mg/kg nicotine once a
day (Fig. 4).
8
Table 1. Nicotine (4 mg/kg, once a day) treatment effects on [ 3 H]nicotine binding
[3H]nicotine
[nM]
Nicotine Day1
Saline Day1
Nicotine Day2
Saline Day2
Nicotine Day7
Saline Day7
Total bound
[dpm]
Non specific
Bound[dpm]
Total added
[dpm]
0.625
1413.0
81.5
47389.2
1.25
2.5
2062.4
2669.6
125.4
209.6
96312.0
192358.0
5
3544.2
435.6
384524.0
10
20
4011.7
5463.0
791.4
1426.9
756837.0
1542820.0
0.625
1.25
2.5
1166.0
1577.7
2158.8
51.6
130.4
221.6
46521.7
94786.8
184135.0
5
2808.3
445.9
370281.0
10
20
3271.6
4341.4
825.8
1722.1
751486.0
1520000.0
0.625
1.25
2.5
990.0
1446.6
2009.5
64.1
151.9
220.7
44497.1
91191.8
185675.0
5
2611.3
399.2
375404.0
10
20
3252.9
4357.0
767.3
1606.5
748429.0
1440000.0
0.625
1.25
2.5
382.2
735.1
1219.5
49.7
101.2
183.8
35323.1
68758.2
142356.0
5
2075.9
362.2
287320.0
10
20
2925.8
4347.7
720.0
1619.1
581227.0
1157550.0
0.625
1.25
2.5
1339.9
1893.7
2509.3
74.8
165.1
282.8
63497.5
126510.0
251218.0
5
3079.8
543.2
499940.0
10
20
3918.9
5193.2
1035.5
2203.2
996149.0
1690000.0
0.625
1.25
2.5
956.0
1588.9
2128.1
56.6
134.1
218.3
43148.8
89771.5
182871.0
5
2409.3
474.1
360453.0
10
20
3298.3
4335.6
888.6
1796.0
692899.0
1458100.0
9
Table 2. Nicotine (2.4 mg/kg, twice a day) treatment effects on [3H]nicotine
binding
[3H]nicotine
[nM]
Nicotine Day1
Saline Day1
Nicotine Day2
Saline Day2
Nicotine Day7
Saline Day7
Total Bound
[dpm]
Non specific
Bound[dpm]
Total Added
[dpm]
0.625
118.4
23.8
51226.2
1.25
2.5
178.8
673.7
28.3
34.1
105377.0
209099.0
5
948.5
83.2
404459.0
10
20
1382.7
2040.1
147.0
333.0
796821.0
1560490.0
0.625
1.25
2.5
1011.4
1346.5
1704.8
9.1
21.0
36.9
49180.1
99502.7
190968.0
5
2093.8
67.8
378562.0
10
20
2474.0
2780.4
165.9
326.5
755365.0
1490540.0
0.625
1.25
2.5
1103.4
1452.6
1811.2
25.5
28.1
43.7
51745.9
104078.0
199794.0
5
2241.1
84.9
396923.0
10
20
2615.6
2972.2
149.1
282.1
766195.0
1535600.0
0.625
1.25
2.5
657.8
998.2
1248.0
12.8
33.8
40.8
51312.4
99660.3
193050.0
5
1724.0
146.5
388828.0
10
20
2022.1
2401.1
150.4
276.5
753325.0
1371360.0
0.625
1.25
2.5
1010.9
1331.9
1709.1
14.8
29.1
41.0
51266.0
103150.0
195983.0
5
2117.9
76.9
391385.0
10
20
2392.0
2724.7
147.0
272.6
766882.0
1460270.0
0.625
1.25
2.5
230.3
401.2
534.2
10.2
13.9
25.9
29856.7
59756.7
114267.0
5
1073.6
41.6
222965.0
10
20
1419.5
1683.6
96.6
182.0
460371.0
880020.0
10
Figure 1. Effect of chronic nicotine treatment (once -daily injection) and its
withdrawal on [3H]nicotine binding to rat striatal membranes. Rats were injected
with (−)-nicotine (4 mg/kg s.c.) once a day for 14 days. Striatum were kept at −70℃
and they were thawed and homogenized. The homogenates of striatum were incubated
with varying concentrations of [3 H]nicotine (0.625-20 nM) at 4℃ for 2 hr. Each point
represents the mean value from three independent experiments.
11
1 Day withdrawal
0.04
Nicotine
Saline
Nicotine
Saline
80
0.03
Bound / Free
Specific Bound (pM )
100
60
40
0.02
0.01
20
0
0
0
10
20
[3H]nicotine (nM )
30
0
40
25
50
75
100
Specific Bound (pM )
2 Day withdrawal
0.032
Nicotine
Saline
Nicotine
Saline
Bound / Free
Specific Bound (pM )
80
60
40
0.024
0.016
0.008
20
0
0
0
10
20
30
40
0
50
15
[3H]nicotine (nM )
30
45
60
75
Specific Bound (pM )
7 Day withdrawal
0.024
Nicotine
Saline
Bound / Free
Specific Bound (pM )
80
60
40
Nicotine
Saline
0.018
0.012
0.006
20
0
0
0
10
20
[3H]nicotine (nM )
30
40
0
15
30
45
Specific Bound (pM )
12
60
75
Figure 2. Effect of chronic nicotine treatment (twice -daily injection) and its
withdrawal on [3H]nicotine binding to rat striatal membranes. Rats were injected
with (−)-nicotine (2.4 mg/kg s.c.) twice a day for 14 days. Striatum were kept at –70℃
and they were thawed and homogenized. The homogenates of striatum were incubated
with varying concentrations of [3 H]nicotine (0.625-20 nM) at 4℃ for 2 hr. Each point
represents the mean value from five independent experiments.
13
1 Day withdrawal
Nicotine
Saline
0.02
45
Bound / Free
Specific bound (pM )
60
30
15
0
Nicotine
Saline
0.015
0.01
0.005
0
0
10
20
30
40
0
15
[3H]nicotine (nM )
30
45
60
Specific Bound (pM )
2 Day withdrawal
0.024
Nicotine
Saline
60
Bound / Free
Specific bound (pM )
80
40
20
Nicotine
Saline
0.018
0.012
0.006
0
0
0
10
20
30
0
40
[3H]nicotine (nM )
15
30
45
60
75
Specific Bound (pM )
7 Day withdrawal
Nicotine
Saline
0.024
Nicotine
Saline
45
Bound / Free
Specific bound (pM )
60
30
15
0.018
0.012
0.006
0
0
0
10
20
30
0
40
[3H]nicotine (nM )
15
30
45
Specific Bound (pM )
14
60
Figure 3. Effect of chronic nicotine treatment (once-daily injection (A) and twicedaily injection (B)) and its withdrawal on Bmax of [3 H]nicotine binding in rat
striatum. Rats were s.c. injected with (–)-nicotine at a dose of either 4 mg/kg, once a
day or 2.4 mg/kg, twice a day for 14 days. The Bmax in striatal tissue was calculated
using the binding data from saturation experiments. Statistically significant difference
from saline assessed by two-way ANOVA. Data are expressed as mean ± SEM of 3-5
independent experiments. *p < 0.001; **p < 0.0005.
15
A
30
Bmax (fmol/mg protein)
i
Saline
Nicotine
*
25
20
15
10
5
0
Day 1
B
Day 2
140
Saline
Nicotine
i
Bmax (fmol/mg protein)
120
Day 7
**
100
80
60
40
20
0
Day 1
Day 2
16
Day 7
*
Bmax (fmol/mg protein)
i
125
4 mg/kg once a day
2.4 mg/kg twice a day
100
75
50
25
0
Day 1
Day2
Day7
Figure 4. Effect of nicotine administration protocol on nAChR density. Rats were
treated for 14 days with 4 mg/kg once a day and 2.4 mg/kg twice a day nicotine
administered. Statistically significant difference from 4 mg/kg assessed by two-way
ANOVA. Data are expressed as mean ± SEM of 3-5 independent experiments. *p <
0.0005.
17
2. Effect of chronic nicotine treatment and its withdrawal on Fos expression in
rat striatum and nucleus accumbens
Figure 7 (see also photomicrographs in Figure 5 and 6) shows the effect of
chronic nicotine treatment (2.4 mg/kg s.c. twice a day) and its withdrawal on the
number of Fos-like immunoreactive nuclei in rat striatum and nucleus accumbens.
After 1 day withdrawal, the number of Fos-like immunoreactive nuclei in the striatum
and nucleus accumbens was decreased by 46.2% and 42.1%, respectively, compared to
that of controls (17.6 ± 1.70 vs. 32.6 ± 1.24, p < 0.0005 and 11.3 ± 0.87 vs. 19.6 ±
1.66, p < 0.005, respectively). After 2 day withdrawal, the number of Fos-like
immunoreactive nuclei in the striatum was still decreased compared to that of controls
(21.3 ± 1.01 vs. 31.1 ± 1.90, p < 0.01), but was increased to control levels in the
nucleus accumbens (15.9 ± 0.14 vs. 16.7 ± 0.23). After 7 day withdrawal, there was no
significant difference in the number of Fos-like immunoreactive nuclei between
nicotine- and saline-treated animals in both striatum and nucleus accumbens (32.1 ±
2.17 vs. 32.4 ± 1.26 and 17.8 ± 1.05 vs. 18.2 ± 1.59, respectively).
18
Saline
Nicotine
Day1
D1S/S
D1N/S
D2S/S
D2N/S
D7S/S
D7N/S
Day2
Day7
Figure 5. Photomicrographs illustrating Fos-like immunoreactive cells in the
striatum. At 200X magnification after 2 hr nicotine (0.4 mg/kg s.c.) challenge. Saline
group were treated saline and nicotine group were nicotine (2.4 mg/kg, twice a day
s.c.) treated for consecutive 14 days. Day is time after withdrawal treatment.
19
Saline
Nicotine
Day1
D1S/N
D1N/N
Day2
D2S/N
D2N/N
Day7
D7S/N
D7N/N
Figure 6. Photomicrographs illustrating Fos-like immunoreactive cells in the
nucleus accumbens. At 200X magnification after 2 hr nicotine (0.4 mg/kg s.c.)
challenge. Saline group were treated saline and nicotine group were nicotine (2.4
mg/kg, twice a day s.c.) treated for consecutive 14 days. Day is time after withdrawal
treatment.
20
Figure 7. Effect of a challenge injection of nicotine (0.4 mg/kg, s.c.) on Fos -like
immunoreactivity in the striatum (A) and nucleus accumbens (B) of rats during
chronic nicotine treatment and its withdrawal. Rats were treated with (–)-nicotine
(2.4 mg/kg s.c.) twice a day for 14 days and received a challenge of nicotine 2 hr
before anesthesia. On 1, 2 and 7 days after the last administration of nicotine, animals
received the acute nicotine challenge. Data are expressed as mean ± SEM of 3-6
observations. *p < 0.005, **p < 0.01.
21
i
40
Fos-like immunoreactive nuclei
A
Saline
Nicotine
30
**
20
*
10
0
Day1
i
25
Fos-like immunoreactive nuclei
B
20
Day2
Day7
Saline
Nicotine
15
*
10
5
0
Day1
Day2
22
Day7
IV. DISCUSSION
The majority of previous studies on the effects of chronic nicotine
administration have shown that nicotine produces an increase in the number of
[3 H]nicotine binding sites in the brain. The fact that chronic agonist administration
produces up-regulation, rather than down-regulation which occurs with many other
receptor systems, has been attributed to the classical categorization of nicotine as both
an agonist and antagonist. Schwartz and Kellar (1985) have shown that repeated
administration of the nicotine and results in nAChR up-regulation.
We examine the changes in number of nAChRs (Fig. 3). The effect of chronic
nicotine treatment and its withdrawal on B max of [3 H]nicotine binding. In rats treated
with 4 mg/kg nicotine once a day, the Bmax was increased by 70.8% compared to that
of controls after 1 day withdrawal (24.6 ± 0.65 vs. 14.7 ± 0.68 fmol/mg protein, p <
0.001). After 2 day withdrawal the Bmax was decreased to control levels (14.7 ± 1.25 vs.
12.4 ± 0.32 fmol/mg protein), and remained unchanged after 7 day withdrawal (13.7 ±
0.81 vs. 11.6 ± 1.26 fmol/mg protein). Similarly, in animals treated with 2.4 mg/kg
nicotine twice a day, the Bmax was increased by 309.4% compared to that of controls
after 1 day withdrawal (86.7 ± 27.9 vs. 21.2 ± 0.28 fmol/mg protein, p < 0.0005).
After 2 day withdrawal the Bmax was decreased to control levels (23.9 ± 0.61 vs. 19.4 ±
0.65 fmol/mg protein , p < 0.0005), and remained unchanged after 7 day withdrawal
(22.5 ± 0.37 vs. 19.4 ± 1.20 fmol/mg protein).
A comparison of the concentrations of nicotine achieved by the different
administration procedures with the extent of receptor up-regulation achieved presents
23
an unusual outcome. In vitro the subunit composition of nAChRs determines the
degree to which receptors are desensitized by various concentrations of nicotine
(Fenster et al., 1997). In striatum examined, the more frequent (but lower individual
dose) treatments resulted in less nAChR up-regulation. It would seem that the more
frequent administration procedures should more closely resemble the constant infusion
protocol than twice-per-day injections; however, with respect to the effect on nAChR
density, this is not the case (Rowell and Li, 1997).
In any event, it appears that two times a day subcutaneous injection are more
effective at producing nAChR up-regulation than more frequent injections at same
dose. Perhaps a longer lasting receptor “inactivation” process, which might be
achieved only at higher nicotine concentrations , is necessary for nAChR up-regulation
with these administration protocols. Long-lasting nAChR inactivation at high nicotine
concentrations has been reported to occur at peripheral nAChRs (Aoshima, 1984;
Robinson and McGee, 1985; Egan and North, 1986; Simasko et al., 1986; Siegel and
Lukas, 1988; Lukas, 1991). There is also evidence that long-term receptor inactivation
occurs in vivo (Sharp and Beyer, 1986; Hulihan-Giblin et al., 1990).
The results of this study on the change of the nAChR density between 4 mg/kg
one time a day and 2.4 mg/kg two times a day injection showed that a daily nicotine
dose of 2.4 mg/kg was required to achieve significant nAChR up-regulation in
striatum. After 1 day withdrawal, the magnitude of B max increase was significantly (p
< 0.0005) higher in animals treated with 2.4 mg/kg nicotine twice a day than in those
treated with 4 mg/kg nicotine once a day (Fig. 4).
Whatever the mechanism, the results of this study indicate that the processes
24
that lead to an increase in nAChR density in brain tissue are rather complex. It appears
that the daily nicotine dose is an important consideration, but the manner in which the
dose is administered is even more important. It does not appear that in vivo
desensitization alone is sufficient to produce nAChR up-regulation. An increase in
central nicotinic receptors has been shown to occur in the brains of cigarette smokers
(Benwell et al., 1988), but it is not clear whether this would be produced by the use of
constant delivery devices such as the nicotine transdermal patch. However, if nicotine
or other nicotinic agonists are to be used effectively for smoking cessation or as
therapeutic agents are the consequences of the delivery protocols on central nAChRs
must be better understood.
In order to identify targets of nicotine, we have studied the expression of the
transcription factor c-fos through quantification of immunoreactive nuclei in two brain
regions after challenge and chronic nicotine treatment. Our study shows that the
number of c-Fos-positive nuclei was increased in specific cerebral regions 2 hr after a
challenge subcutaneous injection of a relatively small dose of nicotine (0.4 mg/kg)
given in chronic nicotine rats (2.4 mg/kg s.c. injected two times a day for 14 days).
Regions consistently showed an attenuated number of c-Fos-positive nuclei following
chronic nicotine. After chronic nicotine protocol, an attenuate of c-Fosimmunoreactive nuclei was also found in striatum and nucleus accumbens. Chronic
nicotine produced a persistent and even amplified c-fos induction.
After 1 day withdrawal, challenge nicotine did not increase the Fos
immunostaining in striatum and nucleus accumbens at this time point (Fig. 5). The
inability of nicotine to induce Fos expression in striatum and nucleus accumbens after
25
1 day withdrawal could be caused by the prolonged inactivation of nAChRs.
Dopaminergic pathways are not necessarily the only ones involved in mediating
nicotine’s effects on Fos protein expression during withdrawal. Cortical and thalamic
glutamatergic inputs to the striatum may modulate the dopaminergic activation of
postsynaptic intracellular mechanisms that lead to changes in Fos expression (Harlan
and Garcia, 1988).
On the 7th day after constant nicotine injection, we expect attenuation of
nicotine’s effects on Fos expression in the striatum and nucleus accumbens was fully
recovered. The postsynaptic effects of nicotine in major dopaminergic target areas as
estimated by Fos expression were increased on the 7th day after nicotine injection, and
challenge nicotine did not activate Fos expression in these areas at 1 day after nicotine
withdrawal. Challenge nicotine treatment induced no changes on Fos expression in the
striatum and nucleus accumbens in rats on the 7th day after continuous nicotine
injection.
These findings agree with microdialysis studies (Benwell et al., 1992; Benwell
and Balfour, 1997) where the effects of acute nicotine challenge on extracellular
concentration of DA in the dorsal striatum and on those of DA, 3,4dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the nucleus
accumbens were attenuated during constant nicotine injection as compared with
controls. This phenomenon could be caused by the continuous presence of an agonist,
in this case nicotine, desensitizing the nAChRs regula ting the dopaminergic neurons.
We thought that the attenuation of nicotine’s effect on DA metabolism occurred with
prolonged exposure to nicotine concentrations approximately similar to these found in
26
the plasma of heavy smokers. Furthermore, such plasma concentrations indicate high
cerebral nicotine concentrations in rat (Deutsch and Cameron, 1992; Rowell and Li,
1997). Lippiello et al. (1995) observed that nicotine tends to stabilize nAChRs in the
high-affinity conformation that is related to the process of functional desensitization.
Thus, our experiments indicate that nAChRs involved in the control of striatum and
nucleus accumbens DA turnover was desensitized during constant nicotine injection.
We have previously suggested that nAChRs involved in the change of density
are effected to in the regulation of the intraneuronal DA metabolism their response to
chronic nicotine administration in rat. It was that we now found in the effect of
nicotine on striatal nAChR and Fos expression of striatum and nucleus accumbens in
rats give further support to this suggestion. Thus, there may be variations in the
functional states or in the subunit compositions of nAChRs mediating nicotine’s
various effects on dopaminergic transmission or differences in receptor distribution in
the limbic and striatal areas and their input areas.
27
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32
ABSTRACT
Effect of Chronic Nicotine Administration and its Withdrawal on
Neuronal Nicotinic Acetylcholine Receptor Density and Fos Expression
Ah Jung Chung
Department of Biochemistry Hanyang University
Thesis Advisor: Profs. Young Gyu Chai and Sang Eun Kim
Chronic nicotine administration produces an up-regulation of nAChR number
and an attenuation of Fos expression in neurons of dopaminergic target areas.
However, little is known about the changes in nAChRs and Fos expression during
withdrawal from chronic nicotine exposure.
The purpose of the present study was to investigate the effect of chronic
nicotine administration and its withdrawal on number of nAChRs and nicotineinduced Fos expression. We also examined the effects of different nicotine
administration schedules on the nAChR density.
Rats received subcutaneous injections of nicotine for 14 consecutive days at the
dose of 4 mg/kg, once a day or 2.4 mg/kg, twice a day. In vitro binding of [3 H]nicotine
to rat striatal membranes was measured using tissues obtained 1, 2 and 7 days after the
last nicotine dose. Also, Fos-like immunoreactive nuclei were measured in the rat
nucleus accumbens and striatum 1, 2 and 7 days after the last nicotine dose using
immunohistochemistry.
In rats treated with 2.4 mg/kg nicotine twice a day, the Bmax of [3 H]nicotine
binding was increased by 309.4% compared to that of controls after 1 day withdrawal.
33
After 2 day withdrawal the Bmax was decreased to control levels, and remained
unchanged after 7 day withdrawal. Similar results were obtained when animals were
treated with 4 mg/kg nicotine once a day. However, after 1 day withdrawal, the
magnitude of Bmax increase was significantly higher in animals treated with 2.4 mg/kg
nicotine twice a day than in those treated with 4 mg/kg nicotine once a day.
After 1 day withdrawal, 46.2% and 42.1% decreased the number of Fos-like
immunoreactive nuclei in the striatum and nucleus accumbens, respectively, compared
to that of controls. After 2 day withdrawal, the number of Fos-like immunoreactive
nuclei in the striatum was still decreased compared to that of controls , but was
increased to control levels in the nucleus accumbens. After 7 day withdrawal, there
was no significant difference in the number of Fos-like immunoreactive nuclei
between nicotine- and saline-treated animals in both striatum and nucleus accumbens.
The results indicate that up-regulation of nAChR number induced by chronic
nicotine exposure may be rapidly recovered after its withdrawal. The inability of
nicotine to induce Fos expression in striatum and nucleus accumbens after 1 day
withdrawal suggests that nAChRs were inactivated during chronic nicotine
administration. This supports the hypothesis that desensitization of neuronal nAChRs
induced by chronic exposure to nicotine initiates up-regulation of nAChR number. The
results also suggest that the manner of nicotine administration may be a more
important factor for nAChR up-regulation than the daily nicotine dose.
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ACKNOWLEDGEMENT
감사의 글
안산에서의 1 년과 서울에서의 1 년, 저에게 짧지만 너무나도 의미 있고 소중
한 시간을 있게 해 주시고 도움을 주신 모든 분들에게 짧은 글이지만 저의 감사의
마음을 전하고 싶습니다.
학부 때는 수업시간에만 뵙던 교수님들을 좀더 가까이서 뵙고 학문과 인생
에
대한 많은 것을 배울 수 있었던 소중한 시간이었습니다. 언제나 묵묵히 저희들
을 지켜봐 주시던 조기승 교수님, 지도 교수님으로써 많은 가르침을 주신 채영규
교수님, 항상 따뜻한 웃음으로 대해주시던 정일엽 교수님, 세심하게 학생들을 배려
해 주시던 김효준 교수님, 언제나 한결같은 모습의 이영식 교수님, 선배님 이시자
교수님으로 항상 저희를 이해해주시던 황승용 교수님, 너무도 감사하다는 말씀을
드리고 싶습니다. 안산에서의 짧았던 1 년 동안 실험실 생활에 적응할 수 있도록
저를 여러 가지로 도와주신 유전공학실험실의 고마운 여러 선배님들과 후배님들,
그리고 자주 찾아 뵙지 못해 항상 죄송스럽던 분자유전학실험실, 분자생물실험실과
면역학실험실, 그리고 언제나 대학원생들을 이끌어 주시던 생화학실험실 선배님들
께 진심으로 감사 드립니다. 학생의 신분으로 나와있던 삼성생명과학연구소에서의
1 년도 저에게는 많은 것을 배울 수 있는 소중한 시간이었습니다. 전혀 다른 분야
에 와서 아무런 지식도 없던 저를 지도하시며 새로운 분야를 알 수 있게 해 주신
김상은 선생님께 가장 감사 드립니다. 처음으로 해보는 직장 아닌 직장생활에 힘들
어 하던 저를 너무도 잘 보살펴주신 핵의학과 연구원여러분, 그리고 항상 따뜻하게
35
맞아주던 3 번 연구실 연구원들, 그리고 저의 보금자리가 된 1 번 연구실 연구원들
모두에게 감사를 드립니다.
그리고 무엇보다도 지금까지 저를 돌봐주신 부모님께 더욱 깊은 감사를 드
립니다. 제가 선택한 길을 지금까지 잘 걸어올 수 있도록 묵묵히 바라봐 주시고 응
원해 주신 아빠, 항상 저 때문에 새벽잠을 설치시고 고생하시는 엄마께 너무 감사
하고 사랑한다는 말씀을 드리고 싶습니다. 또 힘들고 바쁜 직장 생활에서도 동생을
걱정해주던 오빠와 대학원생활로 지치고 힘들 때 위로하며 투정을 받아주던 태훈
오빠에게도 감사의 말을 전하고 싶습니다.
힘들고 어려웠던 일들과 즐겁고 행복했던 대학원 시절의 모든 추억을 있게
해주시고 이 논문을 있게 해 주신 여러분들께 진심으로 감사 드립니다.
2000. 6
36