NEUROGENESIS Y PLASTICIDAD DEL HIPOCAMPO ADULTO

NEUROGENESIS Y PLASTICIDAD DEL HIPOCAMPO ADULTO
Alejandro F. Schinder - Laboratorio de Plasticidad Neuronal
Instituto Leloir – Buenos Aires – Argentina
aschinder@leloir.org.ar
ADULT HIPPOCAMPAL NEUROGENESIS
NEURAL DEVELOPMENT
SYNAPTOGENESIS
NETWORK INTEGRATION
FUNCTION
MODULATION
TOOLBOXES
THE HIPPOCAMPUS: A CORTICAL AREA FUNDAMENTAL FOR LEARNING AND MEMORY
The hippocampus is involved in
learning
Memory (in humans: autobiographic/episodic memory)
spatial navigation
emotional control of behavior
the hippocampal circuit and its “simple” lamellar organization
E. Kropff
E. Kropff
KNOWN HIPPOCAMPAL MECHANISMS TO PROCESS SPATIAL INFORMATION
PLACE CELLS: SPACE CAN BE ENCODED IN THE FIRING PATTERN OF HIPPOCAMPAL NEURONS
RESPONSES RECORDED FROM 4
NEURONS DURING EXPLORATION OF A
LINEAR CAGE
RESPONSES RECORDED FROM 80
NEURONS DURING EXPLORATION OF A
SQUARE CAGE
FROM NAKAZAWA ET AL. 2004 (O’KEFFEE AND DOSTROVSKY ’70s, WILSON AND MCNAUGHTON 90´s)
SPACE CODING BY THE HIPPOCAMPUS: PLACE CELLS
“PLACE cells” recognize particular spots in the environment
“GRID cells” generate a coordinate system for spatial localization
The
hippocampal
trisynaptic
circuit
New granule cells are continuously generated in the adult dentate gyrus
Neural stem cells labeled in the dentate gyrus
of a Nestin-GFP transgenic mosue
Quiescencia, activación, proliferación, diferenciación, maduración neuronal
METHODS TO IDENTIFY ADULT-BORN NEURONS
IT MUST BE DETERMINED THAT A CELL: 1. WAS BORN IN THE ADULT BRAIN
2. IS A NEURON
BROMODEOXYURIDINE
RETROVIRUS
TRANSGENIC MICE
COMPARISON OF METHODS FOR LABELING ADULT-BORN NEURONS
RETROVIRUSES AS MEANS TO IDENTIFY/MANIPULATE ADULT-BORN DENTATE GRANULE CELLS
IN VIVO EXPRESSION OF EGFP IN NEURAL PROGENITOR CELLS USING
A MURINE LEUKEMIA VIRUS - DERIVED VECTOR
6 – 7 week-old
female mice
Adult mouse dentate gyrus
Electrophysiological recordings in hippocampal slices
Confocal microscopy in fixed brain sections
Retroviral transduction renders accurate birth dating of new neurons
Neurons develop in vivo, analysis is performed ex vivo
retroviral
labeling
Only infects
dividing cells!
t=0
1-3d
(neuron’s DOB)
1-2 wk
4 wk
(Time ~
= age of neuron)
NEW NEURONS DEVELOP OVER SEVERAL WEEKS IN THE ADULT MOUSE DENTATE GYRUS
Verónica Piatti
ML
Molecular Layer
14 dpi
GCL
Granule Cell Layer
SGZ
Subgranular Zone
Espósito , Piatti et al, J Neurosci 2005
Verónica Piatti
ML
Molecular Layer
28
21dpi
dpi
GCL
Granule Cell Layer
SGZ
Subgranular Zone
Espósito , Piatti et al, J Neurosci 2005
Slow development and step-wise innervation
Excitatory and inhibitory inputs
glut
glut
Synaptic inputs
glut
GABA
GABA
GABA
GABA
GABA
DCX
3 days
7d
14 d
Expression of early and late neuronal markers
Calbindin`
21 d
Development of
synaptic inputs
GABA
28 d
1) Dendritic GABA (depolarizing)
2) Glutamate in dendritic spines
3) Perisomatic GABA (hyperpolarizing)
Espósito et al, J Neurosci 2005; Piatti et al, J Neurosci 2011
Ge et al, Nature 2006; Overstreet Wadiche et al, J Neurophysiol 2005, J Neurosci 2006
Zhao and Gage, J Neurosci 2006, and others!
42 days
PERSISTENT LABELING OF SPECIFIC CELL TYPES IN GENETICALLY MODIFIED MICE
Cre LoxP SYSTEM
Example: CAG promoter
cag expression)
(ubiquitous
Example: Mash1 prom
(Neural stem cells)
Prom. X
Cre
ER
Allele 1
Prom. Y
flx STOP flx
TOMATO
Allele 2
TAMOXIFEN
Prom. Y
Progeny of neural stem
cells shows red fluorescence
flx
TOMATO
1+2
Cre-LoxP system for targeted mutation or expression
METHOD II
Genetic tagging of adult-born granule cells
6–week–old
mice
Mash1CreERT2; CAG floxStopTomato
mouse
3
8
| | | |
14
|
22
|
28
|
49
|
days after TAM
TAM
Yang et al, J Neurosci 2015
Massive amount of new granule cells in the adult dentate gyrus
Mash1CreERT2; CAG floxStopTomato mouse
Tam induction at > 2 months
TIME-DEPENDENT MATURATION OF PASSIVE MEMBRANE PROPERTIES
Development of neuronal excitability
Neuronal age = 22 days
Neuronal age = 28 days
Neuronal age > 60 days
Mongiat 2009
Retroviral labeling of developing GCs
Yang 2015
Genetic labeling of developing GCs
Cohorts of fast-dividing progenitors
Cohorts of slow + fast dividing progenitors
How does neurogenesis influence the local network?
GABA
INPUT
NETWORK
glu
FFI
OUTPUT
NETWORK
FBI
FFI
ARE NEURONS BORN IN THE ADULT HIPPOCAMPUS ANY DIFFERENT (to those born in development)?
Diego Laplagne
Soledad Espósito
MATURE NEURONS BORN IN THE DEVELOPING vs. ADULT HIPPOCAMPUS
GFP retrovirus
(ventricle)
E15
+
RFP retrovirus
(right DG)
Adult (P42-45)
Laplagne, Espósito et al, PLoS Biol 2006
Laplagne, Kamienkowsi et al, EJN 2007
PAIRED COMPARISONS OF GLUTAMATERGIC AFFERENTS OF DEVELOPMENT AND ADULT-BORN DGCs
Diego Laplagne
Soledad Espósito
DGCs born in the developing vs. adult hippocampus: remarkable similarity of functional inputs
New DGCs are capable of integrating an excitatory drive and spike
Similar firing behavior
Laplagne, Espósito et al, PLoS Biol 2006
Laplagne, Kamienkowski et al, EJN 2007
MATURE Granule cells born in the developing vs. adult hippocampus
display a remarkable functional similarity
=
Laplagne, Espósito et al, PLoS Biol 2006
Laplagne, Kamienkowsi et al, EJN 2007
At what developmental stages are new GCs
relevant for dentate function?
??
? ?? ?
DCX
3 days
7d
14 d
21 d Calbindin
28 d
“RELEVANT”:
Integrate excitatory signal, spike, activate postsynaptic cell
42 d
Spiking is determined by the balance between
weak glutamatergic input and high membrane excitability
MPP
H
GCL
Rm
glu
inputs
Spiking neurons at
maximal PP stimulus (%)
ML
Neuronal Age (days)
Neuronal age
New neurons become
“relevant” by 3 weeks
Mongiat et al, PLoS ONE 2009
Neuron 2007
Whole cell
recording
LTP of glutamatergic inputs is stronger in 4 week-old granule cells
Immature GCs exhibit unique functional properties
4–week–old GCs:
a critical period
High excitability
(Schmidt-Hieber 2004, Mongiat 2009)
Reduced GABAergic inhibition
(Snyder 2001, Espósito 2005, Ge 2006)
Increased synaptic plasticity in
glutamatergic inputs and output
immature
(critical period)
Within CP
mature
Outside CP
(Snyder 2001, Schmidt-Hieber 2004,
Ge 2007, Gu 2012)
glut
Experience determines populations of
input neurons
(Bergami, 2015)
glut
GABA
GABA
What is their ability to process information?
GABA
Immature GCs are more easily activated
than mature GCs
Input activation (axons) triggers spikes in retrovirally labeled DGCs
(immature)
Immature neurons require fewer active inputs to spike
Immature GCs are more easily activated than mature GCs
Input activation (axons) triggers spikes in retrovirally labeled DGCs
(immature)
The difference in the activation of immature and mature GCs is due to GABAergic inhibition
Marín–Burgin et al, Science 2012
Differential inhibitory control enhances activation of immature DGCs
MONOSYNAPTIC EXCITATION
4 wpi
mature
spike
inhibition
o
o
DYSYNAPTIC
FEEDFORWARD INHIBITION
excitation
mature
STRONG FFI
young
WEAK FFI
excitation / inhibition balance @ spike → 4:1 (immature) vs. 2:1 (mature)
Marín–Burgin et al, Science 2012
How do young vs. mature GCs integrate different inputs?
Ca 2+ imaging to assess neuronal populations in the GCL
RFP+ neurons (4 wpi)
% Neurons responding
to both inputs (integration) =
*100
RFP- neurons (mature)
2+
Ca elevation occurs in spiking neurons
Neurons loaded with
calcium-sensitive dye
loose patch
spike
RFP neurons (4 wpi)
failure
Set-up:
ΔF/F0
loose patch
ΔF/F0
DGCs responsive to input 1 only
DGCs responsive to input 2 only
DGCs responsive to both inputs
RFP+ neurons (4 wpi)
Response to input 1
Response to input 2
Data overlay
GCs responding
to both inputs (%)
Immature GCs display enhanced input integration
Within a limited time window
Marín–Burgin et al, Science 2012
The main differences in the activity of young vs mature GCs
are due to network properties
Cortical input
mature
young
WEAK FEEDFORWARD
INHIBITION
STRONG FEEDFORWARD
INHIBITION
mature GC
Scattered spikes
young GC
Frequent spikes
YOUNG • Low spiking threshold
• Refractory to feedforward GABAergic inhibition
GCs
• Low input specificity
• By 8 weeks they are similar to mature GCs
Conclusions I
 Developing GCs undergo a critical period of enhanced excitability that
determines their low input specificity
 Low input specificity of young GCs is due to their poor coupling to inhibitory
networks that control spiking
 Upon maturation, new GCs become highly input specific
 This transition may be relevant to their proposed role in hippocampal
function
HOW CAN WE INVESTIGATE THE OUTPUT OF NEW NEURONS?
TOOLBOX
OPTOGENETICS
AND
CHEMOGENETICS
What is optogenetics?
“Optogenetics is the combination of optical and
molecular strategies to monitor and control designated
molecular and cellular activities in living tissues and cells
using genetically encoded photosensitive proteins.”
2005
Heterologous expression of ChR2 in hippocampal cultured neurons
Heterologous expression of ChR2 in hippocampal cultured neurons
Channelrhodopsin 2
Halorhodopsin
Engineered GPCR
Deisseroth, Nat Methods 2010
DIFFERENT FLAVORS FOR LIGHT-INDUCED DEPOLARIZATION
Deisseroth Nat Methods 2012
HOW NEURONS REACT TO DIFFERENT ChR2 FLAVORS
Deisseroth Nat Methods 2012
Emx: promotor panneuronal
OPTOGENETICS
STRENGTHS
Specificity of neuronal type
LIMITATIONS
Requires invasive stimulation
(implantation of optic fiber)
Neuronal activation or inihibition
Fast kinetics for neuronal control
Activation jitter is broader than
electrical stimuli
Can reach high frequency
Laser may heat tissue
Genetically modified mice available
Long-lasting manipulations may be
difficult
CHEMOGENETICS
What is chemogenetics?
“The term chemogenetics has been used to describe the
processes by which macromolecules can be engineered
to interact with previously unrecognized small
molecules. In neuroscience, receptors that can alter
neuronal excitability are being engineered to bind
artificial ligands. “
DREADDS - Designer Receptors Activated Solely by Designer Drugs
Wess et al., 2013
DREADDS - Designer Receptors Activated Solely by Designer Drugs
Wess et al., 2013
Next generation of DREADDS - ligand-gated ion channels activated by synthetic ligands
Wess et al., 2013
CHEMOGENETICS
STRENGTHS
Specificity of neuronal type
LIMITATIONS
Time course of activation/blocking
dependent on pharmacokinetics
Neuronal activation or inihibition
Slow kinetics
Activation by drug in drinking water
or by i.p. injection (non-invasive)
When activating neurons, uncontrolled
firing patterns (may accommodate)
Allows long-term control
G-coupled receptors may alter neuronal
properties other than firing
Nature Neuroscience 2012
LTP of glutamatergic outputs is stronger in 4 week-old granule cells
Cells with single place fields
Location: GCL
Putative mature GCs
Cells with multiple place fields
Location: GCL/hilus
Putative newborn GCs
The dentate gyrus of the hippocampus is involved
in the discrimination of similar memories
Pattern Separation (dentate gyrus):
Similar inputs produce orthogonal outputs
Output
Input
Adapted from J. Knierim
Cellular players in DG output
GABA
glu
FFI
OUTPUT
NETWORK
FBI
FFI
What is the impact of immature neurons in the local network?
?
What targets are activated by developing adult-born neurons?
Goal: compare local networks recruited by young vs mature GCs
mature
young
NeuN
GFP
Similar activation of young and mature ChR2-GCs by light
447 nm
1 ms
Temprana et al, Neuron 2015
Developmentally-generated ChR2-GCs activate monosynaptic
excitation and disynaptic feedforward inhibition in CA3
EXCITATION
447 nm
1 ms
Vh = -70 mV
EXC
FFI
FEEDFORWARD INHIBITION
CA3
Vh = 0 mV
Developmentally-generated ChR2-GCs activate
disynaptic feedback inhibition onto the GCL
EXCITATION
Vh = -70 mV
447 nm
1 ms
GCL
FEEDBACK INHIBITION
Vh = 0 mV
FBI
Temprana et al, Neuron 2015
Similar activation of CA3 networks by young and mature GCs
young
mature
feedforward
inhibition
excitation
FFI
CA3
CA3 excitation
CA3 Inhibition
Local networks
Poor recruitment of feedback inhibition by young GCs
young
mature
feedback
inhibition
GCL
DG Feedback Inhibition
X
Temprana et al, Neuron 2015
Effect of feedback inhibition by adult-born GCs on the activity of the
principal layer: Field recordings of population spike
stim 2
stim 1
stim 1
GCL
1
stim 2
2
Light-induced reduction of pop-spike by mature ChR2-GC
Reduction of pop-spike by light stimulation of mature GCs
depends on GABAA receptors
Light-evoked reduction (%)
young mature
• Young ChR2-GCs exert poor control of GCL
• Pop-spike reduction by mature ChR2-GC involves feedback
inhibition by GABA interneurons
Temprana et al, Neuron 2015
Mature GCs exert powerful feedback inhibition onto the GCL
How do they affect immature GCs?
Expression of ChR2 in mature GCs
and tdTomato in young GCs
ChR2-GFP
GC-Tom
Feedback inhibition onto young vs MTR GCs
GCL
Immature GCs escape feedback inhibition
Temprana et al, Neuron 2015
A little help from synthetic biology:
use of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)
Acute activation of new GCs by hM3Dq
Retroviral transgene
Retroviral
injection
I
t=0
Vehicle
CNO
I
28 days
CNO
hM3+ ARC+ / hM3+ neurons
(@ 7 h post CNO)
hM3Dq-GFP ARC NeuN
80
60
40
20
0
CNO Veh
Temprana et al,
Neuron 2015
Use of Designer Receptor Exclusively Activated by Designer Drugs
(DREADD HM3Dq) to activate adult-born neurons in vivo
CNO activates hM3-GCs (c-Fos expression)
GFP (hM3-GC)
c-Fos
NeuN
Overlay
Arc+ hM3-GC (%)
Roth and col, Neuron 2009
In vivo activation of PV+ interneurons by mature hM3-GCs
PV+
c-Fos
DAPI
Overlay
Stimulation of mature but not
young GCs elicits activation
of PV interneurons
young
hM3-GC
mature
hM3-GC
Temprana et al, Neuron 2015
GC – interneuron – GC feedback network: The GC – interneuron synapse
PV+ interneurons are direct targets of adult-born GCs
HILUS
Temprana et al, Neuron 2015
TRANSITION
FBI: feedback inhibition
FFI: feed-forward inhibition
DG
CA3
CA3
dentate gyrus
RECRUIT
Mature GCs  recruit CA3 pyr, FFI
Young GCs  recruit CA3 pyr, FFI
FBI (PV+ INs), control GCL activation
not FBI
RECEIVE
strong FBI
escape FBI
Conclusions II
 Distal contacts onto CA3 pyramidal cells are mature, while synaptogenesis
onto proximal GABAergic hilar interneurons is delayed
 During the critical period of high excitability and plasticity, new GCs are
poorly coupled to feedback inhibition (responsiveness + recruitment)
 Upon maturation, new GCs become highly coupled to inhibitory networks
 Parallel channeling of information arriving to the dentate gyrus: highly active
cohorts of young non-specific GCs and highly input-specific mature GCs
 WHAT IS THE POSSIBLE ROLE OF THIS HIGH-COST NETWORK
REMODELING?
Novel input discrimination (pattern separation):
Similar inputs produce orthogonal outputs
Output
Input
Adapted from J. Knierim
A conceptual model on the role of developing GCs
in novel input discrimination
MODULATION OF ADULT NEUROGENESIS
Some factors that modulate adult neurogenesis
A brief (30 min) experience in enriched environment
increases activity in the granule cell layer – ARC expression
Time (min)
0
30
60
90
120
EE
Control
EE
Arc
NeuN
GCs expressing Arc (%)
Perfusion
2.5
2.0
1.5
1.0
0.5
0
Control
EE
Survival of new neurons depends on usage during a critical period
Tashiro, J Neurosci 2007