El circuito del hipocampo de roedores es uno de los modelos de plasticidad mejor caracterizados
El giro dentado del hipocampo adulto genera neuronas durante toda la vida
ARE NEW CELLS OF THE ADULT MAMMALIAN BRAIN REALLY FUNCTIONAL NEURONS?
IN VIVO EXPRESSION OF EGFP IN NEURAL PROGENITOR CELLS
USING A MURINE LEUKEMIA VIRUS - DERIVED VECTOR
Pcmv
LTR

prom EGFP
Retroviruses can integrate in
the genome of dividing cells
WPRE
LTR
Neural Progenitor Cell
The transgene (GFP) is
expressed by the progeny
of transduced cells
IN VIVO RETORVIRAL LABELING OF ADULT-BORN NEURONS
retroviruses
ONLY
label
dividing
retrovirus
cells
7 week-old mice
Electrophysiological analysis
Morphological Analysis
A modified mouse leukemia retrovirus was used to express GFP in dividing cells of the adult DG
van Praag et al 2002
Dendritic spines
Neuronal morphology
1-month-old
4-months-old
EM – synaptic structure
Functional properties
GFP+ neurons fire action potentials and receive functional connections
ADULT NEUROGENESIS:
AN ONGOING DEVELOPMENTAL PROCESS IN A MATURE ENVIRONMENT
Complex neuronal features by 2 months
Morgenstern, Lombardi et al, J Physiol 2008
DEVELOPING NEURONS IN THE ADULT DENTATE GYRUS
7 days
DCX  young neuronal marker
HIGH DCX EXPRESSION
ABSENCE OF CELL POLARITY
SGZ LOCALIZATION
14 days
GFP
DCX
28 days
NeuN
NeuN EXPRESSION BEGINS
MIGRATION TO GCL
POLARITY ESTABLISHED
SIMPLE DENDRITIC TREE
NO SPINES
HIGH NeuN EXPRESSION
MIGRATION HAS ENDED
MATURE NEURONAL MORPHOLOGY
COMPLEX DENDRITIC TREE
PRESENCE OF SPINES
(glu input)
Espósito, Piatti et al, JNeurosci 20
REGISTROS DE WHOLE-CELL PATCH CLAMP DE NEURONAS GFP+
SOME GABAERGIC INTERNEURONS OF THE HIPPOCAMPUS
GCL
Stimulation
Freund and Buzsaki, 1996
RECORDINGS OF POSTSYNAPTIC RESPONSES
ions
Graham Johnson, Graham Johnson Medical Media, Boulder, Colorado
1-7 dpi
8-20 dpi
>21 dpi
NO SYNAPTIC INPUT
GABAERGIC INPUTS
GABA and glu INPUTS
incipient excitability
higher excitability
mature excitability
Time course of afferent connectivity
Silent
GABA dendr.
glu
GABA somatic
(similar to perinatal development)
Espósito y col., JNeurosci 2005
DEVELOPMENT OF NEW NEURONS IN THE ADULT DG
0
1-3d
7d
15d
30d
(Time)
QUESTIONS II
• WHY IS THE HIPPOCAMPUS PRODUCING NEW NEURONS?
• ARE NEW NEURONS DIFFERENT FROM THOSE BORN DURING DEVELOPMENT?
• DO THEY REMAIN AS A DISTINCT NEURONAL POPULATION?
MORPHOGENESIS OF THE DENTATE GYRUS
> 80% of DGCs ARE BORN POSTNATALLY
postnatal
NG
HOW CAN WE SEPARATE UNAMBIGUOUSLY
EACH NEURONAL POPULATION ?
embryonic
NG
adult
NG
E15 P1
~P7
~P14
NEED TO USE DIFFERENT TOOLS FOR
LABELING NEURONS BORN IN
DEVELOPMENT AND ADULTHOOD
GFP retrovirus
(ventricle)
EMBRYO BORN
GFP retrovirus
(right DG)
POSTNATALLY BORN
GFP retrovirus
(right DG)
ADULT BORN
EXPERIMENTAL DESIGN TO COMPARE NEURONS BORN IN THE DEVELOPING vs. ADULT HIPPOCAMPUS
P7 vs. adult: e-phys
GFP retrovirus
(right DG)
RFP retrovirus
(right DG)
=
+
Pup (P7)
Adult (P42-45)
GFP retrovirus
(ventricle)
E15 vs. adult: e-phys
RFP retrovirus
(right DG)
=
+
E15
Adult (P42-45)
DEVELOPMENT
ADULT
NeuN
AGE OF ADULT-BORN NEURONS: 13 WEEKS
PAIRED COMPARISONS OF GLUTAMATERGIC
AFFERENTS OF P7 AND ADULT-BORN DGCs
whole-cell recordings in the presence of BMI
green
red
merge
RFP+
GFP+
depression
50 pA
25 ms
facilitation
Laplagne et al., PLoS Biol 2006
SIMILARITY IN POSTSYNAPTIC GLUTAMATERGIC RESPONSES
EPSC AMPLITUDE DEPENDS ON:
peak
EPSC
number of connections
probability of NT release
postsynaptic receptors
SIMILARITY IN PAIRED PULSE RATIO AND KINETICS
rise time
PPR and Kinetics depend on:
probability of NT release
peak 1
peak 2
receptor composition
PPR = peak 2/peak 1
SIMILARITY IN HIGH-FREQUENCY DEPRESSION (50 Hz)
DEPRESSION DEPENDS ON:
Presynaptic probability of NT release
Speed of presynaptic vesicle recycling
TOPOGRAPHIC DISTRIBUTION OF GABAERGIC INTERNEURONS OF THE DENTATE GYRUS
MOLECULAR
LAYER
GRANULE CELL
LAYER
HILUS
DENDRITIC INHIBITION:
INPUT CONTROL
PERISOMATIC INHIBITION:
OUTPUT CONTROL
SIMILARITY OF PERISOMATIC and DENDRITIC sIPSCs
ALL EVENTS AVG
PERISOMATIC
EVENTS
Vhold= - 80 mV
symm Cl-
DENDRITIC
EVENTS
Vhold= 0 mV
Low Cl- pipette
ALL EVENTS AVG
ADULT-BORN GCs CAN GENERATE A SPIKE IN RESPONSE TO AN EXCITATORY DRIVE
CELL-ATTACHED
RECORDINGS
MPP STIMULATION
Laplagne et al., PLoS Biol 2006
AXONAL PROJECTIONS OF ADULT-BORN GRANULE CELLS
OUTPUT
CA3
H
Toni, Laplagne et al, Nat Neurosci 2008
EM 3-D RECONSTRUCTION OF NEWLY FORMED SYNAPSES
17 dpi
OUTPUT
GFP+ terminal
Dendritic spine
GFP- terminal
28 dpi
75 dpi
1.5 mm
Toni, Laplagne et al, Nat Neurosci 2008
OUTPUT
PNAS 2005
• Retroviral expression of Channelrhodopsin 2 (ChR2) in adult-born neurons
• Use blue light to evoke massive activation of all adult-born GCs in the slice
• Obtain postsynaptic recordings from randomly selected target neurons
Optical presynaptic stimulation
to stimulate many neurons
at a time
Blue light
Electrophysiological recordings
of postsynaptic responses
1st stage - Expression of ChR2-GFP fusion in adult-born GCs
ChR2 overexpression does not alter anatomy or
physiology of adult-born GCs (> 15 weeks old)





Morphology
Membrane potential
Input resistance
Membrane capacitance
Spiking
Toni, Laplagne et al 2008
Activation of adult-born neurons by light
RELIABLE LIGHT-INDUCED
ACTIVATION OF ADULT-BORN GCs
Current clamp:
light-induced spiking
40 mV
1 Hz
1s
50 ms
5 ms
5 Hz
2.5 ms
10 Hz
20 Hz
50 mV
100 ms
30 Hz
Toni, Laplagne et al, Nat Neurosci 2008
3rd stage - Light-induced neurotransmitter release from adult-born
GCs
GABAERGIC INTERNEURON OF THE GCL/HILAR BORDER
SPIKING
hi freq
large AHP
Alexa 594 (patched neuron)
ChR2+ cells
ChR2+
Toni, Laplagne et al 2008
Light-induced neurotransmitter release from adult-born GCs
OUTPUT
10 ms
ChR2+
Individual trials
Average trace (>100 trials)
Lights off
LIGHT-EVOKED POSTSYNAPTIC CURRENTS WERE OBSERVED IN 14 TARGET NEURONS (tested > 120) INCLUDING
GCL/HILAR INTERNEURONS, MOSSY CELLS AND PYRAMIDAL NEURONS
Toni, Laplagne et al, Nat Neurosci 2008
Light-induced responses are mediated by glutamate
Peak PSC (pA)
20
kyn
OUTPUT
dcg
0
-20
control
-40
kyn
wash
dcg
wash
p2
p1
-60
0
10
20
30
40
50
Peak PSC (pA)
time (min)
kyn
bmi
0
-100
p2
p1
-200
-300
0
5
10
15
20
control
bmi
kyn
wash
time (min)
Toni, Laplagne et al, Nat Neurosci 2008
EJEMPLO DE FEEDFORWARD INHIBITION
CONCLUSIONS
 ADULT NEUROGENESIS UTILIZES CELL-AUTONOMOUS AND ENVIRONMENTAL CUES TO
MAINTAIN AN ONGOING DEVELOPMENTAL PROGRAM IN THE DENTATE GYRUS
 ADULT-BORN NEURONS INTEGRATE SYNAPTIC INFORMATION SIMILARLY TO OTHER
GRANULE CELLS GENERATED IN DEVELOPMENT
 ADULT-BORN GRANULE CELLS MAKE FUNCTIONAL SYNAPSES WITH TARGET CELL AND
RELEASE GLUTAMATE AS THEIR MAIN NEUROTRANSMITTER
 NEW NEURONS CAN RECEIVE, PROCESS AND TRANSMIT INFORMATION WITHIN THE
PREEXISTING NETWORK
 THE CONTRIBUTION OF THIS PHENOMENON TO HIPPOCAMPAL FUNCTION IS CURRENTLY
UNDER EXTENSIVE INVESTIGATION
Young neurons display a high intrinsic excitability
Retroviral labeling
of adult born neurons
(GFP)
P42-45
Birth
E15
18 - 56 dpi
Recording of intrinsic excitability,
glu inputs, and spiking
2000
 excitable
1500
1000
8
7
500
0
18-20 21-23 24-26 27-29 42-56 mature
age of neurons (days)
80
6
18-20 dpi
5
21-23 dpi
4
24-26 dpi
3
27-29 dpi
2
mature
1
70
Ithreshold (pA)
number of spikes
Rinput (Mohm)
2500
0
0
60
20
40
60
80
100
120
140
I step (pA)
50
40
30
20
10
0
18-20 21-23 24-26 27-29 42-56 mature
age of ne urons (days)
Mongiat et al 2009
IMMATURE NEURONS CAN SPIKE IN RESPONSE TO AN EXCITATORY DRIVE
spiking neurons (%)
100
80
60
40
20
0
18-20
0.3 mA
p=0.1
0.5 mA
p=0.7
0.7 mA
p=0.9
24-26
27-29
42-56
age of ne urons (days)
0.8
spiking probability
Young neuron
(21-29 dpi)
21-23
young
mature
0.6
0.4
0.2
0.0
0.1
0.3
0.5
0.7
0.9
stimulus intensity (mA)
For = number of active axons
FIRING YOUNG = MATURE
Mongiat et al 2009
SIMILAR SPIKING PROBABILITY IN SIMULTANEOUS RECORDINGS
Mongiat et al 2009
YOUNG NEURONS RECEIVE WEAK GLUTAMATERGIC INPUTS
due to fewer release sites
minis
EVOKED RESPONSES
21-23 dpi
max EPSC (pA)
400
24-26 dpi
300
200
*
*
18-20
21-23
*
*
24-26
27-29
100
0
mature
age (dpi)
27-29 dpi
mature
200
150
100
young (21-29 dpi)
50
0.3
0.5
0.7
0.9
stimulus intensity (mA)
50 ms
2.0
1.5
1.0
0.5
mature
0
0.1
100 pA
peak amplitude (pA)
42-56 dpi
8
2.5
250
frecuency (Hz)
EPSC amplitude (pA)
300
1.1
1.3
6
4
2
0
0.0
young
mature
young
mature
EFFICIENT INPUT-OUTPUT CONVERSION IN YOUNG NEURONS:
SUBTHRESHOLD STIMULI
Young
5 mV
50 ms
Im
50 pA
50 ms
EPSP amplitude (mV)
Vm
Mature
100
young
mature
80
60
40
20
(n > 25)
0
0
50
100
150
200
EPSC amplitude (pA)
Young neurons are about twice as efficient in transducing ionic current
into membrane depolarization
250
EFFICIENT INPUT-OUTPUT CONVERSION IN YOUNG NEURONS:
SUPRATHRESHOLD STIMULI
spike probability
1.0
0.8
0.6
young
mature
*
**
0.4
0.2
0.0
0-100
100-200
200-300
EPSC amplitude (pA)
Activation of fewer axons (synapses) can elicit similar levels of spiking activity
HIGH EXCITABILITY OF YOUNG NEURONS IS DUE TO DELAYED ONSET
OF INWARD RECTIFIER POTASSIUM CHANNELS
10
*
gKir (ns)
8
6
4
2
0
young
Vh:-80mV
80
250
#
200
gNa + (nS)
60
gK+ (nS)
#
40
20
150
100
50
0
0
young mature
young mature
mature
Kir BLOCKADE IN MATURE NEURONS MIMICS FIRING PROPERTIES OF YOUNGE CELLS
6
5
g Kir (ns)
4
3
2
*
*
1
0
- +
mature
mature
mature + Ba2+
50
50
50
40
40
40
30
30
30
20
20
20
10
10
10
0
60 120 180 240
0
60 120 180 240
EPSC amplitude (pA)
*
0.7
*
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0
0.8
0.0
0
60 120 180 240
2+
60
m
at
at
ur
ur
e
e
+
Ba
60
m
60
slope (mV/pA)
young
EPSP amplitude (mV)
- +
young
un
g
Ba
yo
2+
12
5
10
4
number of spikes
6
**
3
2
8
6
4
0
0
e
ur
at
i r2
K
+
0
20
40
60
80
100
current step (pA)
un
g
m
g
.1
2
un
young + Kir2.1
mature
1
yo
young
yo
g Kir (ns)
Kir overexpression induces mature-like firing properties in young neurons
Mongiat et al 2009
CONCLUSIONS
About immature neurons…
 YOUNG NEURONS ARE, IN PRINCIPLE, CAPABLE OF INFORMATION PROCESSING
 GLU INPUTS ONTO IMMATURE GCs ARE WEAK, YET EXCITATION ELICITS A SIMILAR SPIKING
PROBABILITY IN NEURONS AT EITHER DEVELOPMENTAL STAGE
 HIGHLY EFFICIENT IN TRANSDUCING ION FLUXES INTO MEMBRANE DEPOLARIZATION
 MECHANISMS UNDERLYING THESE CHANGES IN EXCITABILITY DURING MATURATION OF NEW
GCs INVOLVE THE LATE EXPRESSION OF Kir CHANNELS IN DEVELOPING NEURONS
WHILE DEVELOPING, NEWBORN DGCs EXHIBIT UNIQUE FUNCTIONAL PROPERTIES
High excitability
(Schmidt‐Hieber Nature 2004, Mongiat PLoS ONE 2009)
Reduced GABAergic inhibition
(Snyder, J Neurophysiol 2001; Espósito, J Neurosci 2005; Ge, Nature 2006)
Increased susceptibility for long‐term synaptic plasticity
(Snyder, J Neurophysiol 2001; Schmidt‐Hieber, Nature 2004; Ge Neuron 2007)
Preferential recruitment during spatial memory
(Kee Nat Neurosci 2007)
When are new neurons young?
For how long (time window)?
Whereabout?
Septotemporal (dorso‐ventral) differences in basal neuronal activity in the granule cell layer Verónica Piatti
Mariela Trinchero
dorsal
ventral
ARC+ cells = active neurons
BASAL ACTIVITY IN THE GCL IS HIGHER IN THE DORSAL HIPPOCAMPUS
(similar data: Snyder ,Hippocampus 2009)
Septotemporal (dorso‐ventral) differences in neuronal maturation
21 dpi
THREE‐WEEK OLD NEURONS
VENTRAL DGCs ARE FUNCTIONALLY IMMATURE AT 21 dpi Lucas Mongiat
Verónica Piatti
Dorsal → Rinput = ~700 M /// mature spiking /// more glutamatergic inputs
sEPSC frequency (Hz)
Ventral → Rinput = ~1900 M /// immature spiking /// poor glutamatergic connectivity
RUNNING INCREASES NETWORK ACTIVITY IN THE TEMPORAL DENTATE GYRUS AND ACCELERATES NEURONAL MATURATION
dorsal
ventral
ARC+ cells = active neurons
21‐day‐old neurons
dorsal
sedentary mice
running mice
ventral
GFP+ cells = new neurons
IS THERE A ROLE FOR INTRINSIC EXCITABILITY IN ACTIVITY‐DEPENDENT MATURATION?
RETROVIRAL EXPRESSION OF Kir IN ADULT‐BORN NEURONS
Non‐conductive Kir control
confocal analysis
Georgina Davies
Soledad Espósito
Lucas Mongiat
electrophysiology
EXPRESSION OF Kir DECREASES INTRINSIC EXCITABILITY IN ADULT‐BORN NEURONS
 Kir expression
↓↓↓
membrane resistance
hyperpolarized
↓↓↓
Subthreshold excitability
SLOW RATE OF MATURATION IN ADULT‐BORN NEURONS WITH REDUCED INTRINSIC EXCITABILITY CONCLUSIONS
‐ Neuronal maturation can be regulated in restricted hippocampal domains in an activity‐
dependent manner
‐ Running‐induced modulation of neuronal activity in the neurogenic niche plays a crucial role on neuronal maturation
‐ The underlying mechanism involves intrinsic neuronal activation ‐The rate of neuronal maturation determines survival