Rôle de la microglie dans le
développement et les pathologies du SNC
Montpellier, Oct 2019
E. Audinat
Institut de Génomique Fonctionnelle
Références:
Développement : Mosser et al., Prog Neurobiol 2017
Pathologie : Butovsky & Weiner, Nat Rev Neuro 2018
Epilepsie : Rassendren & Audinat, J Neurosci Res 2016
Bases d’électrophysiologie : le livre “Physiologie du Neurone”, Tritsch D,
Chesnoy-Marchais et Feltz A Eds. Douin
Microglia cells: the resident macrophages of the CNS
Resting
(dormant)
Activated
Healthy brain Pathology
• The professional phagocytes of the CNS
• Release different inflammatory mediators (cytokines, chemokines, growth
factors…) and orchestrate the inflammatory reaction occurring many CNS
disorders (TBI, Neuropathic pain, stroke, Parkinson, Alzheimer, ALS, epilepsy…)
Morphological changes
Translatome remodeling
Proliferation
Phagocytosis
Release mediators (trophic
factors, cytokines, ROS)
Microglia-synapse interactions
in physiological conditions : adult CNS
Active surveillance of the parenchyma
Nimmerjhan et al. 2005 (see also Davalos et al. 2005)
Tremblay et al. 2010
(see also Wake et al. 2009)
Neuron Microglia Direct contact with synapses
Control of synapse size and number
Parkhurst et al. 2013
control Microglia depletion
“Never resting” microglia monitor synapses and control their morphology/formation
Microglia-synapse interactions
in physiological conditions : adult CNS Microglia control of synaptic activity
1) specific activation of microglia receptors modulate synaptic transmission
Pascual et al.2012 Piccinin et al 2010
“Never resting” and “not just eating” microglia in the adult brain
2) Microglia bulbous endings contact active neurons and reduce their activity
Li et al.2012
In vivo time-lapse imaging of neuronal calcium and microglia morphology in the zebrafish optic tectum
Green: OGB-loaded neurons
Purple: microglia
Microglia-synapse interactions
In physiological conditions : Developing CNS
Synaptic pruning
Paolicelli et al. 2011 Schafer et al. 2012 Microglia PSD95 Microglia
Origin of microglia
Ginhoux et al. 2013
Microglial cells prune supernumerary synapses….
Microglia-synapse interactions
In physiological conditions : Developing CNS
… and help at forming new ones.
Miyamoto et al. 2016 Microglia/Neuron
Origin of microglia
Ginhoux et al. 2013
Microglia-synapse interactions
In physiological conditions : Developing CNS
Besides pruning and forming synapses, can microglia influence
synaptic activity during maturation of neuronal networks ?
Induction of developmental apoptosis (Marin-Teva et al., 2004 ; Bessis et al., 2006)
Neuronal survival (Ueno et al., 2013)
Migration of cortical inhibitory interneurons (Squarzoni et al., 2014)
Pruning of supernumerary synapses (Schafer et al., 2012 ; Paolicelli et al., 2011 ; Zhan et al., 2014)
Synaptogenesis (Miyamoto et al., 2016)
How can microglial cells be at the right place at the right time ?
200 µm
Heterogenous distribution of microglia
during early postnatal development of the brain
CX3CR1+/eGFP mice (Jung et al. 2000)
Thalamic axons
Spiny stellate neurons
Aronoff & Petersen 2008
Formation & maturation of the barrels :
P3
Clustering of Thalamocortical
axons (TCA)
The barrel cortex as a model of neuronal circuit development
• ↗ synaptic density
• Modification of glutamate receptor expression at TC synapses
• setting up of inhibitory network
Period of plasticity
P5
End of the critical window for LTP induction at TC synapses
P10
Inhib. Intern.
150 µm
Increased density of microglial cells in cortical
layers of the developing barrel cortex
CX3CR1+/eGFP mouse (Jung et al 2000)
Arnoux et al. 2013
P5 P5 P5
P7 P6
P5 P5
Scale bar:100µm
Distribution of microglial cells within layer 4 of the
barrel cortex during the first postnatal week
Hoshiko et al. 2012 Microglial cells invade the barrel centers after P5
Two microglia phenotypes in the developing barrel field C
urr
ent density
(pA
/pF
)
Vm (mV)
P5-P9 non-activated
P5-P9 “activated”
Adult controls
Kv1.3
Acute cortical slices from CX3CR1+/eGFP mice
Postnatal age
% o
f m
icro
glia
l cells
expre
ssin
g K
v1.3
What is the signaling mechanism responsible for the
recruitment of microglial cells at thalamo-cortical
synapses?
The fractalkine (CX3CL1) signaling pathway in the CNS
( From Richard M. Ransohoff; Immunity, Nov. 2009)
What is the signaling mechanism responsible for the
recruitment of microglial cells at thalamo-cortical
synapses?
Transgenic mice used (Jung and al, 2000):
- CX3CR1+/eGFP -> mice with functional CX3CR1
- CX3CR1 eGFP/eGFP -> mice lacking CX3CR1
Scale bar: 200 µm
Fractalkine (CX3CL1) immunoreactivity in the
developing somato-sensory cortex
Scale bar:100 µm
CX3CR1 deficiency delays the entry of microglia in the
barrel centers
Knockout mice
for CX3CR1
Heterozygous mice
for CX3CR1
Hoshiko et al. 2012
Scale bar: 100 µm
CX3CR1 deficiency does not impair the overall number
of microglial cells in layer 4 at P7 (but their distribution)
0
5000
10000
15000
20000
hetero homo
5000
10000
15000
CX3CR1+/eGFP
CX3CR1eGFP/eGFP
0
n=12 n=5
Hoshiko et al. 2012
Microglial cells enter the barrel centers during synaptic maturation
CX3CR1 deficiency delays microglia recruitment in the barrel centers
0
20
40
60
80
100
120
00-0
,2
0,2-0,4
0,4-0,6
0,6-0,8
0,8-1
1-1,2
1,2-1,4
1,4-1,6
1,6-1,8
1,8-2 >2
Mean velocity of soma (µm/min)
cum
ula
tive
pro
bab
ility
(%
)
CX3CR1+/-(n=9 exp)
CX3CR1 -/-(n=5 exp)
Mean Soma Velocity (µm/min)
CX3CR1+/-
CX3CR1-/-
Cu
mu
lative
Pro
ba
bili
ty
Reduced directional motility of microglia soma in
developing CX3CR1 knockout mice
Collaboration : Serge Charpak
Arnoux, Audinat 2015
Is there any consequence of CX3CR1 deficiency and of
the delayed recruitment of microglial cells on the
functional maturation of thalamo-cortical synapses?
Development : Conclusion # 1
Microglial cells enter the barrel centers during synaptic
maturation and fractalkine signaling contributes to the
timely recruitment of these immune cells at synaptic
sites
Recordings of spiny stellate cells
minimal stimulations of TC fibers
CX3CR1 deficiency impairs the development of
AMPAR/NMDAR ratio at TC synapses
Hoshiko, Arnoux et al. 2012
The delayed recruitment of microglia in the barrel centers in CX3CR1 knockout
mice is accompanied by an impaired functional maturation of TC synapses
P0 P3 P5 P8-10
Intra-cerebral
injection of liposomes Tests
Clodronate liposomes : another way to delay microglia
invasion of cortical layers
Free clodronate www.clodronateliposomes.org/
No sign of
astrogliosis @ P9
Depletion of microglia with clodronate liposomes also
impairs the functional maturation of TC synapses
The functional maturation of TC synapses is impaired
in the absence of microglia in the barrel centers
Do microglial cells also affect the maturation of
inhibitory circuitry in the barrel cortex ?
Development of TC feed-forward inhibition
in layer 4 of barrel cortex
The TC feed-forward inhibition matures mostly
after the end of the first postnatal week
The development of TC feed-forward inhibition decreases the
integration window that controls the timing and output of
spiny stellate neurons
Chittajallu & Isaac, 2010
P6
P11
P0 P3 P5 P10-11
Intra-cerebral
injection of liposomes Tests
Depletion of microglia impairs the development of
Thalamocortical feed-forward inhibition
The presence of microglia in the barrel cortex favors
the development of the TC feed-forward inhibition
By which mechanism microglial cells influence
the functional maturation of thalamocortical
synaptic circuits?
Development : Conclusion # 2
The presence of microglial cells in the barrel centers is
necessary for the timely maturation of excitatory and
inhibitory synapses of the thalamocortical circuit.
? Microglial activation Nerve injury
Microglial BDNF regulate adult synaptogenesis (Parkhurst et al. 2013)
In context of neuropathic pain, microglial BDNF induces
an increase of neuronal excitability (Coull et al., 2005 ; Ulmann et al., 2008)
An usual suspect : the Brain-Derived Neutrophic Factor
Conditional deletion of BDNF in microglia impairs the
development of the AMPAR/NMDAR ratio at TC synapses
CX3CR1CreER BDNFflox/flox X
P0 P3 P5 P10-11
Tamox Tests
P4
Microglial BDNF controls the functional maturation of TC synapses
Conditional deletion of BDNF in microglia impairs the
development of the TC feed-forward inhibition
CX3CR1CreER BDNFflox/flox X
P0 P3 P5 P10-11
Tamox Tests
P4
Microglial BDNF controls the development of TC feed-forward inhibition
Take home messages
Reciprocal interactions between neurons and microglial cells control the
functional maturation of the thalamocortical network
The presence of microglia in the barrel centers is necessary for the functional
maturation of thalamocortical excitatory synapses and the development of feed-
forward inhibition at early postnatal stages.
(Which of the 2 synapses is controlled by microglia during the development of the
feed-forward inhibition?)
BDNF is one of the mediators by which microglial cells influence the functional
maturation of synaptic networks in the barrel cortex.
(Direct or indirect effect of BDNF on pre- or postsynaptic elements?)
Microglia cells: the resident macrophages of the CNS
Resting
(dormant)
Activated
Healthy brain Pathology
• The professional phagocytes of the CNS
• Release different inflammatory mediators (cytokines, chemokines, growth
factors…) and orchestrate the inflammatory reaction occurring many CNS
disorders (TBI, Neuropathic pain, stroke, Parkinson, Alzheimer, ALS, epilepsy…)
Morphological changes
Translatome remodeling
Proliferation
Phagocytosis
Factor release (trophic factors,
cytokines, ROS)
Aronica, Gorter Neuroscientist 2007
Epilepsy, inflammation and glia
Choi, Koh Yonsei Medical Journal 2008
IL-1alpha, IL-1B, IL-6, NFkB, PLAT, TGFB, GFAP,
VIM, C1q, C3, C/EBPB, C/EBPd, FRD1…
Experimental TLE
- 600 000 patients suffering of different epilepsies in France
- Anti-epileptic drugs (neurocentric) refrain seizures but do not cure epilepsy
and have unwanted side effects
- New therapeutic targets are needed
Epilepsy:
Spontaneous
seizures
Acquired
hyperexcitability Evoked
hyper-excitability
Hours
Status
epilepticus
Release of
Mediators
Phagocytosis
Hypothesis: Purinergic receptors
which play central roles
in microglia functions
determine how these cells
influence hippocampus
remodeling
Days to weeks
“Latent period”
Neuronal death
Re-wiring of the network
Changes in activity
Plusieurs récepteurs purinergiques de types P2X et P2Y
sont exprimés dans la microglie
Fields et al.
Nat Rev Neurosci. 2006
P2X4
P2X7
P2Y6
P2Y12
Kainate
Microglia
phenotype
Status Epilepticus
CX3CR1+/eGFP mice
fluorescent microglia
Hippocampal microglia in a model of
Status epilepticus
Acute hippocampal slices
qPCR whole hippocampus
Immunohistochemistry
Increased expression of pro-inflammatory markers and
neuronal death after Status Epilepticus in CX3CR1+/eGFP
mice (C57bl6)
Control
48h
Post SE
Eosin hematoxylin Fluorojade B
mark
ed c
ells
(cells
/fie
ld)
CA1
CA3
0
10
20
30
40
Control 48h Post
SE
qPCR whole hippocampus
Avignone et al. 2008
Iba1 GFP Merged
PBS
24h
Post
SE
15 µm
Morphological changes of microglial cells in the CA1
region of the hippocampus after Status Epilepticus
Avignone et al. 2008
I density (
pA
/pF
)
Vm (mV)
PBS 3h 24h 48h
-100 0 100 -10
0
10
I d
en
sity (
pA
/pF
)
Vm (mV)
PBS3h24h48h
-100 0 100-10
0
10
I d
en
sity (
pA
/pF
)
Vm (mV)
PBS3h24h48h
-100 0 100-10
0
10
-100 0 100-10
0
10
PBS 3h 24h 48h
*
**
20
25
30
35
40
PBS 3h 24h 48h
Cm
(p
F)
*
**
20
25
30
35
40
PBS 3h 24h 48h
**
1
2
3
4
5
PBS 3h 24h 48h PBS 3h 24h 48h
**
1
2
3
4
5
1
2
3
4
5
Rin
(G
W)
Changes in intrinsic membrane properties of microglial
cells after Status Epilepticus
Acute hippocampal slices from 4-6
week old CX3CR1+/eGFP control
and epileptic mice.
200pA
20ms
PBS 48h after SE
200pA
20ms
200pA
20ms
PBS 48h after SE
Voltage steps from -140mV to +60mV
Holding potential -60 mV.
Avignone et al. 2008
200pA
20ms
Heterogeneity of hippocampal microglia cells
7 days after status epilepticus
P2X7
P2Y6
P2Y12
Production secretion
of, IL-1b, IL-6, TNFa
Phagocytosis
Polarisation
Process extension.
Purinergic receptors of microglial cells
Functions Receptor
P2X4 Production secretion
of bdnf; chemotaxis
Activation
Status Epilepticus induces an increase of purinergic
receptor mRNA expression in the hippocampus
0
1
2
48
12
P2X4P2X7P2Y6
**
PBS 48h3h 24h
P2Y12
**
**
**
**
*
* *
Fo
ldch
an
ge
B
*
0
1
2
48
12
P2X4P2X7P2Y6
**
PBS 48h3h 24h
P2Y12
**
**
**
**
*
* *
Fo
ldch
an
ge
B
*
Avignone et al. 2008
Larger P2X7 receptor-mediated currents in activated
microglia after Status Epilepticus
ATP (1mM)
10 pA
50 s
ATP (1mM)
A740003 (10 µM)
WT P2X7-/- WT+
A740003
0
2
4
6
I AT
P (
pA
/pF
)
PBS 48h after SE
Ca/Mg-free extracellular solution
CsGlu pipettes,
VH -60mV
P2X7
P2Y6
P2Y12
Production secretion
of IL-1b, IL-6, TNFa
Phagocytosis
Polarisation
Process extension.
Purinergic receptor remodeling
upon microglia activation
Function Receptor Activation
LPS
What about
P2X4?
Activation
SE
P2X4 is up-regulated in the hippocampus
48h after Status Epilepticus
Ulmann et al. 2013
P2X4 is up-regulated in microglia
48h after Status Epilepticus
Does P2X4 deficiency impact
microglia activation and neuronal remodeling?
Impaired microglia activation after Status Epilepticus in
P2X4-/- mice
Impaired increase in the number of microglial cells
Num
be
r o
f G
FP
ce
lls / m
m3 (
x1
03)
CX3CR1+/eGFP X P2X4-/-
CX3CR1+/eGFP
0
5
10
15
20
25
PBS Kainate
48h
***
**
*
*
PBS
Kainate
48h
CX3CR1+/eGFP
X P2X4-/- CX3CR1+/eGFP
Normal P2Y12 expression after Status Epilepticus in
P2X4-/- mice
P2Y12 immunohistochemistry (Ab D Julius) qPCR (whole hippocampus)
P2X4+/+ P2X4-/-
Normal P2Y12R-mediated motility of microglia
processes in hippocampal slices of P2X4-/- mice
Z-projection of 5 stacks of images (4 µm thick each)
taken every minute with dynamic speckle illumination
(movie by C Ventalon, V Emiliani, E Avignone) V
elo
city (
µm
/min
)
0
2
4
6
8
PBS 48h post SE
CX3CR1+/-
CX3CR1+/- ; P2X4 -/-
P2X4 deficiency does not impair P2X7 and P2Y6
receptor-mediated response in hippocampal microglia
No change in P2X7, P2Y6 and P2Y12 receptor-mediated
responses in microglia of control and epileptic mice
Impaired up-regulation of Kv1.3 mediated potassium currents
in microglia of P2X4-/- mice after Status Epilepticus
Voltage steps from -140mV to +60mV
Holding potential -60 mV.
Kv1.3
Decreased neuronal death in CA1 after Status
Epilepticus in P2X4-/- mice
CX3CR1+/eGFP CX3CR1+/eGFPxP2X4-/-
Fluoro-Jade B staining
48h post SE
Conclusions
- The reactivity of hippocampal microglia after a status epilepticus is characterized
by larger purinergic responses mediated by P2X7, P2Y6 and P2Y12
- P2X4 expression is also up-regulated in microglia
- P2X4 governs specific aspects of microglial cell reactivity (functional
expression of Kv1.3 and recruitment but not other purinergic responses)
- P2X4 promotes neuronal death after status epilepticus
>>> Specific pathways through which purinergic receptors control
neuronal survival and epileptogenesis remain to be identified
>>> Roles of the BBB and interactions with the peripheral immune
system (monocytes and T cells)