Interaction
Part Neuron-Glia II: Glia Promote
Neuronal Synapse Formation
Three Serial Papers From Ben Barres Lab:
Pfrieger et al., Science, 1997
Ullian et al., Science, 2001
Christopherson et al., Cell, 2005
Methods:
Immunopanning to purify retinal ganglion
cells.
Culture purified postnatal (P8) RGCs in
serum-free medium, plus BDNF, CNTF,
and B-27 (mixture of neurotrophic factors).
Low density (50-100 cells/mm2).
Culture purified RGCs on glial cells
isolated from their target area superior
colliculus.
Synaptic Efficacy Enhanced
by Glial Cells in Vitro
Frank W. Pfrieger, * Barbara A. Barres
Science. 1997 Sep 12;277(5332):1684-7.
This work focuses on synaptic efficacy using electrophysiology recording.
Glia Increase Spontaneous Synaptic Activity
Fig. 1
Density higher?
90% show synaptic activity
50% show synaptic activity
Spontaneous synaptic activity, 5 DIV
Both frequency and
amplitude of synaptic
events recorded from
RGCs increased in
coculture with glia
Both Frequency and Amplitude are Enhanced
Fig. 2
No glia
With glia
Each square is for
the frequency and
average amplitude
in one neuron
Both oligodendrocyte
and astrocyte are
effective
Miniature EPSCs Increased
Fig. 3
Burst activity
Synapse
morphology is
normal
Silent synapse!
Morphological
Seeming synapse
Are more likely
Silent synapses
+Glia
Miniature EPSCs in TTX
Glia Increase Sustainable Release
Fig. 4
Failure rate increases with stimulation frequency
Glia-free
+glia
Summary 1
In the absence of glia, synapses formed
between neurons are morphologically
normal, but functionally inactive.
Glial cells facilitate the functional
maturation of synapses.
Control of Synapse Number by Glia
Ullian EM, Sapperstein SK, Christopherson KS,
Barres BA.
Science. 2001 Jan 26;291(5504):657-61.
This work focuses on synapse number.
Glia Enhances Both Pre- and Postsynaptic Efficacy
-Glia
+Glia
Presynaptic transmitter
release increase
Glutamate
Fig. 1A-D
Postsynaptic glutamate receptor increase
Analysis in Single Autaptic Neuron
-Glia
+Glia
Note the different Y scale
Lower
efficacy
Fig. 1E-J
Higher
failure
rate
Quantal content is a presynaptic index
FM 1-43 Labels Active Presynaptic Boutons
Fluorescence intensity
Fig. 2A-D
Increased Ca channel
expression
Glia Increase Presynaptic Vesicle Release
frequency
Post-
Pre-
More vesicle release when +glia
Amplitude
Fig. 2E-G
Ionomycin bypasses presynaptic Ca channels, forms artificial Ca channels
and induces synaptic vesicle release
Ionomycin-Induced FM 1-43 Labeling
No Glia
With Glia
Fig. 2H-I
Conclusion: downstream effectors after presynaptic
Ca channels play important role!
No Change in Total Presynaptic Protein
Fig. 3a
Postsynaptic puncta glutamate
receptor increased
with glia
What about postsynaptic PSD-95?
Presynaptic
marker
Diffusely
distributed
Few
postsynaptic
PSD-95
puncta
Fig. 3B-E
Clustered
many
Pre- and
postsynaptic
puncta both
increased
with glia
Synaptic Ultrastructure Is Normal
Fig. 4A-B
Number of Synapses Increases In Vivo
after Astrocyte Peak
Fig. 4C-E
S100 labels astrocytes; SV2 labels presynapses
Synapses appear after astrocytes, around postnatal day 6.
Summary 2
Astrocytes increase both pre- and postsynaptic
efficacy, particularly the number of functional
synapses.
Astrocytes increase Ca channel expression, but
that is insufficient to explain the net increase of
synaptic efficacy
Astrocytes induce clustering of presynaptic
proteins, and induces the puncta of PSD-95
proteins.
Astrocytes may promote synapse formation in
vivo.
Brain Teaser
5 Hats and 3 smart man
Cell 120: 421-433, 2005
Thrombospondins Are Astrocyte-Secreted Proteins
that Promote CNS Synaptogenesis
Karen S. Christopherson1, 6, , , Erik M. Ullian6, 7, Caleb C.A. Stokes1,
Christine E. Mullowney1, Johannes W. Hell2, Azin Agah3, Jack Lawler4,
Deane F. Mosher5, Paul Bornstein3 and Ben A. Barres1
ACM increases morphological synapses
Fig. 1
Functional
Astrocyte conditioned medium
does not increase functional events
Morphology
Molecular weight cutoff filter
Synapse-promoting factor
is >100 kDa
ACM increase # of morphological synapses
TSP increases morphological synapses
Fig. 1D-F
Cholesterol not
increase number of
synapses
Thrombospondins (TSP) are large oligomeric extracellular matrix proteins (>500 kDa),
mediating cell-cell and cell-matrix interactions through binding membrane receptors,
cytokines, and other extracellular matrix proteins. TSPs are secreted by astrocytes.
TSP does not promote postsynaptic function
Presynaptically active
Overall
function
silent
I Glu
Fig. 4
mEPSC
Postsynaptically
inactive
Pfrieger et al., Science, 1997
Conditioned medium
does have effect on
frequency of events!
Cholesterol Effect:
Supplement Fig. 1
Cholesterol increases synaptic release and quantal content, although it
does not increase synapse number.
Cholesterol increases
synaptic efficacy
Science 9 November 2001:
Vol. 294. no. 5545, pp. 1354 - 1357
DOI: 10.1126/science.294.5545.1354
Daniela H. Mauch,1 Karl Ngler,1, 3 Stefan
Schumacher,4 Christian Gritz,1, 3 EvaChristina Mller,2 Albrecht Otto,2 Frank W.
Pfrieger1, 3*
Science is full of debate!!!
Summary 3
Thrombospondins secreted from
astrocytes increase presynaptic active
bouton number, but not affect
postsynaptic glutamate receptors.
Cholesterol increases presynaptic release
efficacy, but not number of boutons
although this is debatable.