Ian Wenker

J Neurophysiol 104:1216-1218, 2010. First published Jul 7, 2010; doi:10.1152/jn.00429.2010

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J Neurophysiol 104: 1216 1218, 2010.

First published July 7, 2010; doi:10.1152/jn.00429.2010.

Neuro Forum

An Active Role for Astrocytes in Synaptic Plasticity?

Ian Wenker
Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut

Recently, Henneberger and colleagues blocked hippocampal longterm synaptic potentiation (LTP) induction by clamping intracellular calcium concentration of individual CA1 astrocytes, suggesting
calcium-dependent gliotransmitter release from astocytes plays a role
in hippocampal LTP induction. However, using transgenic mice to
manipulate astrocytic calcium, Agulhon and colleagues demonstrated
no effect on LTP induction. Until the question of how intracellular
calcium causes gliotransmitter release is answered, the role of astrocytes in synaptic plasticity will be incompletely understood.

Address for reprint requests and other correspondence: I. Wenker, University of Connecticut, College of Liberal Arts and Sciences, Department of
Physiology and Neurobiology, 75 North Eagleville Road, Storrs, CT 062693156 (E-mail: ian.wenker@uconn.edu).
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Although glial cells were discovered over 100 years ago, an

understanding of their role in brain physiology has come about
only relatively recently. From their original name glia it was
apparent that they were thought to simply glue the brain
together. Later, it was found that glia comprise a heterogeneous
population of cells that includes oligodendrocytes, astrocytes,
and microglia, each performing unique physiological functions
in the nervous system (Somjen 1988). In particular, astrocytes
are important for neuronal metabolism, synapse formation,
transmitter reuptake, and potassium buffering (Kimelberg 2010). As
these roles for astrocytes became accepted among the scientific
community newer, more active roles for astrocytes have been
proposed, involving astrocytic release of adenosine 5=triphosphate, glutamate, and D-serine (Zhang and Haydon
2005). Although there have been some detailed studies of
gliotransmitter release from astrocytes in culture, there are
few data supporting gliotransmission in intact brain tissue.
Henneberger et al. (2010) tested the hypothesis that astrocytic release of D-serine is essential for generation of long-term
synaptic potentiation (LTP) at the Schaffer collateral (SC)
CA1 pyramidal cell (CA1) synapse of the hippocampus (Fig. 1,
left). This is a reasonable hypothesis based on three previously
established findings. First, D-serine is an agonist of the glycinebinding site of N-methyl D-aspartate receptors (NMDARs)
(Mothet 2000). Second, astrocytes in the CA1 are known to
contain D-serine in their cytoplasm (Schell 1997). Third, astrocytes in culture are known to release D-serine in a calciumdependent manner (Mothet 2005). Also, it was recently shown
that D-serine is the coagonist of NMDARs during LTP in the
supraoptic nucleus of the hypothalamus and strong evidence
for astrocytic D-serine release was demonstrated (Panatier et al.
2006). Henneberger et al. (2010) aimed to clarify the role of
astrocytic D-serine release in hippocampal synaptic LTP.
The use of calcium-clamp on individual astrocytes is
certainly the strongest aspect of the study by Henneberger et al.
(2010). Previous studies have used high concentrations of the
calcium buffer EGTA (e.g., 10 mM) to suppress calcium
transients in astrocytes. However, the use of exogenous calcium buffers can only inhibit rapid calcium transients but likely

does not affect slow changes in free calcium due to homeostatic mechanisms (Henneberger et al. 2010). By making whole
cell patches with an internal solution containing low concentrations of calcium and EGTA, Henneberger et al. (2010) were
able to eliminate slow and transient calcium fluctuations
throughout individual astrocytes by effectively clamping
internal calcium at 50 to 80 nM.
Previous evidence indicates that D-serine release from astrocytes is dependent on increased intracellular calcium (Mothet
2005). Thus the authors clamped calcium to eliminate all
calcium-dependent transmitter release from the patched astrocyte, while simultaneously using standard protocols to induce
and record LTP at the SCCA1 synapse of the hippocampus
(Fig. 1, left). Using this approach the authors demonstrated that
LTP is induced in the region of an astrocyte that is patched
using control internal solution (i.e., little calcium-buffering
capacity), but not when the astrocyte is calcium-clamped.
Interestingly, the effect of LTP inhibition is spatially restricted
to the region of the individual calcium-clamped astrocyte. The
ability to induce LTP persisted when the pipette monitoring
dendritic field potentials is moved away from the calciumclamped astrocyte. This coincides with the observation that
individual astrocytes occupy domains that do not overlap with
neighboring astrocytes. The reader is left with the impression
that each astrocyte regulates synapses within its own local
region of influence, with little overlap from neighboring astrocytes.
The authors next attempt to address the mechanism by which
astrocytes facilitate LTP, i.e., whether astrocytes release Dserine that coagonizes the CA1 NMDARs and leads to LTP
induction. To test this the authors first step is logical: rescue
the loss of LTP with exogenous D-serine. Exogenous D-serine
does rescue LTP near the calcium-clamped astrocyte, suggesting a lack of glycine-binding site activation blocks LTP induction during calcium-clamp experiments. The next step, in this
readers mind, is to demonstrate that D-serine is the coagonist
involved in LTP inductions. In my opinion, this is where the
authors experimental approach becomes problematic.
In an effort to suppress gliotransmission the authors use
fluoroacetate because it is considered a selective gliotoxin
and, in so doing, are able to block LTP. However, fluoroacetate
is selective to astrocytes only because they take it up across
their membranes more readily than their neuronal counterparts
(Hassel et al. 2002). Possible effects on neurons aside, the
effect of fluoroacetate on astrocytes will not be D-serine specific. Fluoroacetate is a metabolic poison and will certainly
cause inhibition of membrane transport processes, most notably that of glutamate. Experiments blocking astrocytic glutamate transport may clarify the effect seen with fluoroacetate.
As discussed in the following text, relatively little is known
about gliotransmitter release in vivo, so the authors use the
most astrocyte-specific inhibitor available; however, with so

Neuro Forum
ACTIVE ROLE FOR ASTROCYTES IN SYNAPTIC PLASTICITY?

Field-potential
Recording

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Presynaptic
Neuron

Postsynaptic
Neuron

Normal
HFS

Presynaptic
Neuron

Ca2+ clamp

Postsynaptic
Neuron

Glycinebinding site

Glutamate

NMDARs

HFS

?
?
clam
p

mGluR
D-serine

[Ca2+]i

Astrocyte

Astrocyte

FIG. 1. Model of astrocyte regulation of long-term synaptic potentiation (LTP) in the hippocampus. Left panel: depiction of the experimental design used by
Henneberger et al. (2010) to clamp astrocyte intracellular calcium and record dendritic field potentials at the Schaffer collateral (SC)CA1 pyramidal cell (CA1)
synapse in the hippocampus. Right panel: depiction of a model for LTP. The currently accepted model of this hippocampal synaptic LTP involves glutamate being
released from the presynaptic neuron and diffusing across the synaptic cleft to activate postsynaptic receptors. N-Methyl D-aspartate receptor (NMDAR)
activation is necessary for LTP induction and requires both glutamate binding and activation of the glycine-binding site. Classically, the latter is thought to be
accomplished by tonic levels of glycine, although Henneberger et al. (2010) propose that D-serine, released from astrocytes, activates the glycine-binding site.
In this model glutamate activates metabotropic glutamate receptors on astrocytes to increase intracellular calcium and cause D-serine release, which in turn binds
the glycine-binding site of the NMDAR. By clamping internal calcium fluctuations of an astrocyte via a patch pipette, Henneberger et al. (2010) demonstrated
elimination of LTP that supports this hypothesis, although another study (Agulhon et al. 2010) that uses a transgenic approach to block intracellular calcium
transients finds opposite results. To more completely understand the role of astrocytes in hippocampal synaptic LTP three questions must be answered in vivo.
1) What mechanism does increased intracellular calcium act on to promote D-serine release? 2) How is D-serine released by astrocytes? 3) Is D-serine the
endogenous cotransmitter involved in synaptic LTP of the hippocampus?

many possible nonspecific effects it is difficult to interpret the

results of these experiments.
In more compelling experiments Henneberger et al. (2010)
used L-erythro-3-hydroxyaspartate (HOAsp), an inhibitor of
D-serine production, loaded into the intracellular astrocyte
pipette to block LTP induction. However, HOAsp effectively
blocks LTP after high-frequency stimulation only in the presence of 2-amino-5-phosphonovaleric acid (APV), an NMDAR
antagonist, which they claim expels previously produced Dserine. In addition, HOAsp is not specific to D-serine production and can affect pyruvate metabolism, and thus the general
metabolism of the patched astrocyte (Strsovsk et al. 2005).
Both experiments using fluoroacetate and HOAsp are not as informative as simply inhibiting D-serine activation of NMDARs using
the antagonist D-amino acid oxidase, a serine-degrading enzyme, as was done in similar studies in hypothalamus slice
preparation and hippocampal cultured neurons (Panatier et al.
2006; Yang et al. 2006). Thus the authors leave the reader to
wonder whether D-serine is the NMDAR coagonist involved in
LTP induction.
A different test of the hypothesis that calcium-induced
release of gliotransmitter by astrocytes is required for LTP at
the SCCA1 synapse of the hippocampus comes from Agulhon
et al. (2010) who report contradictory results when using a
genetic approach to control astrocytic intracellular calcium.
This study uses two transgenic mouse models. One line is an
astrocyte-specific knockin of the MrgA1 receptor (MrgA1),
J Neurophysiol VOL

an exogenous receptor that acutely increases intracellular calcium, via a Gq G protein-coupled receptor, when activated by
its peptide agonist, which is also exogenous to the CNS. Using
MrgA1 mice they demonstrate that increased intracellular
calcium in astrocytes does not affect LTP in the hippocampus.
Although this experiment does not actually contradict the
results of Henneberger et al. (2010), in that they never tested
the consequence of increasing astrocytic intracellular calcium,
it certainly is not very supportive of an astrocytic role in LTP
induction.
The second mouse line used by Agulhon et al. (2010) is a
knockout of inositol triphosphate receptor 2 (IP3R2). IP3R2 is
the receptor thought to be responsible for increasing intracellular calcium in astrocytes, whereas IP3R1 and IP3R3 are
expressed in neurons. The authors claim total elimination of
astrocyte calcium transients, both spontaneous and those induced by synaptic activation. However, unlike the results
obtained using pipette solution to calcium-clamp astrocytes
(Henneberger et al. 2010), the IP3R2 knockout mice demonstrate both short- and long-term potentiations that are identical
in amplitude to those of the wild-type population.
When faced with contradictory results one often looks for
explanation in the different methods used. Both models boast
abolishment of astrocytic calcium transients. Henneberger
et al. (2010) used an internal solution that effectively buffers
free calcium fluctuations, whereas the IP3R2 knockout model
blocks calcium from entering the cytoplasm from intracellular

104 SEPTEMBER 2010

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Ca 2+

Whole-cell
Patch Recording

Neuro Forum
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I. WENKER

J Neurophysiol VOL

cellular calcium lead to transmitter release? Is calcium increase

necessary? If so, what proteins does it act on to cause transmitter release and what is their cellular location? It is important
that the mechanism or mechanisms of gliotransmitter release
are reconciled to form a more complete understanding of how
astrocytes actively participate in hippocampal LTP.
ACKNOWLEDGMENTS

I thank A. Tzingounis and D. Mulkey for support and comments on the

manuscript.
DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

REFERENCES

Agulhon C, Fiacco TA, McCarthy KD. Hippocampal short- and long-term

plasticity are not modulated by astrocyte Ca2 signaling. Science 327:
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Hamilton NB, Attwell D. Do astrocytes really exocytose neurotransmitters?
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Hassel B, Paulsen RE, Johnsen A, Fonnum F. Selective inhibition of glial
cell metabolism in vivo by fluorocitrate. Brain Res 576: 120 124, 1992.
Henneberger C, Papouin T, Oliet SH, Rusakov DA. Long-term potentiation
depends on release of D-serine from astrocytes. Nature 463: 232236, 2010.
Kimelberg HK. Functions of mature mammalian astrocytes: a current view.
Neuroscientist 16: 79 106, 2010.
Malenka RC, Nicoll RA. Long-term potentiation: a decade of progress?
Science 285: 1870 1874, 1999.
Mothet JP, Parent AT, Wolosker H, Brady RO Jr, Linden DJ, Ferris CD,
Rogawski MA, Snyder SH. D-Serine is an endogenous ligand for the
glycine site of the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA
97: 4926 4931, 2000.
Mothet JP, Pollegioni L, Ouanounou G, Martineau M, Fossier P, Baux G.
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synaptic memory. Cell 125: 775784, 2006.
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stores. Could these two approaches have differential ability to

block calcium-dependent transmitter release? The answer is
probably yes. Henneberger et al. (2010) are able to clamp
calcium, whereas Agulhon et al. (2010) block all calcium
fluctuations due to IP3R2 activation. Unfortunately, exactly
how increased intracellular calcium causes release of gliotransmitters is not certain (Fig. 1, right). A recent review (Hamilton
and Attwell 2010) points out that activation of PAR1, but not
P2Y1, receptors causes transmitter release in astrocytes, although both result in increased intracellular calcium. Equally
perplexing, the calcium influx generated by activation of
MrgA1 in astrocytes of MrgA1 mice (described earlier) does
not mediate glutamate-induced inward currents in local neurons, whereas uncaging IP3 to increase intracellular calcium in
the same mice did result in an increased frequency of neuronal
miniature excitatory postsynaptic currents.
It would appear that simply increasing intracellular calcium
is not sufficient for gliotransmitter release. If calcium is truly
required for transmitter release, then it may need to occur in
specific nanodomains, where it is difficult to experimentally
manipulate calcium fluctuations. In this case, knocking out
IP3R2 or clamping intracellular calcium may block generalized
astrocytic calcium oscillations, but may not effectively control
calcium in certain nanodomains. Calcium entry in these locations may occur via mechanisms other than the IP3 pathway,
including transient receptor potential or voltage-gated calcium
channels. Or perhaps the knockout mice have elevated levels of
D-serine present in the synaptic cleft, due to membrane mechanisms altered by lack of IP3R2 function. Nonetheless, it is
also possible that buffering intracellular calcium could have
other unanticipated effects, besides eliminating calcium fluctuations. However, the latter possibility still strongly implies
individual astrocytes play a role in the synaptic plasticity of the
hippocampus.
The past two decades have seen a dramatic advance in the
field of astrocyte physiology, with evidence of astrocytes
playing an active role appearing in many facets of synaptic
function. The present study suggests a new role for astrocytes
in memory processing. Indeed, altering astrocyte physiology
affects LTP induction at the SCCA1 synapse of the hippocampus, but because of lack of evidence that D-serine is
required for LTP induction and contradictory results when
using a transgenic approach (Agulhon et al. 2010) exactly how
this occurs remains unanswered. To further understand how
astrocytes actively regulate synaptic transmission a few questions must be addressed (Fig. 1, right). Is D-serine the cotransmitter involved in LTP? How does elevated astrocytic intra-