1Department of Neuroscience, Howard Hughes Medical Institute, Brown University, Providence, Rhode Island 02912; and 2Program in Developmental and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5 and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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ABSTRACT |
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Sawtell, Nathaniel B., Kimberly M. Huber, John C. Roder, and Mark F. Bear. Induction of NMDA Receptor-Dependent Long-Term Depression in Visual Cortex Does Not Require Metabotropic Glutamate Receptors. J. Neurophysiol. 82: 3594-3597, 1999. We tested the role of group I mGluRs in the induction of long-term depression (LTD) in the visual cortex, using the novel mGluR antagonist LY341495 and mice lacking mGluR5, the predominant phosphoinositide (PI)-linked mGluR in the visual cortex. We find that LY341495 is a potent blocker of glutamate-stimulated PI hydrolysis in visual cortical synaptoneurosomes, and that it effectively antagonizes the actions of the mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (ACPD) on synaptic transmission in visual cortical slices. However, LY341495 has no effect on the induction of LTD by low-frequency stimulation. Furthermore, mice lacking mGluR5 show normal NMDA receptor-dependent LTD. These results indicate that group I mGluR activation is not required for the induction of NMDA receptor-dependent LTD in the visual cortex.
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INTRODUCTION |
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Brief monocular deprivation during early postnatal
life causes a long-term depression (LTD) of synaptic transmission that renders visual cortical neurons unresponsive to stimulation of the
deprived eye. One mechanism for deprivation-induced LTD in visual
cortex is triggered by the residual activity arising from the deprived
eye (Rittenhouse et al. 1999). Brain slice preparations have been established to understand how presynaptic activity can cause
synaptic depression in visual cortex. One reliable method for inducing
homosynaptic LTD in vitro is prolonged low-frequency synaptic
stimulation (LFS) (Kirkwood et al. 1993
). Although it is
clear that activation of postsynaptic glutamate receptors is required
for LFS-induced LTD in visual cortex, there are contradictory data on
which receptor subtypes are involved.
Metabotropic glutamate receptors coupled to phosphoinositide (PI)
metabolism (group I mGluRs) provide an attractive candidate mechanism
for homosynaptic LTD (Bear and Dudek 1991). These
receptors are essential for LTD in the cerebellar cortex (Linden
and Connor 1993
) and are highly expressed in the visual cortex
during the postnatal period when visual deprivation induces LTD
(Dudek and Bear 1989
). Tests of mGluR involvement in
visual cortical LTD have been conducted using the competitive
antagonist
-methyl-4-carboxyphenylglycine (MCPG) (Haruta et
al. 1994
; Hensch and Stryker 1996
; Huber
et al. 1998
). However, interpretation of these studies is
complicated by the recent finding that MCPG is an extremely weak
antagonist of glutamate-stimulated PI hydrolysis in visual cortex
(Huber et al. 1998
). Thus the question remains whether
mGluR activation is required for induction of homosynaptic LTD in
visual cortex.
Here we report new tests of the hypothesis that group I mGluRs play an essential role in the induction of LTD by LFS, using a novel and potent mGluR antagonist LY341495 and mice lacking the major group I mGluR in visual cortex (mGluR5). The data show that mGluRs are not necessary for the induction of one prominent form of LTD in visual cortex.
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METHODS |
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Phosphoinositide hydrolysis assays
Synaptoneurosomes were prepared from rat visual cortices, and PI
hydrolysis was measured and analyzed as described previously (Dudek et al. 1989; Huber et al. 1998
).
Ionotropic glutamate receptor antagonists CNQX (40 µM) and
D,L-AP5 (200 µM) were included in the reactions to ensure
that the glutamate-stimulated PI hydrolysis was due to activation of mGluRs.
Electrophysiology
Visual cortical slices were prepared as described previously
(Huber et al. 1998). Slices were allowed to recover for
1-2 h at room temperature (rat) or 30°C (mouse) in artificial
cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 MgCl2, 2 CaCl2, and 10 dextrose. ACSF was continuously saturated in 95% O2, 5% CO2.
For recordings of synaptic transmission, visual cortical slices were
placed in a submersion recording chamber, maintained at 30°C, and
perfused with ACSF at a rate of 2 ml/min. Extracellular electrodes
(filled with ACSF, 1.0 M
) were placed in layer II/III to monitor
field potentials (FPs) evoked with a stimulating electrode (concentric
bipolar tungsten) placed at the border of layer IV and upper layer V. The amplitude of the maximum negative FP was used to quantify changes
in synaptic responses. Stable baseline responses were elicited at a
rate of 1-4 per min, at 50-60% of the maximal response. LFS
consisted of 900 pulses at 1 Hz. Data were averaged and analyzed as
described previously (Huber et al. 1998
). All
experiments using mice were performed blind to genotype. Genotyping was
done by Therion Corp.
Drug preparation
Glutamate and 1S,3R-aminocyclopentane-1,3-dicarboxylic acid
(ACPD) were obtained from Tocris Cookson (St. Louis, MO). LY341495 was
a generous gift from Eli Lilly. Compounds were dissolved in equimolar
NaOH or H2O aliquoted and stored at 20°C for
no more than 1 wk.
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RESULTS |
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The first series of experiments was designed to determine the
effectiveness of LY341495 as an antagonist of group I mGluRs in the
visual cortex. LY341495 has micromolar potency against quisqualate-stimulated PI hydrolysis in cell lines expressing mGluR1
and mGluR5 (Kingston et al. 1998), and against PI
hydrolysis stimulated by the selective group I agonist
(R,S)-3,5-dihydroxyphenylglycine (DHPG) in rat hippocampal slices
(Fitzjohn et al. 1998
). However, in light of previous
findings that the mGluR antagonist MCPG was effective against ACPD, but
not glutamate-stimulated PI hydrolysis (Huber et al.
1998
), we felt that it was crucial to verify the efficacy of
the antagonist against the endogenous agonist. To address this question
we examined the effects of LY341495 on glutamate-stimulated PI
hydrolysis in synaptoneurosomes prepared from the visual cortex of
postnatal day (P) 21-28 rats.
The rate of PI hydrolysis is determined in this assay by measuring the
inositol monophosphate (IP1) that is generated in
the continuous presence of agonist (Gusovsky and Daly
1988). IP1 accumulation was measured
after a 90 min incubation of synaptoneurosomes in 200 µM glutamate,
the EC50 value in this preparation (Huber et al.
1998
), ± increasing concentrations of LY341495. The data confirm that
LY341495 is an effective antagonist of glutamate-stimulated PI
hydrolysis (Fig. 1;
IC50 < 1 µM; n = 4). LY341495
(100 µM) was sufficient to completely block PI hydrolysis stimulated
by 200 µM glutamate. Therefore 100 µM LY341495 was used in the
subsequent electrophysiological experiments in slices.
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Electrophysiological experiments were conducted using slices prepared
from P21-30 rats, an age when glutamate-stimulated PI hydrolysis is
significantly greater than in adults (Dudek et al. 1989)
and within the critical period for experience-dependent visual cortical
plasticity (Fagiolini et al. 1994
). To confirm that
LY341495 is effective in visual cortical slices, we investigated the
ability of the drug to block the effects of the mGluR agonist ACPD on
synaptic transmission. As previously reported in hippocampal (Baskys and Malenka 1991
; Selig et al.
1995
) and visual cortex slices (Huber et al.
1998
), brief application of ACPD (5 min; 10 µM) rapidly and
reversibly attenuated amplitudes of synaptically evoked FPs (Fig.
2). Preapplication of LY341495 (100 µM)
completely blocked the effects of ACPD on synaptic transmission (Fig.
2A).
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In these same slices, we examined the consequences of LFS given in the presence of LY341495 (Fig. 2A). The magnitude of LTD was unaffected by the drug treatment (78 ± 4% of pre-LFS baseline amplitude values; n = 7), compared with control experiments in which LFS was delivered after testing the effects of ACPD, but in the absence of LY341395 (78 ± 7%; n = 6; P > 0.5; Fig. 2B). These results indicate that LY341495 is an effective antagonist of glutamate-stimulated PI hydrolysis and ACPD-induced synaptic depression, but that it has no effect on LTD induced with LFS in visual cortex slices under our experimental conditions.
In visual cortex and hippocampus, glutamate stimulates PI hydrolysis in
postsynaptic neurons via the activation of mGluR5 (Abe et al.
1992; Romano et al. 1995
; Testa et al.
1994). Therefore to further test the hypothesis that
postsynaptic group I mGluR activation is required for induction of
homosynaptic LTD, we investigated the effects of LFS in visual cortex
of mice (P21-33) with a null mutation of mGluR5 (Lu et al.
1997
).
FP latency, FP half-maximal amplitude, and the stimulus intensity required to elicit half-maximal responses did not differ between wild-type (WT) mice and mGluR5 mutants (data not shown). Similarly, mutant and WT mice displayed no differences in LTD magnitude following LFS (-/-, 83 ± 4%, n = 14 slices, 7 animals; +/-, 81 ± 4%, n = 16 slices, 5 animals; WT, 84 ± 9%, n = 15 slices, 7 animals; Fig. 3A).
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In the rat visual cortex, LTD induction with LFS requires activation of
postsynaptic NMDA receptors under our experimental conditions
(Kirkwood and Bear 1994). Therefore we tested the
effects of the NMDA-receptor antagonist
DL-2-amino-5-phophonovaleric acid (AP5) on LTD
induction in the visual cortex of wild-type and mutant mice (Fig.
3B). In the presence of 100 µM AP5, no significant LTD
was induced by LFS regardless of genotype (WT, 93 ± 7%,
n = 9, P > 0.1; -/-, 96 ± 5%, n = 13, P > 0.5).
After washout of the AP5, stable LTD was induced in the same slices by
a second LFS (WT, 84 ± 1%, n = 5, P < 0.005; -/-, 89 ± 4%,
n = 9, P < 0.05).
Together the results confirm that LFS triggers LTD in visual cortex via activation of NMDA receptors and demonstrate that group I mGluR activation is not required for induction of this form of homosynaptic LTD.
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DISCUSSION |
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Our data show that neither pharmacological blockade of mGluRs nor
genetic ablation of mGluR5 disrupts LTD induced by LFS in slices of
visual cortex. Although contrary to some earlier reports using MCPG in
visual cortex (Haruta et al. 1994; Hensch and
Stryker 1996
), these results are consistent with a recent study
in which LY341495 did not affect NMDA receptor-dependent LTD in the CA1 region of the hippocampus (Fitzjohn et al. 1998
). LTD in
CA1 hippocampus is also normal in mGluR5 knock out mice (Huber
and Bear 1998
). We believe that the controversy surrounding the
role of mGluRs in synaptic plasticity has been largely attributed to
the questionable efficacy of MCPG (Huber et al. 1998
)
and the fact that multiple forms of LTD can coexist at the same
synapses (Oliet et al. 1997
). The results presented
here, using effective pharmacological blockade and gene knock out
technology, are conclusive evidence that mGluR activation is not
required for induction of at least one form of LTD in the visual
cortex. We cannot rule out the possibility that a second,
mGluR-dependent form of LTD also exists in visual cortex, which might
be revealed under different experimental conditions. However, if such a
form of LTD does exist in visual cortex, it is mechanistically distinct
from the NMDA receptor-dependent form.
In a recent study, the contributions to visual cortical plasticity of
group I mGluRs and the mechanisms of homosynaptic LTD were questioned
on the basis of findings that infusion of MCPG failed to prevent the
LTD caused by monocular deprivation in visual cortex in vivo but did
block the synaptic depression caused by LFS in slices (Hensch
and Stryker 1996). However, the conclusions of that study must
be reevaluated in light of the evidence that 1) MCPG has
little effect on glutamate-stimulated PI turnover mediated by group I
mGluRs (Brabet et al. 1995
; Huber et al.
1998
), and 2) neither MCPG (Huber et al.
1998
) nor blockade of mGluRs (present results) inhibit
induction of the NMDA receptor-dependent form of LTD. In future studies
it will be important to use this knowledge to dissect the individual
and combined roles of homosynaptic LTD and mGluRs in visual cortical plasticity.
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ACKNOWLEDGMENTS |
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We thank the Eli Lilly Co. for their generous gift of LY341495.
This work was supported in part by the National Institutes of Health, the National Science Foundation, and the Medical Research Council.
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FOOTNOTES |
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Address reprint requests to M. F. Bear.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 June 1999; accepted in final form 20 August 1999.
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REFERENCES |
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