1Department of Neuroscience, Howard Hughes Medical Institute, Brown University, Providence, Rhode Island 02912; and 2Program in Developmental and Fetal Health, Samuel Lunenfeld Research Institute, Toronto, Ontario M5G 1X5, Canada
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ABSTRACT |
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Huber, Kimberly M., John C. Roder, and Mark F. Bear. Chemical Induction of mGluR5- and Protein Synthesis-Dependent Long-Term Depression in Hippocampal Area CA1. J. Neurophysiol. 86: 321-325, 2001. Recent work has demonstrated that specific patterns of synaptic stimulation can induce long-term depression (LTD) in area CA1 that depends on activation of metabotropic glutamate receptors (mGluRs) and rapid protein synthesis. Here we show that the same form of synaptic modification can be induced by brief application of the selective mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). DHPG-LTD 1) is a saturable form of synaptic plasticity, 2) requires mGluR5, 3) is mechanistically distinct from N-methyl-D-aspartate receptor (NMDAR)-dependent LTD, and 4) shares a common expression mechanism with protein synthesis-dependent LTD evoked using synaptic stimulation. DHPG-LTD should be useful for biochemical analysis of mGluR5- and protein synthesis-dependent synaptic modification.
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INTRODUCTION |
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Homosynaptic long-term
depression (LTD) is a widely expressed form of synaptic plasticity in
the brain. The best understood type of LTD is induced in hippocampal
area CA1 by low-frequency synaptic stimulation (LFS) via an
N-methyl-D-aspartate (NMDA) receptor-dependent
rise in postsynaptic intracellular Ca2+ and the
activation of a protein phosphatase cascade (Bear and Abraham
1996). Under the appropriate circumstances, pharmacological activation of NMDA receptors (NMDARs) can also induce this type of LTD.
This "chem-LTD" approach has been useful for the biochemical characterization of the mechanism, revealing, for example, that NMDAR-dependent LTD is associated with dephosphorylation of the GluR1
subunit of the postsynaptic
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
(Lee et al. 1998
).
Recent work has shown that mechanistically distinct types of LTD can
also be induced in CA1 by other types of synaptic stimulation. For
example, paired-pulse stimulation repeated at 1 Hz for 15 min (PP-LFS)
induces LTD that is independent of NMDARs and requires activation of
metabotropic glutamate receptors (mGluRs) (Huber et al.
2000; Kemp and Bashir 1999
). This
mGluR-dependent form of LTD is of particular interest because it also
requires rapid translation of preexisting mRNA (Huber et al.
2000
). A "chem-LTD" approach could be particularly useful
for dissecting this novel mechanism. Indeed, reports from several
groups indicate that transient activation of group 1 mGluRs with the
selective agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) can induce LTD
(Camodeca et al. 1999
; Fitzjohn et al.
1999
; Huber et al. 2000
; Palmer et al.
1997
). However, it is clear that not all protocols are
equivalent; for example, some are effective only under conditions of
low Mg2+ and are partially dependent on NMDARs
(Palmer et al. 1997
; Schnabel et al.
1999
).
Here we characterize a chemical induction protocol that reliably
produces protein synthesis-dependent LTD (Huber et al.
2000). We show that mGluR5 is required for LTD induction and
provide novel evidence that this chemically induced LTD shares a common saturable expression mechanism with LTD induced using PP-LFS. We
anticipate that the method we describe here will be useful for
understanding how mGluR activation regulates mRNA translation and the
expression of synaptic LTD.
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METHODS |
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All animals were used in accordance with procedures approved by
the Brown University Institutional Animal Care and Use Committee. Hippocampal slices were prepared from postnatal day
21-30 (P21-30) Long Evans rats (Charles River,
Cambridge, MA) and mGluR5 knockout mice (Lu et al. 1997)
as described previously (Huber et al. 2000
). For most
experiments, CA3 was removed immediately after sectioning. Slices
recovered for 1-2 h at room temperature (rats) or at 30°C (mice) in
artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 MgCl2, 2 CaCl2, and 10 dextrose, saturated with 95%
O2-5% CO2. For recording, slices were placed in a submersion recording chamber and perfused with
30°C ACSF at a rate of 2 ml/min.
Synaptically evoked field potentials (FPs) were recorded from area CA1
as described previously (Huber et al. 2000). Sharp microelectrode and whole cell voltage-clamp recordings were made using
Axoclamp 2B and Axopatch 1D amplifiers (Axon Instruments), respectively. Sharp electrodes (80-120 M
) were filled with 3 M
K-acetate and 10 mM KCl; patch pipettes (3-7 M
) were filled with
(in mM) 134 K-gluconate, 6 KCl, 4 NaCl, 10 HEPES, 0.2 EGTA, 4 MgATP,
0.3 TrisGTP, and 14 phosphocreatine. The pH of the internal solution
was adjusted to 7.25 with KOH, and the osmolarity was adjusted to 300 mOsm with H2O or sucrose. Only experiments in which there was less than a 15% change in series resistance were included in the analysis. Waveforms were filtered at 2 kHz and acquired
and digitized at 10 kHz on a PC using Experimenter's Workbench
(DataWave Systems, Boulder, CO).
Baseline responses were collected every 10-30 s using a stimulation intensity (10-30 µA; 0.2 ms) yielding 50-60% of the maximal response. Experiments in which there was a >5% drift in the response magnitude during the 20-min baseline period before DHPG or LFS were excluded from further analysis. All experiments with mGluR5 KO mice used wildtype littermates as controls and were performed blind to the genotype, later determined by Therion (Troy, NY). LFS consisted of 900 pulses at 1 Hz. PP-LFS consisted of 900 pairs of stimuli (50-ms interstimulus interval) delivered at 1 Hz. In saturation experiments, stimulus duration was increased from 0.2 to 0.4 ms during PP-LFS.
The group data were analyzed as follows: 1) the initial slopes of the FPs and excitatory postsynaptic potentials (EPSPs), or the amplitude of the excitatory postsynaptic currents (EPSCs), for each experiment were expressed as percentages of the preconditioning or DHPG baseline average, 2) the time scale in each experiment was converted to time from the onset of conditioning or DHPG, and 3) the time-matched, normalized data were averaged across experiments and expressed in the text and figures as the means ± SE. Significant differences between groups were determined using an independent t-test or ANOVA performed on a 5-min average taken 1 h after LFS or DHPG application.
R,S-DHPG and D-2-amino-5-phosphonopentanoic acid
(D-AP5) was purchased from Tocris (St. Louis, MO); all
other chemicals were from Sigma Chemical (St Louis, MO). DHPG was
prepared as a 100 times stock in H2O, aliquoted
and stored at 20°C. Fresh stocks were made once a week. A 10 times
stock of AP5 was prepared in ACSF and stored at 4°C. These stocks
were diluted in ACSF to achieve their final concentrations. Picrotoxin
was dissolved directly into ACSF immediately before use.
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RESULTS |
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Application of DHPG for 5 min produced an acute, dose-dependent
depression of evoked FPs (Fig.
1A). At concentrations 50 µM, the FP did not fully recover after drug wash out. Instead, the
synaptic responses stabilized at a depressed level (50 µM: 69 ± 5%, means ± SE, of pre-DHPG baseline; n = 11;
100 µM: 48 ± 1%; n = 4). In all subsequent
studies 50 µM, DHPG (5 min) was used to induce what we will refer to
as DHPG-LTD. Application of another group 1 mGluR agonist, quisqualic
acid (5 min; 5 µM), also resulted in LTD (81 ± 2%;
n = 4), confirming that the effect is not peculiar to
DHPG. Two-pathway experiments (n = 4), in which only
one input was stimulated during DHPG, indicated that DHPG-LTD does not
require concurrent synaptic stimulation (stimulated: 62 ± 4%;
unstimulated: 68 ± 5%, P > 0.2; Fig.
1B). DHPG-LTD also showed evidence of saturation; two
applications of 50 µM DHPG were sufficient to produce maximal
depression (Fig. 1C).
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Intracellular recordings confirmed that the DHPG-LTD of FPs reflects
diminished synaptic transmission. Both sharp electrode recording of
EPSPs and whole cell voltage-clamp recording of EPSCs (recorded at 70
mV) revealed stable LTD (EPSP: 61 ± 5%; n = 6; Fig. 1D; EPSC: 69 ± 5%; n = 5; Fig.
1E). In contrast, there were no significant long-term
changes in membrane potential, input resistance, or membrane
excitability measured 1 h after DHPG (data not shown). Thus
DHPG-LTD is a long-lasting modification of synaptic transmission.
The competitive NMDAR antagonist AP5 (50 µM) had no effect on the magnitude of DHPG-LTD as compared with interleaved control slices (AP5: 83 ± 3%, n = 5; control: 85 ± 3%, n = 4; P > 0.3; Fig. 2A). LTD induced with 5 µM quisqualic acid was also unaffected by AP5 (79 ± 2%; n = 3). Therefore LTD induced by pharmacological activation of group 1 mGluRs under these experimental conditions does not require concurrent NMDAR activation.
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To assess the involvement of mGluR5, the major group 1 mGluR in area
CA1 pyramidal neurons (Romano et al. 1995), DHPG-LTD was
attempted in mice lacking this receptor. DHPG-LTD was absent in the
mGluR5 homozygous mutants (98 ± 3% measured 1 h after DHPG application; n = 8; Fig. 2A). An
intermediate amount of LTD was observed in heterozygous mutants
(84 ± 4%; n = 6), as compared with wild-type
littermate controls (77 ± 2%; n = 9; Fig.
2B). A one-way ANOVA revealed a significant effect of
genotype [F(2,19) = 10.33, P < 0.001]. A subsequent Tukey test revealed that both the wild-type and
heterozygotes were significantly different from homozygotes
(P < 0.025). Although there is a trend for DHPG-LTD in
the heterozygotes to be less than wildtypes, this is not significant (P = 0.5). Thus DHPG-LTD strictly relies on mGluR5, and
the presence of one allele for mGluR5 is sufficient for LTD induction.
In contrast to DHPG-LTD, normal NMDAR-dependent LTD, induced with LFS,
was observed in the homozygous mutants (87 ± 2%;
n = 6; P > 0.6; Fig. 2C) as
compared with the wild type mice (89 ± 5%; n = 6). These results indicate that there are two distinct routes of LTD
induction in area CA1: one that relies on NMDARs and another on mGluR5.
The results from the mGluR5 knockouts indicate that the induction mechanisms of NMDAR-dependent LTD and DHPG-LTD are different. The next experiment was designed to test whether these two forms of LTD utilize similar expression mechanisms. Repeated episodes of LFS were delivered to saturate NMDAR-dependent LTD (Fig. 3A). DHPG then was then applied, and the magnitude of LTD was measured by renormalizing FP slope values to a pre-DHPG baseline. If NMDAR-dependent LTD and DHPG-LTD utilize a common expression mechanism, then previous saturation of NMDAR-dependent LTD should reduce or occlude DHPG-LTD. However, DHPG still significantly depressed synaptic responses (81 ± 5% of pre-DHPG baseline; n = 5; P < 0.05; Fig. 3B), suggesting that NMDAR-dependent LTD and DHPG-LTD use distinct expression mechanisms.
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The same approach was used to assess whether DHPG-LTD employs the same saturable expression mechanism as synaptically evoked mGluR-dependent LTD. PP-LFS in the presence of the NMDAR antagonist D-AP5 (50 µM) was used to saturate mGluR-dependent LTD, and DHPG (50 µM) was then applied to the slice (Fig. 3C). In contrast to the previous occlusion experiment, DHPG application after saturation of LTD with PP-LFS did not induce any further LTD (100 ± 5% of pre-DHPG baseline; n = 5; P > 0.5; Fig. 3D). These results provide strong evidence that mGluR-LTD induced with DHPG and PP-LFS share common expression mechanisms.
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DISCUSSION |
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A number of different protocols have been introduced to induce
homosynaptic LTD in CA1 (Berretta and Cherubini 1998;
Camodeca et al. 1999
; Dudek and Bear
1992
; Fitzjohn et al. 1999
; Huber et al.
2000
; Kemp and Bashir 1999
; Oliet et al.
1997
; Overstreet et al. 1997
; Palmer et
al. 1997
). Although mGluR involvement has been suggested for
many of these, the constellation of findings is confusing and not
entirely consistent with a single mGluR-dependent form of LTD. For
example, it has been reported that application of 100 µM DHPG for 10 min to adult hippocampal slices elicits little LTD unless slice
excitability is increased by removing Mg2+ from
the extracellular medium (Palmer et al. 1997
;
Schnabel et al. 1999
). The resulting LTD is partially
blocked by NMDAR antagonists. Moreover, PP-LFS in adult hippocampal
slices can apparently elicit LTD via activation of either group 1 mGluRs or activation of AMPA/kainate receptors (Kemp
and Bashir 1999
). In contrast, we recently demonstrated that in
P21-30 rats, both PP-LFS and DHPG (50 µM, 5 min) induce LTD that is 1) independent of NMDAR activation,
2) blocked entirely by mGluR antagonists, and 3)
dependent on a transient phase of mRNA translation (Huber et al.
2000
). The latter finding is of particular importance, as this
mGluR-LTD model should be useful for elucidating the regulation and
function of dendritic protein synthesis, which may be defective in
fragile-X mental retardation (Jin and Warren 2000
).
Because of the diverse effects of DHPG and PP-LFS, it could not be
assumed that previous findings under different experimental conditions
would apply to our model. Therefore it was necessary to characterize
the protein synthesis-dependent form of mGluR-LTD. We have shown here
that DHPG-LTD is a saturable form of synaptic plasticity, that it
requires mGluR5, that it is mechanistically distinct from
NMDAR-dependent LTD, and, importantly, that it shares a common
saturable expression mechanism with the LTD evoked using PP-LFS.
Because DHPG-LTD does not require concurrent synaptic stimulation, it
is a form of "chem-LTD" (Lee et al. 1998) that should be useful for biochemical and biophysical studies.
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FOOTNOTES |
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Address for reprint requests: M. F. Bear (E-mail: mbear{at}brown.edu).
Received 23 January 2001; accepted in final form 23 March 2001.
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REFERENCES |
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