Department of Physiology, Marshall University School of Medicine, Huntington, West Virginia 25755-9340
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ABSTRACT |
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Grover, Lawrence M. and
Chen Yan.
Evidence for Involvement of Group II/III Metabotropic Glutamate
Receptors in NMDA Receptor-Independent Long-Term Potentiation in Area
CA1 of Rat Hippocampus.
J. Neurophysiol. 82: 2956-2969, 1999.
Previous studies
implicated metabotropic glutamate receptors (mGluRs) in
N-methyl-D-aspartate (NMDA)
receptor-independent long-term potentiation (LTP) in area CA1 of the
rat hippocampus. To learn more about the specific roles played by
mGluRs in NMDA receptor-independent LTP, we used whole cell recordings
to load individual CA1 pyramidal neurons with a G-protein inhibitor
[guanosine-5'-O-(2-thiodiphosphate), GDPS]. Although loading
postsynaptic CA1 pyramidal neurons with GDP
S significantly reduced
G-protein dependent postsynaptic potentials, GDP
S failed to prevent
NMDA receptor- independent LTP, suggesting that postsynaptic
G-protein-dependent mGluRs are not required. We also performed a
series of extracellular field potential experiments in which we applied
group-selective mGluR antagonists. We had previously determined that
paired-pulse facilitation (PPF) was decreased during the first 30-45
min of NMDA receptor-independent LTP. To determine if mGluRs might be
involved in these PPF changes, we used a twin-pulse stimulation
protocol to measure PPF in field potential experiments. NMDA
receptor-independent LTP was prevented by a group II mGluR antagonist
[(2S)-
-ethylglutamic acid] and a group III mGluR antagonist
[(RS)-
-cyclopropyl-4-phosphonophenylglycine], but was not
prevented by other group II and III mGluR antagonists [(RS)-
-methylserine-O-phosphate monophenyl ester or
(RS)-
-methylserine-O-phosphate]. NMDA receptor-independent LTP was
not prevented by either of the group I mGluR antagonists we examined,
(RS)-1-aminoindan-1,5-dicarboxylic acid and
7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester. The
PPF changes which accompany NMDA receptor-independent LTP were not
prevented by any of the group-selective mGluR antagonists we examined,
even when the LTP itself was blocked. Finally, we found that tetanic
stimulation in the presence of group III mGluR antagonists lead to
nonspecific potentiation in control (nontetanized) input pathways.
Taken together, our results argue against the involvement of
postsynaptic group I mGluRs in NMDA receptor-independent LTP. Group II
and/or group III mGluRs are required, but the specific details of the
roles played by these mGluRs in NMDA receptor-independent LTP are
uncertain. Based on the pattern of results we obtained, we suggest that
group II mGluRs are required for induction of NMDA
receptor-independent LTP, and that group III mGluRs are involved in
determining the input specificity of NMDA receptor-independent LTP by
suppressing potentiation of nearby, nontetanized synapses.
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INTRODUCTION |
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An N-methyl-D-aspartate (NMDA)
receptor-independent form of long-term potentiation (LTP) can be
induced in area CA1 of rat hippocampus by high-frequency (100-200 Hz)
tetanic stimulation delivered in the presence of NMDA receptor
antagonists (Cavus and Teyler 1996; Grover
1998
; Grover and Teyler 1990
,
1992
, 1994
). Induction of this form of
LTP requires a postsynaptic Ca2+ signal
(Grover and Teyler 1990
), nifedipine-sensitive (L-type) voltage-dependent calcium channels (VDCCs) (Cavus and Teyler
1996
; Grover and Teyler 1990
; Shankar et
al. 1998
), and postsynaptic action potential firing
(Grover 1998
; Grover and Chen 1999
). We suggested (Grover 1998
; Grover and Chen
1999
) that postsynaptic action potential firing during tetanic
stimulation provides the depolarizing signal for gating of L-type
VDCCs, which mediate the postsynaptic calcium influx needed for LTP
induction. In addition to these requirements, NMDA
receptor-independent LTP in area CA1 requires metabotropic glutamate
receptors (mGluRs) because LTP induction is blocked by the mGluR
antagonist (R,S)-
-methyl-4-carboxyphenylglycine (MCPG)
(Grover 1998
; Little et al. 1995
).
Three major groups of mGluRs have been identified based on sequence
homology, receptor pharmacology, and intracellular signaling pathway
(Conn and Pin 1997; Ozawa et al. 1998
;
Pin and Duvoisin 1995
). Group I mGluRs (mGluR1 and
mGluR5) stimulate phospholipase C activity, leading to the formation of
inositol trisphosphate and diacylglycerol, which in turn promote the
release of intracellular Ca2+ and the activation
of protein kinase C. mGluRs belonging to group II (mGluR2 and mGluR3)
and group III (mGluR4, mGluR6, and mGluR7) are negatively coupled to
adenylate cyclase and thereby inhibit the formation of cAMP. In
hippocampal area CA1, in situ hybridization and immunohistochemical
studies indicate that mGluR subtypes are selectively expressed in
specific cell elements. Postsynaptic CA1 pyramidal neurons express
group I mGluRs (predominantly mGluR5) (Fotuhi et al.
1994
; Lujan et al. 1996
; Shigemoto et al.
1997
). A population of GABAergic interneurons located at the
border of the stratum oriens and the alveus also express group I mGluRs (mGluR1; Fotuhi et al. 1994
; Hampson et al.
1994
; Lujan et al. 1996
;
Shigemoto et al. 1997
). The presynaptic axons of CA3
pyramidal neurons primarily express group III mGluRs (mGluR7;
Bradley at al 1996
; Shigemoto et al.
1997
), although pharmacological evidence also suggests
presynaptic expression of group II mGluRs (Vignes et al.
1995
). In addition, CA1 glial cells express group II receptors (mGluR3; Fotuhi et al. 1994
; Shigemoto et al. 1997
).
Recently, several mGluR antagonists with selectivity between the
three groups have become available. To better understand the role of
mGluRs in NMDA receptor-independent LTP, we examined a series of these
group-specific mGluR antagonists. In addition, we used the whole cell
recording technique to load individual CA1 pyramidal neurons with the
G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPS) to
test for the potential involvement of postsynaptic mGluRs in NMDA
receptor-independent LTP. Finally, because paired-pulse facilitation
(PPF), an index of presynaptic function (Dobrunz and Stevens
1997
; Hess et al. 1987
; Otmakhov et al.
1993
), is reduced during the first 30-45 min of NMDA
receptor-independent LTP (Grover 1998
), we attempted to
determine if a presynaptic mGluR (group II or III mGluR) might
contribute to the tetanization-induced change in PPF. Therefore, we
measured PPF changes during NMDA receptor-independent LTP and looked
for any alteration of these changes by group-selective mGluR antagonists.
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METHODS |
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Slice preparation
Male and female Sprague-Dawley rats (6-16 wk old) were sedated by inhalation of a CO2/air mixture and decapitated. The skull was opened and the brain was removed and placed into chilled, modified artificial cerebrospinal fluid (modified ACSF) with a composition of (in mM) 124.0 NaCl, 26.0 NaHCO3, 3.0 KCl, 0.5 CaCl2, 5.0 MgSO4, and 10.0 D-glucose, bubbled with 95% O2/5% CO2 (pH 7.35). The brain was trimmed down to a block containing the hippocampus, glued to the stage of a vibrotome (Campden Vibroslice), immersed in chilled modified ACSF, and sectioned at 400 µm. Sections containing the hippocampus in a transverse orientation were collected. Individual hippocampal slices were cut free from surrounding structures using two 25- gauge needles.
Hippocampal slices were transferred to a holding chamber where they were stored at room temperature. The holding chamber was filled with standard ACSF composed of (in mM) 124.0 NaCl, 26.0 NaHCO3, 3.4 KCl, 2.0 CaCl2, 2.0 MgSO4, 1.2 NaH2PO4, and 10.0 D-glucose, pH 7.35, gassed with 95% O2/5% CO2. Slices were incubated in the holding chamber for a minimum of 1 h before use in experiments.
For electrophysiology, slices were transferred to a small volume (200 µL) interface recording chamber heated to 35°C. The recording chamber was perfused at a rate of 1.0 to 1.2 ml/min with standard ACSF. Upper surfaces of the slices were exposed to a warmed, humidified 95% O2/5% CO2 atmosphere. Before beginning an experiment, slices were allowed a minimum 30 min recovery period after transfer to the recording chamber.
Drugs were first dissolved in DMSO or NaOH and then were diluted (100-1000 fold) in ACSF for bath application to the slices. NMDA receptor and mGluR antagonists were obtained from Tocris Cookson. All other reagents were from Fisher, Sigma or RBI.
Electrophysiology
Whole cell recordings were obtained from the somata of CA1
pyramidal neurons by the method of Blanton et al. (1989). Patch electrodes (4-6 M
) were filled with a solution of 140 mM cesium gluconate or potassium gluconate, 10 mM sodium HEPES, 3 mM
MgCl2, 3 mM sodium ATP, and 0.2 mM sodium guanosine
5'-trisphosphate (GTP) or 0.5 mM lithium GDP
S. Positive pressure was
applied to the back of the patch electrodes as they were lowered into
the somatic layer of area CA1, and the electrode resistance was
continuously monitored. When electrode resistance increased, positive
pressure was released and gentle negative pressure was applied to form a high-resistance seal (>1 G
, typically 2-5 G
) with the cell membrane. The membrane patch was then ruptured to obtain the whole cell
recording configuration.
Membrane potentials were measured with an Axoclamp 2B (Axon
Instruments) operating in continuous current clamp mode. Access resistance was measured and compensated using the Axoclamp bridge balance circuitry. Cell input resistance was monitored throughout experiments by passing small hyperpolarizing and depolarizing currents
into the cell (up to ±200 pA). Cells were discarded if either access
or input resistances showed large, abrupt, irreversible changes.
Resting membrane potentials were maintained at a constant level near
the normal CA1 pyramidal cell resting potential (65 to
70 mV) by
injecting current through the recording electrode.
Field potentials were recorded from a patch electrode, filled with ACSF, and placed into the middle of the apical dendritic region in the stratum radiatum.
Excitatory postsynaptic potentials (EPSPs) were evoked by stimulating electrodes placed in the mid stratum radiatum to activate Schaffer collateral/commissural fibers. In most recordings, two stimulating electrodes were placed in the stratum radiatum, one on each side of the recording site, to activate two sets of afferent fibers. In the remaining experiments, a single stimulating electrode was used. Constant voltage test stimuli were delivered every 30 s when a single stimulating electrode was used, and were delivered every 15 s (alternating between the two electrodes) when two stimulating electrodes were used.
Inhibitory postsynaptic potentials (IPSPs) were evoked by a single
stimulus or short trains of stimuli (2-3 stimuli delivered at 200 Hz).
Monosynaptic GABAB receptor-mediated IPSPs were isolated by application of -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (15 µM 6,7-dinitroquinoxaline-2,3-dione, DNQX), NMDA (50-100 µM D,L-2-amino-5-phosphonopentanoic acid, AP5), and
GABAA (10 µM bicuculline methiodide) receptor antagonists.
For LTP experiments, test stimulus intensities were set by first determining the intensity that would consistently evoke an orthodromic action potential (in whole cell experiments) or a clearly discernible population spike (in field potential experiments). Test stimuli were delivered at half the intensity needed to evoke firing. Evoked synaptic potentials were low-pass filtered (1-2 kHz for whole cell responses, 2-3 kHz for field responses), amplified (gain of 10-100 for whole cell, 100-1000 for field), digitized (10-40 kHz), and stored on an Intel Pentium processor-based personal computer. Individual synaptic potentials were measured by computing the slope of the initial rising (whole cell) or falling (field) phase of the EPSP.
In most field potential recordings, test stimuli were presented as
pairs separated by a 50 ms interval. Using this stimulus protocol, the
EPSP evoked by the second stimulus is facilitated relative to the first
response (PPF). In these experiments, PPF was quantified as a ratio
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LTP was induced by four 200-Hz stimulus trains (0.5 s duration)
delivered at 5 s intervals. Stimulus intensity during
tetanization was set to twice the test stimulus intensity. Tetanic
stimuli were always delivered in the presence of the competitive NMDA receptor antagonist AP5 (100 µM) to block NMDA receptor dependent LTP
(Grover and Teyler 1990, 1992
,
1994
). In some slices, the noncompetitive NMDA receptor
antagonist MK-801 (dizocilpine, 20 µM) was applied with AP5. Tetanic
stimulation was delivered only after a stable baseline recording period
(minimum of 5 min in whole cell experiments and 10 min in field
potential experiments).
Data analysis
EPSP slopes were normalized by comparison to the mean EPSP slope value obtained during the baseline recording period; EPSP measurements are reported as percentage change from the mean of the baseline. IPSP amplitudes were normalized relative to the original IPSP, recorded 4 min after obtaining the whole cell configuration; IPSPs are reported as percentage of the original value. Grouped data are given as mean ± SE. Statistical significance was determined by use of the Student's t-test (for independent or dependent samples, as appropriate) with P < 0.05 (two-tailed) considered significant. In whole cell recordings, statistical comparisons were made for averaged responses recorded over the 19-20 min posttetanus time period; in field potential recordings, comparisons were made for averaged responses recorded over the 40-45 min posttetanus time period.
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RESULTS |
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Role of postsynaptic G-proteins in NMDA receptor-independent LTP
To assess the possible contribution of postsynaptic mGluRs to NMDA
receptor-independent LTP, we used whole cell patch clamp recordings to
load individual CA1 pyramidal neurons with GDPS, an inhibitor of
G-protein coupled receptors. Because the site of the whole cell
recordings (somatic membrane) was located at a distance from the
presumed apical dendritic location of the mGluRs activated during LTP
induction, we needed to first determine the amount of time required for
somatically applied GDP
S to diffuse into the dendrites and reach a
sufficiently high concentration to inhibit G-protein dependent
receptors. We determined this length of time by recording monosynaptic
GABAB receptor-mediated IPSPs evoked in the
middle of the apical dendritic layer (mid stratum radiatum). Control
cells were recorded with a potassium gluconate-based pipette solution
containing GTP. In control cells, GABAB IPSPs increased in amplitude up to 10 min after breakthrough into the whole
cell mode, but then remained stable for the remainder of the 30 min
whole cell recording period (at 30 min, IPSPs averaged 137 ± 41%
of original amplitude). In contrast, when the pipette solution
contained GDP
S instead of GTP, GABAB IPSPs
showed a steady decline in amplitude, beginning shortly after
breakthrough (Fig. 1, A and
B2). In most GDP
S-loaded neurons, IPSPs reached a stable
minimum 25-30 min after breakthrough (at 30 min, IPSPs averaged
39 ± 7% of original amplitude, P < 0.02 compared with GTP-loaded neurons). From this experiment we concluded
that a 30 min whole cell recording period was sufficient time for
GDP
S to diffuse to a mid-dendritic location and reach a
concentration high enough to significantly inhibit G-protein-coupled
receptors.
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To determine if a postsynaptic G-protein-coupled receptor is required
for NMDA receptor-independent LTP, we examined a second group of
neurons loaded with GDPS using the procedure described above. This
second group of GDP
S-loaded neurons were tetanized in the presence
of AP5 to test for NMDA receptor-independent LTP. NMDA
receptor-independent LTP was also examined in a control group of
neurons. For control neurons, the pipette solution contained GTP
instead of GDP
S. Two afferent pathways were stimulated in both
control and GDP
S experiments; one pathway (tetanized) was stimulated
with four 200 Hz stimulus trains (0.5 s in duration, 5 s
inter-train interval), and the second pathway (not tetanized) was used
as a control for nonspecific changes in EPSPs. Control cells
(Fig. 2A) showed NMDA
receptor-independent LTP in the tetanized pathway (mean increase in
EPSP = 58 ± 26%) but not in the control pathway (EPSP
change =
6 ± 19%; P < 0.03 versus
tetanized pathway), as described in Grover (1998)
.
Neurons loaded with GDP
S also showed NMDA receptor-independent LTP
(Fig. 2B), which was indistinguishable from that
observed in control neurons (compare Fig. 2A with
2B); EPSPs evoked by test stimulation of the tetanized
pathway were increased 63 ± 24% (not significantly different
from control cells, P > 0.89), whereas EPSPs
evoked in the control pathway were not altered (
7 ± 10%
change; P < 0.03 versus tetanized pathway).
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Group-specific mGluR antagonists and NMDA receptor-independent LTP
The lack of difference in LTP magnitude between GDPS-loaded
neurons and control neurons suggests that postsynaptic G-proteins, including postsynaptic mGluRs, are not required for NMDA
receptor-independent LTP. To test this conclusion, we took advantage
of the anatomic segregation of mGluR subtypes in the CA1 area of the
hippocampus (reviewed in INTRODUCTION). The mGluR subunits
expressed by postsynaptic CA1 pyramidal neurons belong to group I. In
contrast, group II and III mGluRs in area CA1 are expressed in other
cell populations (presynaptic CA3 pyramidal neurons and glial cells).
If postsynaptic mGluRs are not required for NMDA receptor-independent
LTP, then group I mGluR antagonists should not inhibit the LTP but
group II or III mGluR antagonists may inhibit the LTP. We therefore examined a series of mGluR antagonists possessing selectivity for group
I, II, or III mGluRs. We used extracellular field potential recordings
to monitor changes in EPSPs during these experiments. In addition, we
used a twin-pulse stimulation protocol to measure PPF to determine if
the previously identified (Grover 1998
) changes in PPF
during NMDA receptor-independent LTP are affected by mGluR antagonists, in particular mGluR antagonists which inhibit
presynaptically expressed mGluRs.
To have a basis for comparing the effects of mGluR antagonists on NMDA
receptor-independent LTP, we interspersed control experiments where
slices were tetanized in the presence of NMDA receptor antagonists AP5
or AP5 + MK-801 only, throughout this series of experiments. As
reported in Grover (1998), 200 Hz tetanization in the
presence of AP5 or AP5 + MK-801 led to an enduring increase in EPSP
slope (Fig. 3, A and
C1), which was accompanied by a decrease in the PPF ratio
(Fig. 3, B1 and C1). Although the EPSP
potentiation remained constant over a 45 min posttetanus recording
period, PPF reverted toward pretetanus levels during this same time
period (Grover 1998
). Neither EPSP slope nor PPF was
altered in control nontetanized pathways (Fig. 3, A, B2, and
C2).
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Group I mGluR antagonists
We examined two antagonists selective for group I mGluRs:
7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (Casabona et al. 1997; Hermans et al.
1998
) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA)
(Moroni et al. 1997
; Pellicciari et al.
1995
). NMDA receptor-independent LTP was easily obtained in
the presence of CPCCOEt (100 and 250 µM, Figs.
4 and 12), with no difference in magnitude from control slices (Fig. 12). Likewise, the posttetanic decrease in PPF seen in control slices (Fig. 3) was unaffected by
CPCCOEt. Control pathways from CPCCOEt-treated slices showed neither
LTP nor posttetanic changes in PPF. A second group I mGluR antagonist,
AIDA (250 µM), was also examined. Like CPCCOET, AIDA did not prevent
NMDA receptor-independent LTP (Figs. 5
and 12). Application of AIDA to slices was accompanied by a reversible depression of EPSPs, with tetanized and control pathways being affected
equally (Fig. 5). The EPSP depression was associated with an increase
in PPF ratio, suggesting that the EPSP depression was caused by a
decrease in glutamate release (Dunwiddie and Haas 1985
;
Manabe et al. 1993
). Slices tetanized in AIDA showed an immediate posttetanic decrease in PPF ratio (Fig. 5B1), but
it was difficult to compare the time course of the PPF change with that
seen in control slices and CPCCOEt- treated slices because of the
opposing effect of AIDA application on PPF. The inability of the group
I mGluR antagonists CPCCOEt and AIDA to inhibit NMDA receptor-independent LTP is consistent with the result obtained in our
whole cell recording experiments. Together, these experiments argue
against the involvement of a group I (postsynaptic) mGluR in this form
of LTP.
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We initially supposed that the depressant effects of AIDA application on baseline synaptic transmission were caused by the drug itself. However, similar effects were later noted with other mGluR antagonists possessing different pharmacological profiles (see Figs. 8, 10, and 11). We eventually determined that the EPSP depression was not a drug effect but that it was caused by the NaOH used to solubilize these compounds, because NaOH alone could reproduce these effects (Fig. 6). Although NaOH did depress excitatory synaptic transmission, NaOH did not prevent NMDA receptor-indepedent LTP and NaOH did not prevent the posttetanic decrease in PPF (Fig. 6).
|
Group II mGluR antagonists
We examine two group II-selective mGluR antagonists:
(2S)--ethylglutamic acid (EGLU) (Jane et al. 1996
)
and (RS)-
-methylserine-O-phosphate monophenyl ester (MSOPPE)
(Thomas et al. 1996
). EGLU, at 100 and 250 µM but not
at 25 µM, prevented NMDA receptor-independent LTP (Figs.
7 and 12). Although EPSP potentiation was
blocked at the two higher concentrations, the posttetanic decrease in
PPF was not altered by EGLU (Fig. 7B1). We obtained a
similar result previously (Grover 1998
) in experiments
with the mGluR antagonist MCPG, which also abolished EPSP potentiation
without affecting posttetanic changes in PPF.
|
Although the group II mGluR antagonist EGLU was able to prevent NMDA receptor-independent LTP, a second group II antagonist, MSOPPE (250 µM), failed to block this LTP (Figs. 8 and 12). The PPF ratio was decreased immediately after tetanization in slices treated with MSOPPE; however, as was the case with AIDA, it was difficult to compare the time course of the PPF change with control slices because of the effect of MSOPPE application on baseline excitatory synaptic transmission. Like AIDA, MSOPPE was dissolved in NaOH prior to dilution into ACSF. We therefore attribute the depression of the EPSP slopes and the associated increase in PPF ratio to the NaOH vehicle rather than to the MSOPPE.
|
Group III mGluR antagonists
We examined two group III-selective mGluR antagonists:
(RS)--cyclopropyl-4-phosphonophenylglycine (CPPG) (Jane et
al. 1996
; Toms et al. 1996
) and
(RS)-
-methylserine-O-phosphate (MSOP) (Thomas et al.
1996
). Both of these compounds had significant effects on NMDA
receptor-independent LTP. At the highest concentration tested (10 µM), CPPG blocked NMDA receptor-independent LTP (Figs. 9 and 12). CPPG, like EGLU, blocked LTP
of EPSP slope but did not abolish the posttetanic decrease in PPF (Fig.
9B1). Lower concentrations of CPPG (1 and 2.5 µM) failed
to block LTP (Fig. 12) and also did not alter the PPF changes (data not
shown). A second group III antagonist, MSOP, appeared to reduce the
magnitude of NMDA receptor-independent LTP (at 100 and 250 µM; Fig.
10), but this effect did not reach significance (Fig. 12). MSOP did not prevent the posttetanic decrease in PPF; however, MSOP, like AIDA and MSOPPE, had reversible effects on
baseline PPF (Fig. 10B), making it difficult to compare the time course of the posttetanic change in PPF with that seen in control
slices. MSOP, like AIDA and MSOPPE, also depressed the baseline EPSP
slope (Fig. 10A). As with AIDA and MSOPPE, we attribute the
reversible effects of MSOP on the EPSP slope and PPF ratio to the NaOH
vehicle used with this compound.
|
|
Both group III antagonists, in at least one
concentration, had an additional effect not observed with the
group I and II antagonists: control (nontetanized) pathways showed a
persistent LTP-like increase in EPSP slope (Figs.
10-12).
Significant potentiation of control pathways was observed with 2.5 µM
CPPG, and 25 and 100 µM MSOP (Fig. 12). We were initially concerned
that the potentiation seen in control pathways could be an artifact
caused by overlapping stimulation of the same afferent fibers by both
stimulating electrodes. Two considerations argue against this
explanation. First, identical methods were used for placement of
stimulating electrodes in all experiments, yet potentiation of control
pathways was seen only when slices were perfused with a group III mGluR
antagonist. Second, potentiation of control pathways occurred despite
the confirmation, by electrophysiological criteria, of pathway
independence (see example shown in Fig. 11). The potentiation of
control pathways that were seen when slices were tetanized in CPPG or
MSOP was not caused simply by application of the drugs because slices
treated with either CPPG or MSOP, but not tetanized, showed no
potentiation; 40-45 min after washout of 10 µM CPPG, the EPSP slope
was changed by only 4 ± 4% (n = 5); 40-45
min after washout of 100 µM MSOP, the EPSP slope was changed by only
3 ± 10% (n = 11). We note that this
observation-the "spreading" of potentiation to nontetanized synapses
has precedent in the CA1 area (Engert and Bonhoeffer 1997
; Schuman and Madison 1994
).
|
|
Summary of mGluR antagonist effects on NMDA receptor-independent LTP
Figure 12 summarizes and compares the magnitude of NMDA receptor-independent LTP (EPSP change measured 45 min posttetanus) obtained in control slices and in slices treated with group-selective mGluR antagonists. Changes in EPSP slope in both tetanized (Fig. 12A) and control (Fig. 12B) pathways are shown. Tetanization in the presence of the group I antagonists AIDA and CPCCOEt produced results indistinguishable from tetanization under control conditions (AP5/AP5 + MK-801); tetanized pathways showed LTP whereas control pathways were unaltered. One of the group II antagonists (EGLU) inhibited NMDA receptor-independent LTP in a concentration-dependent manner, with significant inhibition seen at 100 and 250 µM concentrations. There was a tendency for control pathway EPSPs to be reduced at the 45 min posttetanus time point, although this effect was significant for only one of the three concentrations examined (100 µM; Fig. 12B). A second group II antagonist (MSOPPE) failed to affect NMDA receptor-independent LTP even at a high concentration (250 µM). Two group III mGluR antagonists were examined, one of which, CPPG, significantly inhibited NMDA receptor-independent LTP, but only at the highest concentration tested (10 µM). The second group III antagonist (MSOP) failed to prevent LTP in tetanized pathways. As described in the preceding section, tetanization in the presence of both CPPG (at 2.5 µM) and MSOP (at 25 and 100 µM) led to potentiation of EPSPs in the control (nontetanized) pathways. Collectively, these results indicate that group II and/or group III mGluRs contribute, in some critical way, to NMDA receptor-independent LTP. However, these results provide no support for an involvement of group I mGluRs in NMDA receptor-independent LTP.
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DISCUSSION |
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NMDA receptor-independent LTP and postsynaptic G-proteins
We used the whole cell recording technique to load individual CA1
pyramidal neurons with the G-protein inhibitor GDPS. In a control
experiment, we found that the G-protein dependent,
GABAB receptor-mediated IPSP was suppressed in
GDP
S-loaded cells. In contrast, NMDA receptor-independent LTP was
fully intact in GDP
S-loaded neurons. This finding suggests that
postsynaptic G-protein coupled receptors, including postsynaptic
mGluRs, are not required for induction of NMDA receptor-independent
LTP. This conclusion is dependent on the adequacy of our methods, the
major limitation being uncertainty over the GDP
S concentration
obtained in the intracellular compartment where synaptically activated
postsynaptic mGluRs are located. In particular, it is possible that we
might not have obtained a high enough concentration of GDP
S in this compartment to adequately inhibit the postsynaptic mGluRs. Our control
experiment examining GDP
S inhibition of GABAB
receptor-mediated IPSPs argues against this possibility.
There are two factors that limit the concentration of GDPS in the
synaptic region of the postsynaptic neuron. The first factor is
diffusion of GDP
S from the patch pipette into the neuron cell body,
which depends primarily on the molecular weight of the diffusing substance and the access resistance of the whole cell pipette (Pusch and Neher 1988
). The second factor is diffusion
from the cell body into the dendrites, which depends on the length and diameter of the dendritic process. However, consideration of these two
factors cannot account for the difference in GDP
S inhibition of
GABAB IPSPs compared with NMDA
receptor-independent LTP. Although there was a difference in whole
cell access resistance in these two experiments (31 ± 2 M
in
the IPSP experiment and 18 ± 2 M
in the LTP experiment,
P < 0.005), this difference would have caused better
diffusion of GDP
S into cells in the LTP experiment, yet in this same
experiment GDP
S was without effect. In these experiments, the
postsynaptic potentials (IPSPs and EPSPs) were both evoked at the same
distance from the neuron cell bodies (mid stratum radiatum) and we
allowed an identical time period (30 min) for GDP
S loading. In our
IPSP experiment, 30 min was sufficient for suppression of synaptically
evoked, G-protein dependent postsynaptic responses. Because we allowed
30 min for pretetanus whole cell recording in our LTP experiment,
postsynaptic mGluRs should have been at least partially inhibited at
the time of tetanization, yet the LTP was unaltered. Based on these
considerations, we conclude that postsynaptic mGluRs are unlikely to be
required for induction of NMDA receptor-independent LTP. Because mGluR
subtypes are anatomically segregated in the various cell populations of
the CA1 area, with postsynaptic pyramidal neurons expressing group I
mGluRs and presynaptic pyramidal neurons and glial cells expressing
group II and III mGluRs, we were able to test this conclusion using a
different experimental approach that examined group selective mGluR
antagonists for inhibition of NMDA receptor-independent LTP.
Group selective mGluR antagonists and NMDA receptor-independent LTP
Six group-selective mGluR antagonists were examined for their
ability to inhibit NMDA receptor-independent LTP: AIDA and CPCCOEt (group I selective), EGLU and MSOPPE (group II selective), and CPPG and
MSOP (group III selective). Neither of the group I antagonists inhibited NMDA receptor-independent LTP. This lack of efficacy is
consistent with the results of our GDPS experiments and further argues against a role for postsynaptic (group I) mGluRs in NMDA receptor-independent LTP in hippocampal area CA1.
NMDA receptor-independent LTP was inhibited by one of the group II
antagonists (EGLU) and one of the group III antagonists (CPPG),
indicating the involvement of a group II and/or group III mGluR.
However, LTP was not prevented by a second group II antagonist (MSOPPE)
nor a second group III antagonist (MSOP). This inconsistent pattern of
results may reflect any of the following factors: 1) The
pharmacological activity of the group II and III antagonists that we
used is incompletely characterized. The effectiveness of these
compounds has largely been established based on antagonism of
prototypical agonists for group II [e.g.,
(1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid] and group III
(e.g., L-2-amino-4-phosphonobutyric acid) mGluRs (Jane
et al. 1996; Thomas et al. 1996
; Toms et
al. 1996
), and so the mGluR subtype selectivity for these
compounds is not known. For instance, MSOPPE might be ineffective
against a required group II mGluR or MSOP might be ineffective against
a required group III mGluR. 2) Some of these compounds
may have low potency against a specific mGluR subtype that is required
for NMDA receptor-independent LTP. We might therefore have applied a
particular compound at too low a concentration. 3) Many
of the antagonists used in this study lose selectivity when applied at
high concentrations. For example, CPPG exhibits 30-fold selectivity for
group III mGluRs as compared with group II mGluRs, but can potently
inhibit group II mGluRs when used at a sufficient concentration
(Jane et al. 1996
). 4) NMDA
receptor-independent LTP may require mGluRs belonging to both groups
II and III.
In two previous studies (Grover 1998; Little et
al. 1995
), we found that MCPG, a less specific antagonist of
mGluRs, was able to prevent NMDA receptor-independent LTP. MCPG is an
antagonist of group I and II mGluRs (Conn and Pin 1997
;
Pin and Duvoisin 1995
). Evidence for MCPG as an
antagonist of group III mGluRs is weak (Conn and Pin
1997
; Pin and Duvoisin 1995
) but not completely lacking (Roberts 1995
). Considered as a whole
(Grover 1998
; Little et al. 1995
; present
study), our evidence best supports a required role for group II mGluRs
in NMDA receptor-independent LTP because the LTP was blocked by EGLU
and MCPG. The negative findings with MSOPPE could reflect a lack of
efficacy at the particular mGluR subtype required or may simply reflect
the low potency of the compound. The positive findings with CPPG, on
the other hand, could reflect the relatively high potency of this
compound against group II mGluRs (Toms et al. 1996
) in
addition to its effectiveness against group III mGluRs. At present, we
have little evidence supporting a role for group I mGluRs in NMDA
receptor-independent LTP. The ability of MCPG to block this LTP would
be consistent with a role for group I receptors; however, other group I
selective antagonists (AIDA and CPCCOEt) were not effective. Moreover,
postsynaptic neurons loaded with GDP
S showed LTP despite the
demonstrated effectiveness of the loading procedure against another
G-protein coupled postsynaptic receptor and the known postsynaptic
localization of group I mGluRs in hippocampal area CA1.
One possibility that our data cannot exclude is the involvement of a
presynaptic mGluR5. AIDA and CPCCOEt are more potent antagonists of
mGluR1 than mGluR5 (Casabona et al. 1997; Moroni et al. 1997
). It could therefore be argued that we applied
these group I antagonists at too low a concentration to inhibit any presynaptic mGluR5 receptors involved in NMDA receptor-independent LTP. However, the mGluR expression pattern in hippocampal area CA1
argues against this possibility because mGluR5 is predominant localized
to postsynaptic pyramidal neurons (Shigemoto et al. 1997
; Takumi et al. 1998
).
Roles of mGluRs in NMDA receptor-independent LTP
In a previous study (Grover 1998) we found that
PPF, which provides a relative index of the probability of transmitter
release, is altered during the first 30-45 min posttetanus, and we
have confirmed this finding in the present study. Because our
experiments with group selective mGluR antagonists suggest the
potential involvement of a presynaptically expressed mGluR, it seemed
possible that activation of this presynaptic mGluR might contribute to
the posttetanic decrease in PPF. However, our results argue against
this possibility because the two compounds (EGLU and CPPG) that
effectively inhibited NMDA receptor-independent LTP and that act
against group II and III mGluRs failed to prevent the posttetanic
decrease in PPF. The mechanism underlying the posttetanic decrease in
PPF therefore remains unclear. Whatever the mechanism, the posttetanic
change in PPF may be mechanistically unrelated to NMDA
receptor-independent LTP because the PPF changes can persist under
conditions where the LTP is blocked.
As previously suggested (Grover and Teyler 1992), mGluRs
appear to be involved in regulating the pathway specificity of NMDA receptor-independent LTP. However, the details of this regulation appear to be considerably different from what we initially suspected; in the absence of pharmacological intervention, NMDA
receptor-independent LTP is input-specific (i.e., observed only in
tetanized pathways and not in control pathways). However, when slices
are treated with group III selective antagonists, input specificity is
lost. Group III mGluRs may therefore play a role in suppressing
potentiation in nontetanized input pathways. Potentiation in
nontetanized pathways could be triggered by the spatially extensive
postsynaptic Ca2+ signal that is generated during
tetanization because cytoplasmic Ca2+
concentration
mediated by influx through VDCCs
is elevated across a
large extent of the dendritic tree during tetanization (Miyakawa et al. 1992
). Additionally, nontetanized synapses may be
stimulated by glutamate "spilling over" from tetanized synapses
(Kullmann and Asztely 1998
). Activation of group III
mGluRs might therefore be required to suppress potentiation at
nontetanized synapses. Although "spillover" is most prominent at
lower-than-physiological temperatures (Kullmann and Asztely
1998
), the multiple high-frequency stimulus trains we used to
induce NMDA receptor-independent LTP might facilitate spillover even
at the near-physiological temperature (35°C) that we used. We
speculate that activation of group III mGluRs on nontetanized synapses
provides a "stop" signal to prevent LTP. In contrast, group II
mGluRs may provide a "go" signal because activation of group II
mGluRs seems to be necessary for LTP induction. Although both group II
and group III mGluRs at nontetanized synapses might be activated by
"spilled over" glutamate, input specificity could be maintained if
there were differences in anatomic localization between group II and
III mGluRs. Current evidence indicates that group II mGluRs are located
on axon preterminal membranes whereas group III mGluRs are located
directly on axon terminals (Shigemoto et al. 1997
;
Takumi et al. 1998
). Our hypothesis concerning the roles
of group II and III mGluRs in NMDA receptor-independent LTP is
summarized diagrammatically in Fig. 13.
|
Although other explanations might be offered to explain the pattern of results we obtained, the "stop" and "go" signals that we postulate do lead to testable predictions. For instance, if group III mGluRs constitute a stop signal for NMDA receptor-independent LTP, then it should be possible to prevent LTP by treating slices with a group III agonist. In addition, if group II mGluRs provide a go signal, then it should be possible to promote nonspecific LTP at nontetanized synapses by treating slices with a group II agonist.
Our results suggest the involvement of mGluRs with presynaptic or glial
localization. Can we reconcile this finding with our previous studies
(Grover 1998; Grover and Chen 1999
;
Grover and Teyler 1990
) that indicated a critical
postsynaptic role in NMDA receptor-independent LTP induction? If
induction of this form of LTP requires both postsynaptic and
presynaptic (or glial) elements, then some type of intercellular
messenger may be required. A variety of signals that could serve this
role have been identified and may play roles during LTP in hippocampal
area CA1, including arachidonic acid or one of its metabolites
(Williams and Bliss 1989
; Williams et al.
1989
), nitric oxide (Hawkins et al. 1998
;
Holscher 1997
), platelet activating factor (Kato
et al. 1994
) and neurotrophic factors (Kang et al.
1997
; Patterson et al. 1992
). It will be important to determine whether any of these signals are involved in the
NMDA receptor-independent type of LTP.
Metabotropic glutamate receptors are likely to play diverse roles in
synaptic plasticity. Group I receptors are involved in NMDA
receptor-dependent LTP (Aiba et al. 1994;
Lu et al. 1997
; Manahan-Vaughan 1997
),
where they directly contribute to the generation of a postsynaptic
Ca2+ signal (Wilsch et al. 1998
). In
addition, group I mGluRs can indirectly contribute to the postsynaptic
Ca2+ signal through up-regulation of NMDA receptors
(Fitzjohn et al. 1996
; Harvey and Collingridge
1993
; Holohean et al. 1999
; Pisani et al.
1997
). In NMDA receptor-independent LTP, the role of mGluRs is
quite different because group II and III mGluRs are coupled to a
different signaling pathway that does not directly increase intracellular Ca2+ concentration.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-34650.
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FOOTNOTES |
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Address reprint requests to L. M. Grover.
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 14 May 1999; accepted in final form 30 August 1999.
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REFERENCES |
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