Evidence for Involvement of Group II/III Metabotropic Glutamate Receptors in NMDA Receptor-Independent Long-Term Potentiation in Area CA1 of Rat Hippocampus

Lawrence M. Grover and Chen Yan

Department of Physiology, Marshall University School of Medicine, Huntington, West Virginia 25755-9340


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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), GDPbeta S]. Although loading postsynaptic CA1 pyramidal neurons with GDPbeta S significantly reduced G-protein dependent postsynaptic potentials, GDPbeta 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)-alpha -ethylglutamic acid] and a group III mGluR antagonist [(RS)-alpha -cyclopropyl-4-phosphonophenylglycine], but was not prevented by other group II and III mGluR antagonists [(RS)-alpha -methylserine-O-phosphate monophenyl ester or (RS)-alpha -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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-alpha -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) (GDPbeta S) 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) 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 GDPbeta 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 GOmega , typically 2-5 GOmega ) 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 alpha -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
<FR><NU>slope EPSP<SUB>2</SUB></NU><DE>slope EPSP<SUB>1</SUB></DE></FR>.
PPF provides a relative index of the probability of neurotransmitter release from the presynaptic terminal (Dobrunz and Stevens 1997; Hess et al. 1987; Otmakhov et al. 1993); increased probability of release is associated with a decrease in PPF, whereas decreased probability of release is accompanied by an increase in PPF.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 GDPbeta S, 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 GDPbeta 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 GDPbeta S instead of GTP, GABAB IPSPs showed a steady decline in amplitude, beginning shortly after breakthrough (Fig. 1, A and B2). In most GDPbeta 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 GDPbeta 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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Fig. 1. GABAB receptor-mediated inhibitory postsynaptic potentials (IPSPs) were suppressed by inclusion of guanosine-5'-O-(2-thiodiphosphate) (GDPbeta S) in the whole cell recording solution. GABAB IPSPs were isolated by perfusion of slices with 6.7-dinitroquinoxaline-2,3-dione (15 µM) and D,L-2-amino-5-phosphonopentanoic acid (AP5, 50-100 µM, to block excitatory postsynaptic potentials (EPSPs), and bicuculline (10 µM), to block GABAA receptor mediated ISPSs. A: after an initial run-up period, GABAB ISPSs remained stable during whole cell recordings with a pipette solution containing guanosine 5'-trisphosphate (GTP). In contrast, GABAB IPSPs were greatly reduced during whole cell recording when a pipette solution containing GDPbeta S was used. IPSP peak amplitudes were normalized relative to the initial value. Time scale is relative to the time of breakthrough into the whole cell configuration. IPSPs were not recorded for the first 4 min after breakthrough. At 30 min after breakthrough, IPSPs were significantly smaller in cells recorded using GDPbeta S (39 ± 7% of original amplitude) compared with cells recorded with GTP (137 ± 41% of original amplitude; P < 0.02). B: representative IPSPs recorded at 5, 10, and 30 min after breakthrough. B1: IPSPs recorded using a pipette solution containing GTP. The IPSPs increased in amplitude from 5 to 10 min and then remained stable up to 30 min after breakthrough. B2: IPSPs recorded using a pipette solution containing GDPbeta S. The IPSPs were unchanged in amplitude between 5 and 10 min but declined substantially by 30 min after breakthrough.

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 GDPbeta S using the procedure described above. This second group of GDPbeta 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 GDPbeta S. Two afferent pathways were stimulated in both control and GDPbeta 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 GDPbeta 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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Fig. 2. Inclusion of GDPbeta S in the whole cell recording solution did not affect N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation (LTP). A: control cells were recorded with GTP pipette solution. Tetanization (200 Hz) was delivered at 0 min. The tetanized pathways showed NMDA receptor-independent LTP (58 ± 25%), but the control pathways were not altered (-6 ± 19%; significantly different from tetanized pathway, P < 0.03). NMDA receptor antagonist AP5 (100 µM) was applied as indicated (horizontal bar). B: cells recorded with GDPbeta S in the whole cell pipette showed NMDA receptor-independent LTP equivalent to control cells. Whole cell recordings began 30 min before 200 Hz tetanization. Application of AP5 is indicated by the horizontal bar. LTP averaged 63 ± 24% in the tetanized pathways (not different from control cells, P > 0.89); control pathways were unchanged (-7 ± 10%; significantly different from tetanized pathway, P <0.03). Inset: EPSPs recorded from one GDPbeta S-loaded neuron before (1 and 3) and after (2 and 4) 200 Hz tetanization; the EPSP from the tetanized pathway was increased (1 and 2) whereas the EPSP in the control pathway was slightly decreased (3 and 4).

Group-specific mGluR antagonists and NMDA receptor-independent LTP

The lack of difference in LTP magnitude between GDPbeta S-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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Fig. 3. NMDA receptor-independent LTP evoked under control conditions and measured with field potential recordings. A: slices were tetanized (200 Hz) in the presence of AP5 (100 µM) or AP5 + dizocilpine (MK-801) (100 µM and 20 µM, respectively). Tetanization led to a sustained increase in EPSP slope (mean increase = 34 ± 6% at 40-45 min posttetanization). In approximately half of the slices examined, a second, nontetanized pathway was tested. In agreement with previous findings (Grover 1998; Grover and Teyler 1992), tetanization of one pathway had no effect on EPSPs evoked in a nontetanized control pathway (-2 ± 3% change in EPSP slope at 40-45 min posttetanization). B1: paired-pulse facilitation (PPF) was tested in approximately half of the tetanized pathways. As previously reported (Grover 1998), tetanization resulted in an immediate decrease in PPF. Over the 45 min posttetanus period, PPF returned toward the initial pretetanus level. B2: PPF was measured in all slices where a second control pathway was tested. There were no posttetanic changes in PPF in the control pathways. C: sample EPSPs from one of the slices studied in the current experiment. C1: EPSPs evoked by paired stimulation of Schaffer collaterals before and 30 min after tetanic stimulation. At 30 min posttetanus, the first and second EPSPs of the pair were increased but the first EPSP was increased more, resulting in a decrease in PPF ratio (from 1.85 to 1.71). C2: in the control pathway of the same slice, there was little change in either the first or second EPSP (pretetanus PPF ratio, 1.70; posttetanus PPF ratio, 1.74). Error bars in A and B show ± SE; at most time points the error bars are smaller than the symbols used for plotting.

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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Fig. 4. The group I metabotropic glutamate receptor (mGluR) antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCEOt) did not prevent NMDA receptor-independent LTP. A: slices were tetanized in the presence of 250 µM CPCCOET + 100 µM AP5. At 40-45 min posttetanization, tetanized pathway EPSPs were potentiated by 30 ± 7% whereas control pathway EPSPs were unaltered (-2 ± 11% change). B1: posttetanic decrease in PPF in the tetanized pathway was not prevented by CPCCOEt. B2: PPF in the control pathway was constant throughout the experiment.



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Fig. 5. Group I mGluR antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) did not prevent NMDA receptor-independent LTP. A: slices were tetanized in the presence of 250 µM AIDA + 100 µM AP5. At 40-45 min posttetanization, tetanized pathway EPSPs were potentiated by 36 ± 11% whereas control pathway EPSPs were unaltered (0 ± 14% change). AIDA application was associated with a reversible decrease in EPSP slope in both tetanized and control pathways. The EPSP depression was not caused by AIDA itself but was caused by the NaOH vehicle (0.25 mM final concentration) used to dissolve AIDA (see Fig. 6). B1: slices tetanized in AP5 + AIDA showed an immediate posttetanic decrease in PPF in the tetanized pathway. However, AIDA application was associated with an increase in PPF and the time course of the posttetanic PPF change overlapped with the recovery from this increase in PPF, making it difficult to determine the exact time course of the tetanization-induced change in PPF. B2: PPF in the control pathway was not affected by tetanization but was increased by AIDA application. The increase in PPF ratio during AIDA application, like the decrease in EPSP slope, was attributable to the NaOH vehicle because an identical effect was seen with NaOH alone (Fig. 6). The increase in PPF during AIDA application suggests that the EPSP depression was caused by presynaptic inhibition of glutamate release.

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).



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Fig. 6. Application of NaOH (1 mM, equal to the highest concentration applied to slices; see Fig. 8) reproduced the depressant effect of AIDA but did not prevent NMDA receptor-independent LTP. Data shown here are from one slice; similar results were obtained in four additional slices. A: EPSP slopes were depressed during application of NaOH but NMDA receptor-independent LTP was still induced. B: PPF was increased by NaOH application, suggesting that the depression of the EPSP slope was caused by a reduction in the probability of glutamate release. Comparison between the PPF ratios for tetanized and control pathways indicated that NaOH did not alter the typical posttetanic change in PPF.

Group II mGluR antagonists

We examine two group II-selective mGluR antagonists: (2S)-alpha -ethylglutamic acid (EGLU) (Jane et al. 1996) and (RS)-alpha -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.



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Fig. 7. Group II mGluR antagonist (2S)-alpha -ethylglutamic acid (EGLU) blocked NMDA receptor-independent LTP without affecting the posttetanic decrease in PPF. A: NMDA receptor-independent LTP was prevented by application of 250 µM EGLU. Tetanized pathway EPSP slopes were unchanged (-7 ± 13%; 40-45 min posttetanization), as were the control pathway EPSP slopes (-17 ± 10%; 40-45 min posttetanization). B1: EGLU failed to affect the posttetanic decrease in PPF in the tetanized pathway (compare with changes shown in Fig. 3B1). B2: there were no changes in PPF ratio in the control pathway. NaOH was used a vehicle for EGLU; the final NaOH concentration applied to slices in A and B was 0.25 mM.

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.



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Fig. 8. Group II mGluR antagonist (RS)-alpha -methylserine-O-phosphate monophenyl ester (MSOPPE) did not inhibit NMDA receptor-independent LTP. A: NMDA receptor-independent LTP was not affected by application of 250 µM MSOPPE. Tetanized pathway EPSP slopes were potentiated (54 ± 22% at 40-45 min posttetanizaton) with no change in control pathway EPSP slopes (5 ± 8%). Application of MSOPPE caused a reversible depression of the EPSP slope in both tetanized and control pathways similar to that seen with AIDA (Fig. 5). This depression, most likely presynaptic in origin because it was accompanied by an increased PPF ratio (B1 and B2), can be attributed to the NaOH vehicle used to dissolve MSOPPE. B1: PPF ratio was increased during application of MSOPPE. Although there was clearly an immediate posttetanic decrease in PPF in the tetanized pathway, the time course of this change was difficult to estimate because of the overlapping increase in PPF caused by NaOH. B2: PPF was increased during the time when MSOPPE was applied to the slice, but there was no tetanization-related change in PPF in the control pathway. The final NaOH concentration applied to slices in A and B was 1 mM.

Group III mGluR antagonists

We examined two group III-selective mGluR antagonists: (RS)-alpha -cyclopropyl-4-phosphonophenylglycine (CPPG) (Jane et al. 1996; Toms et al. 1996) and (RS)-alpha -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.



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Fig. 9. Group III mGluR antagonist (RS)-alpha -cyclopropyl-4-phosphonophenylglycine (CPPG) blocked NMDA receptor-independent LTP without affecting the posttetanic decrease in PPF. A: NMDA receptor-independent LTP was prevented by application of 10 µM CPPG. Tetanized pathway EPSP slopes were unchanged (1 ± 10%; 40-45 min posttetanization), as were the control pathway EPSP slopes (-3 ± 13%; 40-45 min posttetanization). B1: CPPG failed to affect the posttetanic decrease in PPF in the tetanized pathway (compare with changes shown in Fig. 3B1). B2: there were no changes in PPF ratio in the control pathway. NaOH was used a vehicle for CPPG; the final NaOH concentration applied to slices in A and B was 0.1 mM.



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Fig. 10. Group III mGluR antagonist (RS)-alpha -methylserine-O-phosphate (MSOP) did not block NMDA receptor-independent LTP. A: NMDA receptor-independent LTP occurred in slices tetanized in AP5 + MSOP (250 µM). At 40-45 min posttetanus, tetanized pathway EPSP slopes were increased by 18 ± 9%. Control pathway EPSPs were also increased in slices tetanized in AP5 + 250 µM MSOP (16 ± 15%; 40-45 min posttetanization). As with other compounds applied to slices using an NaOH vehicle, slices treated with 250 µM MSOP showed a reversible decrease in EPSP slope. B: during MSOP application, there was an increase in PPF ratio for both the tetanized (B1) and control (B2) pathways, suggesting a decrease in the probability of glutamate release from presynaptic terminals. B1: immediate posttetanic decrease in PPF in the tetanized pathway was not prevented by MSOP. B2: in the control pathways, there was an increase in PPF ratio during drug application but there was no apparent effect of tetanization on PPF in the control pathway. The final NaOH concentration applied to slices in A and B was 0.25 mM.

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).



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Fig. 11. Representative example illustrating the potentiation observed in control (nontetanized) pathways when slices were tetanized in the presence of CPPG or MSOP. A: nonspecific potentiation of both tetanized and control (47 and 31% increase in EPSP slope at 40-45 min posttetanus, respectively) pathways in a slice that was tetanized in the presence of AP5 + 250 µM MSOP. Although potentiation of the control pathway was more reliable when lower concentrations of MSOP were used, it was observed in half of the slices tetanized in 250 µM MSOP. Control pathway potentiation was observed in 5 of 8 slices tetanized in 25 µM MSOP, 5 of 7 slices tetanized in 100 µM MSOP, and 3 of 6 slices tetanized in 250 µM MSOP. B: PPF in the tetanized pathway was increased during MSOP application (as shown in Fig. 10). In addition, there was a posttetanic decrease in PPF ratio in the tetanized pathway of this slice. PPF was increased in the control pathway during MSOP application but there was no tetanus-induced change in PPF ratio. C and D: tests to determine if tetanized and control pathways were independent. C1: paired stimuli (50 ms interstimulus interval) applied to the tetanized pathway revealed substantial PPF both before tetanization (1) and 40-45 min after tetanization (2). C2: paired stimuli (50 ms interstimulus interval) applied to the control pathway revealed substantial PPF both before tetanization (1) and 40-45 min after tetanization (2). D: cross-facilitation between pathways was not observed. D1: a single stimulus was delivered to the tetanized pathway followed 50 ms later by a single stimulus to the control pathway. The dotted line superimposed on the second (control pathway) EPSP shows control pathway response no preceding stimulus was given to the tetanized pathway. The exact match between the dotted and solid lines shows that stimulation of the tetanized pathway failed to cross-facilitate the control pathway. D2: a single stimulus was delivered to the control pathway followed 50 ms later by a single stimulus to the tetanized pathway. The dotted line superimposed on the second (tetanized pathway) EPSP shows the tetanized pathway response when no preceding stimulus was given to the control pathway. The exact match between the dotted and solid lines shows that stimulation of the control pathway failed to cross-facilitate the tetanized pathway. D3: tetanized and control pathways were stimulated simultaneously, resulting in a composite EPSP (solid line) which closely matched the predicted EPSP (dotted line) obtained by algebraic addition of the EPSPs evoked by stimulating each pathway individually (dotted lines in D1 and D2). The composite EPSP slopes for both the measured (solid line) and predicted (dotted line) responses match exactly. There was a small difference in peak amplitudes between the measured and predicted responses, with the measured response amplitude being slightly smaller. This difference in EPSP amplitude may reflect the nonlinear summation of EPSP in hippocampal pyramidal neurons (Langmoen and Andersen 1983; Urban and Barrionuevo 1998). Data in A-D are from the same slice. Scale bars in C-D are 1 mV, 10 ms.



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Fig. 12. Summary of EPSP changes in tetanized (A) and control (B) pathways measured 40-45 min after 200 Hz tetanization in AP5/AP5 + MK-801 or AP5 + mGluR antagonist. Results from slices tetanized in AP5/AP5 + MK801 are shown by black bars. Results from slices tetanized in AP5 + one of the group I mGluR antagonists are shown by light gray bars. Results from slices tetanized in AP5 + one of the group II mGluR antagonists are shown by dark gray bars. Results from slices tetanized in AP5 + one of the group III mGluR antagonists are shown by open bars. AP5 concentration was 100 µM for all slices; MK-801 was applied at 20 µM. mGluR antagonist concentrations (in µM) are indicated by numerals below the bars. A: NMDA receptor-independent LTP of tetanized pathways was significantly inhibited by EGLU (100-250 µM) and CPPG (10 µM). B: significant, long-lasting, nonspecific potentiation of control pathway EPSPs was observed with an intermediate concentration of CPPG (2.5 µM) and with lower concentrations of MSOP (25 and 100 µM). Control pathway EPSPs tended to be decreased at 40-45 min posttetanus in EGLU, but this effect was significant for only the intermediate concentration (100 µM). Asterisks in A and B indicate significant difference (P < 0.05) relative to slices tetanized in AP5/AP5 + MK-801.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 GDPbeta S. In a control experiment, we found that the G-protein dependent, GABAB receptor-mediated IPSP was suppressed in GDPbeta S-loaded cells. In contrast, NMDA receptor-independent LTP was fully intact in GDPbeta 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 GDPbeta 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 GDPbeta S in this compartment to adequately inhibit the postsynaptic mGluRs. Our control experiment examining GDPbeta S inhibition of GABAB receptor-mediated IPSPs argues against this possibility.

There are two factors that limit the concentration of GDPbeta S in the synaptic region of the postsynaptic neuron. The first factor is diffusion of GDPbeta 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 GDPbeta 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 MOmega in the IPSP experiment and 18 ± 2 MOmega in the LTP experiment, P < 0.005), this difference would have caused better diffusion of GDPbeta S into cells in the LTP experiment, yet in this same experiment GDPbeta 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 GDPbeta 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 GDPbeta S 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 GDPbeta 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.



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Fig. 13. Hypothetical roles for group II and group III mGluRs in NMDA receptor-independent LTP. NMDA receptor-independent LTP is triggered by the combination of an increase in postsynaptic intracellular Ca2+ concentration (mediated by influx through voltage-gated Ca2+ channels and occurring across a large extent of the postsynaptic dendritic volume) in conjunction with an appropriate pattern of mGluR activation. In this model, group II mGluRs provide an essential "go" signal whereas group III mGluRs provide an inhibitory "stop" signal. Group III mGluRs are assumed to be located in the axon terminal membrane adjacent to the active zones where glutamate release occurs, and group II mGluRs are assumed to be located in preterminal axon regions (Shigemoto et al. 1997; Takumi et al. 1998). A: during tetanic stimulation, glutamate is released from the terminals of tetanized axons. Glutamate concentration is highest in the synaptic cleft but some glutamate is assumed to "spill out" of the synapse, with the concentration progressively decreasing at greater distances (indicated by the two concentric circles). At tetanized synapses, the go signal provided by group II mGluR activation is strong enough to override the stop signal from inhibitory group III mGluRs. B: at nearby nontetanized synapses, there may be some activation of group II mGluRs but the stop signal from inhibitory group III mGluRs ordinarily dominates. However, if group III mGluRs are blocked pharmacologically, then LTP induction at nearby, nontetanized synapses is able to proceed (as in Figs. 10-12). C: at distant, nontetanized synapses, the glutamate concentration due to "spillover" is too low to activate either group II or group III mGluRs, and the lack of an essential go signal from group II mGluRs causes LTP to fail at these synapses. The scheme depicted here can allow LTP to be restricted to specific synapses despite a diffuse postsynaptic Ca2+ signal and the extracellular spillover of glutamate to nearby nontetanized synapses.

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.


    ACKNOWLEDGMENTS

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-34650.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society