Journal of Physiology (1995), 485.1, pp. 121-134

Dual modulation of synaptic inhibition by distinctmetabotropic glutamate receptors in the rat hippocampus

Jean-Christophe Poncer *, Haruhiko Shinozaki t and Richard Miles

Laboratoire de Neurobiologie Cellulaire, Institut Pasteur, 25 rue du Dr Roux,75724 Paris cedex 15, France and t The Tokyo Metropolitan Institute of Medical Science,

3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113, Japan

1. The effects of metabotropic glutamate receptor (mGluR) activation on synaptic inhibitionwere examined using whole-cell recordings of spontaneous and miniature inhibitorysynaptic currents from CA3 pyramidal cells in rat hippocampal slices.

2. The mGluR agonist (1S, 3R)trans-i-aminocyclopentane-1,3-dicarboxylic acid (tACPD)increased spontaneous IPSC (spIPSC) frequency by up to 5-fold. At doses above 5/AM theincrease was transient (15-45 s) and was followed by a decline to control frequency. Inthese conditions, elevating external K+ from 2 to 8 mm could still increase spIPSCfrequency.

3. Miniature IPSCs (mIPSCs) were recorded in the presence of 1 UM TTX, 5 iM Mg2+ andnominally zero Ca2+. At concentrations above 50/uM, tACPD induced a sustained,reversible reduction in mIPSC frequency by up to 43 %.

4. Quisqualate, at doses as low as 50 nM, increased spIPSC frequency, but did not affectmIPSC frequency at concentrations up to 10 /M.

5. The specific mGluR2 and 3 agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine(DCG-IV, 3/#M) reduced mIPSC frequency by 40+4% but did not increase spIPSCfrequency.

6. The putative mGluR antagonist L-2-amino-3-phosphonopropionate (L-AP3, 1 mM) blockedthe effect of tACPD on mIPSC but not spIPSC frequency. The broad-spectrum antagonist(RS)-a-methyl-4-carboxyphenylglycine (MCPG, 500 /M) blocked both responses.

7. mGluR activation also had dual effects on IPSCs evoked by focal extracellular stimulation.Application of 5,uM tACPD increased the mean amplitude of evoked IPSCs by 112 + 9%,largely by reducing the proportion of response failures. In contrast, IPSC amplitude wasreduced to 44 + 1 % of control values by 3/M DCG-IV.

8. These results suggest hippocampal inhibitory cells express two distinct mGluR subtypes.One receptor (possibly mGluRi or 5) is located on somato-dendritic membrane andenhances cell excitability. Another (mGluR2 or 3) is present at inhibitory terminals andreduces the probability ofGABA release.

Many neurotransmitters act on receptors coupled to Activation by the same transmitter of differentG-proteins (Hille, 1992). Depending on their location, the G-protein-coupled receptors at these two sites on a singleactivation of these receptors may have two different cell may result in functionally additive or opposingactions on a postsynaptic neurone. Somato-dendritic effects. For instance, activation of GABAB receptors bothreceptors can modulate cellular excitability by changing hyperpolarizes hippocampal pyramidal neurones andthe properties of their ionic conductances (see Nicoll, reduces excitatory transmission presynaptically (Dutar &1988). Receptors located on presynaptic terminals control Nicoll, 1988a). In contrast, muscarinic receptor activationsynaptic efficacy by actions on transmitter release excites pyramidal neurones but reduces transmittermechanisms (see Thompson, Capogna & Scanziani, 1993). release from their terminals (Dutar & Nicoll, 1988 b).

* To whom correspondence should be addressed at Institut fur Hirnforschung der Universitat Zurich, August Forel-Strasse 1,8029 Zurich, Switzerland.

3620 121

J -C. Poncer, H. Shinozaki and R. Miles

Metabotropic glutamate receptors (mGluRs) form a familyof at least seven receptors coupled, via G-proteins, tovarious intracellular targets (see Nakanishi, 1992;Saugstad, Kinzie, Mulvihill, Segerson & Westbrook, 1994).These receptors may act postsynaptically by modulatingionic conductances or other receptors (see Schoepp & Conn,1993; Glaum & Miller, 1993). Activation of presynapticmGluRs reduces transmitter release at both excitatory(Forsythe & Clements, 1990; Baskys & Malenka, 1991;Sladeczek, Momiyama & Takahashi, 1993) and inhibitorysynapses (Hayashi et al. 1993).

We previously reported that synaptically releasedglutamate activates mGluRs on hippocampal inhibitorycells and leads to a short term excitation of these cells(Miles & Poncer, 1993). The mGluR agonist tACPD is alsoreported to suppress synaptic inhibition by an unknownmechanism (Desai & Conn, 1991). In the present study, weexamined the possibility that mGluRs exert a dualinfluence on hippocampal inhibitory cells. We show thatactivation of mGluRs both excites inhibitory cells andreduces GABA release from their terminals. Pharmac-ological and kinetic differences between these two effectssuggest that two distinct receptors are involved.

METHODSNeonatal Sprague-Dawley rats (5-15 days old) were decapitated.One hippocampus was quickly removed and immersed in ice-coldoxygenated solution. Transverse hippocampal slices 150 ,mthick were prepared using a vibro-slicer (Campden). Slices wereheld in a submerged chamber at 31 °C and bathed with aphysiological saline containing (mM): NaCl, 125; KCl, 2;NaHCO3, 26; glucose, 10; CaCl2, 2; MgCl2, 3; gassed with a 5%C02-95% 02 mixture (320 mosmol, pH 7 3).Recordings were made at room temperature (20-25°C) in achamber of volume 0-5 ml continuously perfused at a rate of2-3 ml min'. Pyramidal cells were identified visually underNomarski optics using an upright microscope (Optiphot-2, Nikon).Electrodes were made from thick-wall (0-32 mm) borosilicateglass of external diameter 1-5 mm (Clark ElectromedicalInstruments). They had a resistance of 1-5-4 MQ2 when filledwith (mM): CsCl, 125; Hepes, 10; EGTA, 10; MgCl2, 2; Na2ATP,3 (290 mosmol, pH adjusted to 7-3 with KOH).

Tight-seal (> 2 GQ) whole-cell recordings (Edwards, Konnerth,Sakmann & Takahashi, 1989) were obtained from pyramidal cellbodies in the CA3 area. Strong positive pressure was appliedduring the approach to a pyramidal cell. The pressure was gentlyreleased when a deformation of the cell membrane was evident.The formation of a membrane seal and transition to whole-cellrecording were performed as commonly described. Synapticcurrents were recorded with either an EPC-7 (List) or anAxopatch 200A amplifier (Axon Instruments), filtered at 3 kHzand stored on a DAT recorder (Biologic) for further analysis.Pyramidal cells were clamped at a holding potential of -70 mV.Series resistance typically ranged from 3 to 8 MQ and was notcompensated. Since the amplitude of most synaptic currents waslower than 500 pA, the DC series resistance error was not greaterthan 4 mV. Recordings were rejected when series resistance

varied by more than 20%. The mean input resistance of eighty-three pyramidal cells was 410 + 36 M.Q and their meancapacitance was 32-0 + 1 9 pF.In all recordings EPSCs were blocked using the excitatory aminoacid receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX, 40 FM) and D,L-2-amino-5-phosphonovaleric acid(D,L-APV, 100 uM, Tocris Neuramin). All spontaneous synapticcurrents occurring in these conditions were suppressed bypicrotoxin (20 uM, Sigma). Quisqualic acid (Quis, Sigma), L(+)-2-amino-4-phosphonobutyric acid (L-AP4, Tocris), (2S,1'R,2'R,3'R)-2(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and (1S,3R) trans-1-aminocyclopentane-1,3-dicarboxylic acid (tACPD, Tocris) wereused to activate metabotropic glutamate receptors. These drugs,as well as the antagonists L-2-amino-3-phosphonopropionate (L-AP3, Tocris) and (RS)-a-methyl-4-carboxyphenylglycine (MCPG,Tocris) were applied by bath perfusion.In some experiments we examined the effects of mGluR agonistson the frequency of miniature IPSCs. Miniature IPSCs wererecorded in the presence of 1-2 AM tetrodotoxin (TTX, Sigma),5 mM Mg2+ and nominally zero Ca2+. The voltage-dependent Na+current elicited by a 50 mV voltage jump in the recorded cell wastypically suppressed within 20-60 s of switching to this solution.However, IPSC frequency continued to decline for another3-5 min before reaching a steady state. Therefore measurementswere begun 5-10 min after the Na+ current was completelysuppressed. In four recordings of more than 40 min duration thefrequency and mean amplitude of miniature IPSCs remainedstable within 5% of their initial values.

Current records of 2-3 min duration were acquired onto aMacintosh computer at 5 kHz using a 12-bit A/D converter(National Instruments). IPSCs were detected using a version of apreviously described program operating under the LabViewenvironment (Miles & Poncer, 1993; Ankri, Legendre, Faber &Korn, 1994). Briefly, the current signal was smoothed anddifferentiated. An event was identified when the treated signalexceeded a threshold for a specified duration. The rising phase ofeach event was determined from the original record. Detectionparameters were optimized on twenty randomly chosen sectionsof record. The time from the start of the record, amplitude, timeto peak and single-exponential decay time constant were thenautomatically measured for each detected event. Amplitudedistributions before and after agonist application were comparedusing Kolmogorov-Smirnov statistics on cumulative plots ofIPSC amplitudes (Van der Kloot, 1989). IPSC decay was fitted bya mono-exponential function using the KaleidaGraph software(Synergy Software, Reading, PA, USA).

Inhibitory synaptic currents were evoked by focal stimulationdelivered through a second patch pipette filled with standardphysiological solution. The stimulating pipette was placed within40-100 jum of the recorded pyramidal cell either in stratumpyramidale or stratum lucidum. Stimuli of intensity 4-10 1uAand duration 75-100 #ts were delivered at 0-67 Hz from anisolated pulse stimulator (A-M Systems, Everett, USA). 'Minimalstimulation' was achieved by setting stimulation intensity justabove threshold so that frequent failures were observed. In suchconditions, stimulation no longer evoked synaptic currents whenthe pipette was withdrawn by 5-10 #sm from its originalposition. Evoked responses were digitized at 6 kHz using a TL-1DMA interface (Axon Instruments) and pCLAMP software.Amplitude measurements were made semi-automatically usingCLAMPDAT.

122 J. Phy-sol. 485. 1

Modulation of synaptic

RESULTStACPD transiently increases spontaneous IPSCfrequencySpontaneous IPSCs were recorded from forty-five CA3pyramidal cells. Their mean frequency was 1-7 + 0-2 Hzin control conditions. When recorded at -70 mV with125 mm CsCl internal solution, their mean amplitude was

60-7 + 1.0 pA (mean + S.E.M.).

tACPD reversibly increased IPSC frequency withoutsignificantly affecting the holding current of the recordedpyramidal cell (+2-5 0 9 pA, n = 12 cells). Atconcentrations above 5 /uM, this effect was transient(15-45 s, Fig. 1A). With our perfusion system, theincrease occurred with a delay of 65 + 8 s after switchingto tACPD-containing solution and was followed by a

rapid decline to control frequency. This transient response

could reflect either a block by depolarization of inhibitoryneurones or a desensitization of the receptors. However,

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inhibition by mGluRs 123

in such conditions, an elevation of external K+concentration from 2 to 8 mm increased IPSC frequencymore than 4-fold, suggesting some inhibitory neuronescould still be recruited by depolarization. Thisdepolarizing effect occurred with a delay of 62 + 7 s afterswitching to 8 mm KCl solution (n = 4). We observed thatthe proportion of large and fast decaying IPSCs markedlyincreased both during the early phase of tACPDapplication and when external K+ was raised (Fig. lBand C). This suggests some inhibitory neurones that were

made to fire by tACPD might still be driven to dischargeduring prolonged application of the agonist.

We examined responses at different doses of tACPD(Fig. 2). When applied at low doses (1-5 /sM), tACPDincreased IPSC frequency by up to 220% (n = 9 cells) andthis response was maintained over 2-5 min of application.This effect was transient at concentrations above 5 /Mand was maximal at 50 ,UM. Figure 2C shows both the

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Figure 1. tACPD transiently increases the frequency of spontaneous IPSCsA, recording from a CA3 pyramidal cell in the presence of 100 /M D,L-APV and 40/M CNQX. Perfusionof tACPD (50 FM) caused a transient (15-45 s) increase in spIPSC frequency of 491 %. At steady state,elevation of external K+ concentration from 2 to 8 mm induced a sustained increase in spIPSCfrequency. B, cumulative IPSC amplitude histograms for each of the 4 sections shown on the currenttrace above. C, plot relating IPSC decay time constant to amplitude for sections a (-) and b (C1). tACPDincreased mostly the proportion of large and fast IPSCs (n = 5 cells).

J. Physiol. 485.1

124

maximal and steady-state responses to tACPDapplication. The EC50 value derived from the maximalresponse was 5-4 /CM.

These results show that mGluR activation leads to atransient and reversible excitation of some hippocampalinhibitory neurones.

tACPD, at higher doses, decreases miniature IPSCfrequencyActivation of mGluRs by tACPD depresses excitatorysynaptic transmission by a presynaptic mechanism (Baskys& Malenka, 1991; Sladeczek et al. 1993). These effectsoccur at tACPD concentrations much higher (100-200 ,uM)than those we used to increase spontaneous IPSC

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J. Physiol. 485.1

frequency. We examined whether mGluR activation alsoaltered GABA release using miniature IPSC recordings.Miniature IPSCs were recorded from forty-four pyramidalcells in a solution containing 1-2 /SmM 1TX, 5 mM MgCl2and nominally zero Ca2+. These events were presumed toresult from the spontaneous exocytosis of single GABAquanta from presynaptic terminals. Their frequency wasnot significantly affected by addition of the calciumchannel blocker Cd2+ (CdCl2, 50-100 ,UM) suggesting theseevents did not depend on Ca2" entry (-5-7 + 2-6 %, n = 3cells).

When recorded at a potential of -70 mV with 125 mMCsCl internal solution, miniature IPSCs had a mean

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Figure 2. Dose dependence of tACPD effect on spontaneous IPSC frequencyspIPSCs were recorded from CA3 pyramidal cells in the presence of 100 /M APV and 40 /M CNQX.A, IPSC frequency measured during 15 s intervals. Application of 5/SM tACPD increased IPSCfrequency by more than 4-fold. An application of 50/M tACPD 13 min later led to a transientincrease of duration 10-15 s followed by a decline to control frequency. B, sections of traces from thisexperiment. Second and fourth traces were taken at steady state, within the final 10 s of tACPDapplication. C, relation between tACPD concentration and increase in IPSC frequency. 0, frequencyat steady state (the final 75 s of a 150 s application). 0, frequency during 30 s around maximalresponse. Experimental points were fitted with a sigmoidal curve of the form y= 1 + Emaxi[1 + (EC50/x)'] where EC50 = 5 40 uM and Emax is the maximum increase in spIPSC frequency. Eachpoint represents the mean + S.E.M. from 3 to 8 experiments.

f-C. Poncer, H. Shinozaki and R. Miles

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Modulation of synaptic

amplitude of 32-4 + 1P9 pA and occurred at a frequency of1'1 + 0'1 Hz. Their decay could be well fitted by a singleexponential function with a time constant between 10and 35 ms.

At concentrations up to 25 uM, tACPD did not affectminiature IPSC frequency (n = 7 cells). However, athigher concentrations, tACPD decreased the frequency ofminiature IPSCs in a dose-dependent and reversiblemanner (Fig. 3, n = 17 of 25 cells). We tested the effects ofdifferent doses of tACPD. The relation between tACPDconcentration and the reduction in frequency was wellfitted by a sigmoidal curve (Fig. 3C). The maximalreduction was 43% and the EC50 was 71-4 /M (n =6cells). This effect occurred with a delay of 168 + 11 s after

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inhibition by mGluRs 125

switching to tACPD-containing solution. This delay wassignificantly higher than that for the tACPD effect onspontaneous IPSC frequency. In addition, the reduction infrequency was maintained during 4 min of tACPDapplication, even at the highest doses (500 uM, Fig. 3A).

tACDP has been reported to depress synaptic inhibitionby a reduction in efficacy of postsynaptic GABA receptors(Liu, Disterhoft & Slater, 1993; Glaum & Miller, 1993). Adecrease in IPSC amplitude might appear to reduce thefrequency of detected miniature IPSCs. In order to testfor a decreased postsynaptic sensitivity to GABA, we

examined the distributions of miniature IPSC amplitudesbefore and during application of tACPD (Fig. 4). Nosignificant difference in these distributions could be

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Figure 3. At higher doses, tACPD decreases miniature IPSC frequencymIPSCs were recorded in the presence of 100 ,UM APV, 40 uSM CNQX, 1 /M TTX, 5 mm MgCl2 andnominally zero calcium. A, mIPSC frequency averaged during 1 min intervals. Exposure to 25 /SMtACPD did not change mIPSC frequency. On perfusion with 500 /SM tACPD, mIPSC frequency was

reduced by 45 2% and recovered within 150 s of return to control solution. B, sections of traces fromthis experiment. C, plot relating tACPD concentration to reduction in mIPSC frequency. Data pointswere fitted by a sigmoidal curve of the form y= 1 - Emax/[1 + (EC50/x)'], where EC50 = 71'4 4UM,

and Emax is the maximum decrease in mIPSC frequency.

J. Physiol. 485.1

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detected. The mode of distribution histograms as well asthe mean amplitude and decay time constant of miniatureIPSCs were also unchanged (Fig. 4C, n = 5 cells). Theseobservations suggest that tACPD, at high concentrations,reduces synaptic inhibition by a presynaptic mechanismrather than through an action on GABA receptors.

Quisqualate and DCG-IV discriminate betweenmGluR actions on spontaneous and miniature IPSCfrequencySeveral molecular species of mGluRs exist which are

linked to various intracellular pathways and possess

distinct pharmacological profiles (see Nakanishi, 1992;Schoepp & Conn, 1993). The differences in both the delayand the effective concentrations of tACPD for the actionson spontaneous and miniature IPSCs suggested theseeffects could be mediated via distinct mGluR subtypes.

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J. Physiol. 485.1

We examined this hypothesis by comparing the actions ofother mGluR agonists on both spontaneous and miniatureIPSCs.

Quisqualate has a higher efficacy than tACPD at some(mGluR1 and 5) but not other (mGluR2, 3 and 4) mGluRsubtypes (Schoepp & Conn, 1993). We found quisqualate,like tACPD, increased spontaneous IPSC frequency in a

dose-dependent and reversible manner (Fig. 5). Atconcentrations as low as 50 nM, IPSC frequency wasincreased by 55 + 7% (n = 8 cells, Fig. 5). At higherconcentrations (up to 0-5 fSM), in contrast to tACPD,quisqualate still increased IPSC frequency by up to 150%(n = 6 cells). However, at these high doses, quisqualateelicited a maintained inward current in the recordedpyramidal cell (Fig. 5C). This current was partly reducedby elevating CNQX concentration from 40 to 100,uM,suggesting it may have been due to displacement of

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Figure 4. tACPD does not affect properties of miniature IPSCsA, amplitude distributions histograms of mIPSCs recorded before and during a 6 min application of300,u4M tACPD. The mean frequency was 0-64 Hz in control and declined to 0'38 Hz on tACPDapplication but the shape and mode of the distribution were not changed. B, cumulative distributionsof IPSC amplitudes were not significantly different (Kolmogorov-Smirnov test). C, averages of 20mIPSCs collected before and during tACPD application. Their mean amplitude and decay timeconstant were not significantly changed (Student's t test).

J -C. Poncer, H. Shinozaki and R. Miles

CNQX by quisqualate at AMPA receptors (Honore, 1991).We also examined the effects of quisqualate on miniatureIPSCs. At concentrations up to 10 /,M, quisqualate inducedno detectable change in miniature IPSC frequency(Fig. 5C, n = 6 cells). Their mean amplitude andamplitude distribution also remained unchanged (datanot shown).

The compound (2S,1 'R,2'R,3'R )-2-(2,3-dicarboxycyclo-propyl) glycine (DCG-IV) is suggested to be a specificagonist of mGluR2 and 3 subtypes (Ohfune, Shimamoto,Ishida & Shinozaki, 1993; Hayashi et al. 1994). Weexamined its effects on both spontaneous and miniatureIPSCs (Fig. 6). At a concentration of 3/CM, DCG-IVdecreased miniature IPSC frequency by 40-2 + 4-2%(n = 4 of 5 cells tested). The latency of this response wascomparable to that observed with tACPD (185 + 15 s).Like tACPD, DCG-IV did not affect the amplitudedistribution of miniature IPSCs (not shown). Applicationof 3 /LM DCG-IV for 4 min caused neither a transient nor asustained increase in spontaneous IPSC frequency

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(Fig. 6C). Instead, it caused a delayed decrease in both thefrequency (-44-5 + 1-9%) and amplitude (-13-8 + 1-5%)of spontaneous IPSCs (n = 5 cells), consistent with apresynaptic site of action of this agonist.

Finally, the phosphonic glutamate derivative L-2-amino-4-phosphonobutyrate (L-AP4) has a high affinity formGluR4, 6 and 7 subtypes (Tanabe, Nomura, Masu,Shigemoto, Mizuno & Nakanishi, 1993; Nakajima et al.1993; Saugstad et al. 1994). No effect of L-AP4(100-300 /LM) was apparent on either spontaneous orminiature IPSC frequency (n=4 cells for each). Theseresults show that different mGluR agonists have differenteffects on spontaneous and miniature IPSCs, consistentwith the involvement of different receptor subtypes.

L-AP3 suppresses only presynaptic actions oftACPDWe also examined the effects ofmGluR antagonists on themodulation of synaptic inhibition. L-2-amino-3-phosphonopropionate (L-AP3) has been suggested topossess partial antagonist properties at some but not all

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Figure 5. Quisqualate increases spontaneous IPSC frequency without affecting miniatureIPSC frequency

A, application of 50 nM quisqualate (Quis) reversibly increased the frequency of spIPSCs recorded in thepresence of 100 /M APV and 40 /M CNQX. B, relation between Quis concentration and increase in IPSCfrequency measured during the last 75 s of a 150 s application (means S.E.M. from 4-8 experiments).C, mIPSCs recorded in the presence of 100 uM of both APV and CNQX. Quis (10 #M) elicited a reversibleinward current but did not significantly change mIPSC frequency (+2-9 2-0 %, n = 6 cells).

J. Physiol. 485.1 Modulation of synaptic inhibition by mGluRs

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mGluRs (see Schoepp & Conn, 1993). The reduction ofminiature IPSC frequency induced by 300 uM tACPD wastotally abolished in the presence of 1 mM L-AP3 (Fig. 7B,n = 3 cells). In contrast, L-AP3 did not reduce the effect oftACPD on spontaneous IPSC frequency (Fig. 7A, n = 3cells). L-AP3 had no detectable effect on the amplitude ofeither spontaneous or miniature IPSCs.

The compound (RS)-ax-methyl-4-carboxyphenylglycine(MCPG) has recently been described as a broad-spectrummGluR antagonist (Eaton et al. 1993). We observed that500 uM MCPG totally suppressed the effect of quisqualate(50 nM) on spontaneous IPSC frequency withoutsignificantly affecting their mean amplitude (Fig. 8A,n = 3 cells). This antagonist also abolished the reductionof miniature IPSC frequency induced by 500 uM tACPD(Fig. 8B, n = 3 cells). Again, MCPG did not significantlychange miniature IPSC mean amplitude. These resultsfurther confirm that these two effects of mGluR

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J. Physiol. 485.1

activation on synaptic inhibition present distinctpharmacological profiles.Dual modulation of evoked inhibitory synaptictransmissionOur observations on changes in the frequency ofspontaneous and miniature IPSCs suggest mGluRactivation may either enhance or depress synapticinhibition. In further experiments, we asked whetherthese effects were evident as changes in amplitude ofevoked IPSCs.

Monosynaptic IPSCs were evoked by focal extracellularstimulation delivered through a second patch pipetteplaced close to the recorded cell (see Methods). We firstattempted to examine the effects due to an increase ininhibitory cell excitability. Stimulation intensity wasthus set just above threshold in order to elicit some IPSCswith frequent failures of response. Evoked IPSCs had alatency of 3.3 + 0-1 ms and an amplitude between 20 and

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Figure 6. DCG-IV mimics the effects oftACPD on miniature but not spontaneous IPSCsA, mIPSC frequency was reversibly reduced on application of 3 uM DCG-IV. B, the means + S.E.M. ofdata from 4 pyramidal cells. The mean reduction in frequency was 40 2 + 4 2%. There was no

detectable change in mIPSC amplitude. El, control conditions; *, +3/uM DCG-IV. * Statisticallysignificant difference according to Student's t test (P < 005). C, DCG-IV (3/uM) applied for 4 min didnot increase but rather decreased spIPSC frequency and mean amplitude by 44 0 and 6-1 %,respectively.

J -C. Poncer, H. Shinozaki and R. Miles

08 9 10

Modulation of synaptic

300 pA. The mean amplitude of the response wasreversibly increased by 112 + 9% (n = 4 cells) onapplication of 5 jM tACPD (Fig. 9A). We examined thiseffect further by constructing amplitude distributionhistograms of the responses (Fig. 9B). The apparentincrease in the amplitude of the evoked responses mostlyreflected a reduction in the occurrence of failures. Onaverage, tACPD application decreased the proportion offailures by 60-4 + 0 4% (n = 4 cells). This shows that theincrease in excitability of inhibitory cells by tACPDresults in an enhanced ability for a fixed stimulus totrigger an IPSC.

We wished also to test the effects of activatingpresynaptic mGluRs on evoked IPSCs, independent ofchanges in inhibitory cell excitability including a possibledepolarization block. Figure 6 shows that DCG-IVreduced the frequency of miniature IPSCs but did not

inhibition by mGluRs 129

excite inhibitory cells. Therefore we compared IPSCsevoked in control conditions and after 3 min of DCG-IVapplication (Fig. 10). DCG-IV (3 ,M) reversibly suppressedevoked IPSCs to 55X7 + 1.1 % of their control amplitude(Fig. 10A, n = 8 cells). Amplitude distribution histogramsrevealed both an increase in the proportion of failures anda shift in IPSC amplitudes towards lower values (Fig.lOB).

DISCUSSIONThese results suggest that activation of mGluRs has twoeffects on hippocampal inhibitory cells. One is anenhancement of cellular excitability while the other is areduction of GABA release from inhibitory terminals(Fig. 11). Several observations suggest that these twoactions involve distinct mGluR subtypes. They differedboth in their effective tACPD concentration and in the

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Figure 7. L-AP3 only antagonizes the tACPD effect on miniature IPSC frequencyA, left panel, mIPSC frequency was reduced to 43% of control by 300 /M tACPD. The same applicationrepeated in the presence of 1 mM L-AP3 was ineffective. Each point represents IPSC frequencyaveraged during 60 s intervals. Right panel, mIPSC frequency reduction by 300 /SM tACPD and mIPSCmean amplitude before (L) and during (U) L-AP3 application (n = 3 cells). B, the effect of 5 #M tACPDon spIPSC frequency was not significantly affected by L-AP3 (n = 3 cells). Left, each point representsIPSC frequency averaged over 30 s intervals. * Statistically significant difference (Student's t test,P< 0 05).

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delav- before the effect w\-as apparent. Inhibitory cellexcitation was trainsient, while the reduction of (kABArelease was sustainied. The actions of specific agonists andaIntaCo'Inists supported furthler the dlistinction l)etweenm(luR sutAypes inivolved in both effects. Activation ofmCluRs may- either enlhance or redluce the efficacy ofinhilbitory svnalptic transmvission.

Distinct mGluRs influence hippocampal inhibitoryneuronesPIharmnacolocdical (lifferences l)et ween mGlluR subtypespermilit a tentative attrib)ution of the somiato-dendriticexcitation andcl reduction of GABA release to differentreceptors. \V=e found L-AP4, wA-hichl exclusively activatesmnGluR 4, 6 anid( 7 (Nakanishi, 1992; Sauostad ci al. 1994),had no effect oni eitlher spontaneous oir mniniature IPSCfrequency. Quisqcualate, which is described as a highlypotent agonist at mG-luR1 ancd 5 only (Nakanishi, 1992;Schoepp & Conn, 1993), increasecl spontaneous IPSCfrequency at nainomiiolar concentrations. In contrast, weolb,served that the specific mnGluR29 ancd 3 agoniist DCG-IV

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(Hayashi et al. 1993) reduced miniature IPSC frequency-without increasing inhibitory cell excitability. Thissuggests that clifferent mGluR subty-pes are located onsomato-dendritic memnbrane and on terminals ofinhibitory cells.

The tw-o effects also differed in their latency. tACPDincreasecd spontaneous IPSC frequency witlh a delay of afew seconds w-hile several tens of seconds were neededbefore miiniature IPSC frequency was reducedl. Thisdifference in latency suggests the effects may be mediatedvia distinct metabolic pathways. im(GluRl and 5 activatephosplholipa,se C wN-hiile mGluR2 and 3 are negativelycoupled to adenylate cyclase (Tanabe et al. 1992).Consistent -with our observations, activation of Cea2+-dependent K+ channels by acetylcholine, which involvesphosplhoinositide production, occur's writh a dlelay of 1-3 s(Horn & AMarty, 1988). In contrast, changes in transmitteirrelease involvins cAMIP formation occur w-ith a delay ofseveral tens of seconds (Llano & Gerschenfeld, 1993;Chavez-Noriegca & Stevens, 1994).

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Figure 8. The antagonist MCPG blocks the effects of mGluR activation on both spontaneousand miniature IPSCsA, left panel, eachi point represents spI1SC fiejLuency averagedl over 30 s intervals. Righlt panel, Quis-iluclele(l inicrease in sl)IPS(' fre(jtuenicy andI 1PS(' imieani amiplittulde b)eftoe (D) ancl Cdlur1inlg (U) AMPTapl))licati)n (i = 3 cells). B, efte(t of 500.(iO tAt'PD oni nIPS( frecquency before ancl after MCPG'application (i = .3 cells). * Stati.sticall- significant dlifference (Sttu(lenit's I test, IP< 0a).

J.-C. Poncer, H. Shinozaki auid R. illiles

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Finally, excitation of inhibitory cells by tACPD wastransient at high concentrations while the reduction inminiature IPSC frequency was sustained. This transientexcitation observed on prolonged application of theagonist may reflect a block by depolarization of someinhibitory neurones. Such an effect has been observed insome CA3 and CAI inhibitory cells upon tACPDapplication (Miles & Poncer, 1993; McBain, DiChiara &Kauer, 1994). However, we observed that spontaneousIPSC frequency could still be increased by raising theextracellular potassium concentration. Since these IPSCswere similar in amplitude and kinetics to those initiatedby tACPD, it is possible that some cells excited by tACPDmay still be recruited by depolarization. This raises thepossibility that a desensitization occurring at thereceptors or their intracellular effectors may contribute tothe transient nature of this response. Similar transientresponses occur during activation of some mGluRs (Staub,Vranesic & Kn6pfel, 1992; Herrero, Miras-Portugal &Sanchez-Prieto, 1994; McBain et al. 1994). However, dualrecordings from inhibitory cells and their postsynaptictargets will be needed to determine which inhibitory cellsinitiate large and fast IPSCs and to resolve contributions

from these two mechanisms to the transient excitation bytACPD.

Mechanism of the reduction in GABA releaseModulation of transmitter release may reflect changes inionic conductances at presynaptic terminals affecting thecalcium influx during an action potential (e.g. Klein,Camardo & Kandel, 1982; Mintz & Korn, 1989).Alternatively, modulation may occur at some stagesubsequent to calcium entry in the processes leading totransmitter release (Scholtz & Miller, 1992; Scanziani,Gahwiler & Thompson, 1993).

In our experiments, miniature IPSCs were recorded in thepresence of high Mg2+ and nominally zero Ca2+. Theirfrequency was not affected by the non-specific calciumchannel blocker CdCl2 but was reduced by both tACPDand DCG-IV. These results suggest that the activation ofpresynaptic mGluRs interferes with GABA releaseprocesses at a stage subsequent to calcium entry. Thesefindings contrast with the mGluR-mediated inhibition ofexcitatory transmission in visual cortex which appears toinvolve the blockade of a 4-AP-sensitive potassiumconductance (Sladeczek et al. 1993). However, the mGluR

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Figure 9. Effect of 5/SM tACPD on evoked IPSCsStimulation intensity was set just above threshold so that some IPSCs were triggered with some

response failures. A, averages of 170 responses collected before, during and after tACPD application.tACPD reversibly increased IPSC amplitude by 331 %. B, the distributions of the response amplitudesbefore and during tACPD application. The noise distribution is shown by the thick Gaussian curve

normalized to the height of the corresponding bin of the histogram. The increase in amplitude of theaveraged response was largely due to a reduction in the frequency of transmission failures.

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Figure 10. Suppression of evoked IPSCs by 3/EM DCG-IVStimulation intensity was just above threshold. A, averages of 170 responses collected before, duringand after DCG-IV application. Stimuli delivered in the presence of DCG-IV were started after 180 s ofapplication. DCG-IV reversibly reduced the mean amplitude of the response by 52 %. B, distribution ofresponse amplitudes. Noise distribution is shown as in Fig. 9. DCG-IV mostly enhanced the proportionof failures.

subtype involved may differ since the receptors presenton excitatory terminals were activated by L-AP4(Forsythe & Clements, 1990; Baskys & Malenka, 1991)and this compound had no effect in our experiments.

The precise mechanism of this Ca2+-independentinhibition of GABA release remains unknown. mGluR2 or

3, which may be responsible for this effect, are negativelycoupled to cAMP formation (Tanabe et al. 1992). Atcerebellar and hippocampal synapses, activation ofadenylate-cyclase enhances transmitter release (Llano &Gershenfeld, 1993; Chavez-Noriega & Stevens, 1994).Changes in protein kinase A activity subsequent to cAMPformation are thought to alter vesicle-associated proteinphosphorylation and so affect exocytosis probability (seeGreengard, Valtorta, Czernik & Benfenati, 1993).

mGluR1/5

mGluRs modulate the activity of a subpopulation ofinterneuronesmGluR activation has been suggested to excite a subset ofinhibitory cells (Miles & Poncer, 1993; McBain et al. 1994).This observation is supported by morphological studiesshowing that mGluRla is only expressed by a subset ofinhibitory cells which also contain somatostatin (Baude,Nusser, Roberts, Mulvihill, McIlhinney & Somogyi, 1993).Our present results suggest that, in the CA3 region,inhibitory neurones expressing somato-dendritic mGluRsgenerate IPSCs of large amplitude and fast kinetics. Inaddition, we have shown that activation of mGluRexpressed on CA3 inhibitory terminals depresses GABArelease. The present data do not resolve whether the sameor different inhibitory cell groups express both receptors.

Figure 11. Proposed scheme for the dual influence ofmGluR activation on synaptic inhibitionOne subtype (mGluRl or 5) is present on the somato-dendriticmembrane of interneurones and enhances their excitability.Another subtype (mGluR2 or 3) is present at inhibitoryterminals and reduces GABA release.

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J. Physiol. 485.1 Modulation of synaptic inhibition by mGluRs 133

Dual recordings from inhibitory neurones and theirpostsynaptic targets are needed to determine whether thesame subpopulation of neurones express two types ofmGluRs.

Finally, there is increasing evidence for a diversity ofhippocampal inhibitory cells (Gulyas, Miles, Hajos &Freund, 1993; Buhl, Halasy & Somogyi, 1994). Activationof muscarinic receptors, as well as mGluRs, enhancesinhibitory cell excitability and reduces GABA releasefrom their terminals (Pitler & Alger, 1992; Behrends &Ten Bruggencate, 1993). Thus, acetylcholine andglutamate have similar effects on hippocampal synapticinhibition. It remains to be clarified whether thesetransmitters act on the same or different subsets ofinhibitory cells.

Role of a dual modulation of synaptic inhibitionOur data suggest mGluRs are present at the somato-dendritic and terminal membrane of inhibitory neurones.These different positions imply they will be activated indifferent ways.

Somato-dendritic mGluRs will be activated bytransmitter released from excitatory synapses ontoinhibitory cells. The distance from the release site tomGluRla, localized by immunocytochemistry, is about50 nm (Baude et at. 1993). In contrast, presynapticmGluRs will be activated when an inhibitory terminaland an excitatory terminal make close contact with thesame pyramidal cell. This occurs, for instance, ondendritic spines (Gulyas et al. 1993) where the shortestdistance between the excitatory release site and thepresynaptic mGluRs will be about 500-1000 nm (seeHalasy & Somogyi, 1993).

Somato-dendritic mGluRs can be activated duringrepetitive stimulation of excitatory afferents to inhibitorycells (Miles & Poncer, 1993) thus enhancing recurrentinhibition. Physiological conditions under whichpresynaptic mGluRs will be activated are less clear.Sustained activity of neighbouring excitatory afferents isprobably required to allow glutamate diffusion to mGluRson inhibitory terminals. Such trans-synaptic interactions,even though limited by uptake mechanisms, occur at bothglycinergic and GABAergic synapses (Faber & Korn,1988; Isaacson, Solis & Nicoll, 1993). The effects of theseinteractions may be spatially limited to recently excitedspines. They would tend to reduce inhibitory control ofspine depolarization by subsequent EPSCs and so promotelong-term changes in excitatory transmission (see Koch &Zador, 1993). Our data suggest this effect would occurafter a delay of several tens of seconds (Fig. 3).

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AcknowledgementsWe wish to thank J. P. Pin for his generous help and N. Ankriand H. Korn for kindly sharing event detector software. Thiswork was supported by The Human Frontier Science Programand The Ministere de la Recherche et de l'Espace.

Received 18 July 1994; accepted 7 November 1994.