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Neuropharmacology 43 (2002) 215–221 www.elsevier.com/locate/neuropharm
Differential regulation of synaptic transmission by mGlu2 and mGlu3 at the perforant path inputs to the dentate gyrus and CA1 revealed in mGlu2 -/- mice James N.C. Kew ∗, Marie-Claire Pflimlin, John A. Kemp, Vincent Mutel F. Hoffmann-La Roche Ltd, Preclinical CNS Research, CH-4070 Basel, Switzerland Received 8 April 2002; received in revised form 31 May 2002; accepted 5 June 2002
Abstract Group II metabotropic glutamate (mGlu) receptors can act as presynaptic autoinhibitory receptors at perforant path inputs to the hippocampus under conditions of high frequency synaptic activation. We have used mGlu2 -/- mice to examine the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, DCG-IV, and by synaptically released glutamate. Field excitatory postsynaptic potentials evoked by stimulation of either the perforant path inputs to the dentate gyrus mid-moleculare or the CA1 stratum lacunosum moleculare were inhibited by DCGIV with IC50 values and maximum percentage inhibition of: 169 nM (60%) and 41 nM (72%) in wild-type mice and 273 nM (19%) and 116 nM (49%) in mGlu2 -/- mice, respectively. Activation of presynaptic group II mGlu autoreceptors by synaptically released glutamate, as revealed by a LY341495-mediated increase in the relative amplitude of a test fEPSP evoked after a conditioning burst, was observed in both the dentate gyrus and the stratum lacunosum of wild-type, but not mGlu2 -/- mice. These observations demonstrate that activation of mGlu3 receptors can regulate synaptic transmission at perforant path synapses but suggest that mGlu2 is the major presynaptic group II autoreceptor activated by synaptically released glutamate. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Metabotropic glutamate receptor; Autoreceptor; Synaptic transmission
1. Introduction Whilst fast excitatory glutamatergic transmission is mediated via the ionotropic AMPA and NMDA receptors, glutamate also activates a family of G-proteincoupled receptors, termed metabotropic glutamate (mGlu) receptors, which can modulate neuronal excitability and synaptic transmission (Anwyl, 1999). To date, eight mGlu receptors have been identified (mGlu1– 8) and these have been divided into three groups according to their sequence homology: group I receptors, mGlu1 and 5; group II receptors, mGlu2 and 3 and group III receptors, mGlu4, 6, 7 and 8 (Conn and Pin, 1997). Whereas group I mGlu receptors are primarily localised Corresponding author. Tel.: +44-1279-622150; fax: +44-1279875389. Present address: Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. E-mail address: [email protected] (J.N.C. Kew). ∗
postsynaptically, group II and III receptors are typically presynaptic and can regulate neurotransmitter release (Cartmell and Schoepp, 2000). Group II mGlu receptors are highly expressed in the terminal fields of the perforant path input from the enthorinal cortex in the mid-molecular layer of the dentate gyrus and the CA1 stratum lacunosum moleculare as revealed by both immunohistochemistry (Neki et al., 1996; Petralia et al., 1996; Shigemoto et al., 1997; Ohishi et al., 1998) and radioautography (Mutel et al., 1998; Schaffhauser et al., 1998) studies. Application of group II mGlu receptor selective agonists inhibits synaptic transmission in these pathways (Ugolini and Bordi, 1995; Macek et al., 1996; Kilbride et al., 1998; Kew et al., 2001) and activation of these receptors by synaptically released glutamate has been observed following high frequency burst stimulation (Kew et al., 2001). In situ hybridisation studies have indicated that mGlu2 is largely neuronal whilst mGlu3 is expressed in both neurones and glia (Ohishi et al., 1993a; b) and immunocyto-
0028-3908/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 8 4 - 9
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chemistry studies have confirmed the neuronal localisation of mGlu2 (Neki et al., 1996; Ohishi et al., 1998). A recent study using an mGlu3-selective antibody, that cross reacts weakly with mGlu2, in both wild-type and mGlu2 -/- mice has confirmed the mixed neuronal / glial distribution of the receptor (Tamaru et al., 2001). Moderate labelling was observed in the CA1 stratum lacunosum moleculare, with strong immunoreactivity evident throughout the dentate gyrus molecular layer, where ultrastructural analysis identified labelled glial processes, dendritic spines and axons / axon terminals (Tamaru et al., 2001). In this study, we have utilised mGlu2 -/- mice (Yokoi et al., 1996) to investigate the relative contributions of mGlu2 and mGlu3 to the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, (2S,2⬘R,3⬘R)-2-(2⬘3⬘dicarboxycyclopropyl)glycine (DCG-IV) (Brabet et al., 1998) and by synaptically released glutamate.
2. Methods Mice were anaesthetised in a 3.5% halothane/96.5% oxygen mixture and decapitated, as approved by the local institutional animal welfare committee. Hippocampal slices (400 µm) were cut from 4–5 month old mGlu2 -/- mice, or wild-type littermates derived from heterozygote matings, with a Sorvall tissue chopper. Slices were maintained in a submerged chamber and perfused at 35 °C with a simple salt solution containing (mM): NaCl, 124; KCl, 2.5; MgSO4, 2; CaCl2, 2.5; KH2PO4, 1.25; NaHCO3, 26; glucose, 10; sucrose, 4; gassed with 95% O2/5% CO2 (pH 7.4, 307 mOsm). The perforant path inputs to either the mid-molecular layer of the dentate gyrus or the CA1 stratum lacunosum moleculare were stimulated (0.033 Hz, 100 µs) at a strength adjusted to evoke field excitatory post synaptic potentials (fEPSPs) equal to 40 or 60% of the relative maximum amplitudes without superimposed population spike, respectively. For stimulation of the medial perforant path input to the dentate gyrus, stimulating and recording electrodes were placed in the middle third of the molecular layer. For stimulation of the perforant path input to the CA1 stratum lacunosum moleculare, stimulation and recording electrodes were placed in the distal region of the CA1 stratum lacunosum moleculare. Field EPSPs evoked in this region are characterised by a high fibre volley:fEPSP ratio which we used as an exclusion criteria for each slice (see Otmakhova and Lisman (1998). Responses were recorded with a glass micropipette (1–3 M⍀) containing 2M NaCl and were averaged over a 2 min period (i.e. each data point is the mean of four stimuli). fEPSP amplitudes are expressed as the percent of mean stable baseline values recorded for 10 min before the start of the experiment. Paired stimulation
or burst conditioning was initiated after stable baseline recordings at 0.033 Hz. For paired stimulation (50 ms interstimulus interval), paired-pulse facilitation data is expressed as a ratio of amplitudes fEPSP2/fEPSP1 for each stimulus pair. Burst conditioning stimulation consisted of a conditioning train (5 or 7 stimuli at 100Hz) followed after a 200 ms interval by a test stimulus. Amplitudes of the first fEPSP evoked by the conditioning train or the test fEPSP are expressed as the percent of mean stable baseline values recorded for 10 min before application of drug. The relative amplitude of the test fEPSP is expressed as a percentage of that of the first fEPSP evoked by the respective conditioning train (fEPSPtest/fEPSP1) and was calculated as the mean of the last four observations recorded pre- or post- drug application. All data are expressed as mean±standard error (SE). Drugs were applied by bath perfusion. LY341495 (2-amino-2-(2-carboxycyclopropan-1-yl)-3(dibenzopyran-4-yl)propanoic acid) was synthesised at F. Hoffmann-La Roche (Basel, Switzerland). DCG-IV ((2S,2⬘R,3⬘R)-2-(2⬘3⬘-dicarboxycyclopropyl)glycine) was purchased from Tocris Cookson (Bristol, UK). Statistical significance was determined by two-way ANOVA for repeated measures at a significance level of 0.05. Inhibition curves were fitted according to the Hill equation with baseline: Y ⫽ ( 100 ⫺ Ybase ) / ( 1 ⫹ ( x / IC50 )n) ⫹ Ybase where Ybase is maximum inhibition level and n is the slope factor.
3. Results To investigate the relative modulation of synaptic transmission by mGlu2 and mGlu3 receptor activation, we assayed the effects of the selective group II mGlu receptor agonist, DCG-IV, on fEPSPs elicited via stimulation of the perforant path inputs to the dentate gyrus mid-molecular layer and the CA1 stratum lacunosum moleculare in slices from wild-type and mGlu2 -/- mice. In slices from wild-type mice, fEPSPs elicited in the dentate gyrus and stratum lacunosum moleculare were inhibited by DCG-IV in a concentration-dependent manner. IC50s and maximum percentage inhibitions were 169 nM, 60% and 41 nM, 72%, respectively (Fig. 1). In slices from mGlu2 -/- mice a relatively small inhibition of dentate gyrus fEPSPs was observed, IC50 ⫽ 273 nM, 19%, whilst a much larger inhibition of stratum lacunosum moleculare fEPSPs, IC50 ⫽ 116 nM, 49%, was evident. DCG-IV (1 µM)-mediated inhibition in mGlu2 -/mice was blocked by 20 min pre-application of the group II selective mGlu receptor antagonist, LY341495 (1 µM), in both the dentate gyrus and stratum lacunosum moleculare (data not shown). To determine whether the action of DCG-IV in the stratum lacunosum moleculare
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Fig. 1. DCG-IV inhibits perforant path synaptic transmission in wild-type and mGlu2 -/- mice. Concentration-dependent inhibition of fEPSPs evoked by stimulation of the perforant path and recorded in either the dentate gyrus mid-moleculare (A), or the CA1 stratum lacunosum moleculare (C) in hippocampal slices from wild-type (filled circles) and mGlu2 -/- (open circles) mice. Data points are mean±SE fEPSP amplitudes expressed as a percentage of the pre-drug control value (n ⫽ 4–5). Fitted curves yielded IC50 values of 169 nM (slope: 1.8, maximum percentage inhibition: 60%) and 273 nM (slope: 1, 19%) for wild-type and mGlu2 -/- slices, respectively, in (A), and values of 41 nM (slope: 1.4, 72%) and 116 nM (slope: 1.1, 49%), respectively, in (C). Representative fEPSPs recorded in the dentate gyrus mid-moleculare (B) and CA1 stratum lacunosum moleculare (D) from wild-type (left) and mGlu2 -/- (right) slices are shown under control conditions and in the presence of 1 µM DCG-IV. Scale bars, 0.1 mV, 5 ms.
of mGlu2 -/- mice was mediated pre- or postsynaptically, we examined its effects on paired-pulse facilitation. Paired pulses (50ms interstimulus interval) were elicited at 0.033 Hz. Application of DCG-IV (1 µM) inhibited evoked fEPSPs and this inhibition was accompanied by an enhanced paired-pulse facilitation (Fig. 2), consistent with a presynaptic mode of action. To compare the effects of synaptically released glutamate on transmission in both pathways we employed a protocol consisting of a burst conditioning stimulus followed after 200 ms by a test stimulus. We have previously demonstrated that application of LY341495 to rat hippocampal slices resulted in an increase in the relative amplitude of test fEPSPs evoked 200 ms after a conditioning burst, but not after a single conditioning stimulus, in both pathways (Kew et al., 2001). The medial perforant path input to the dentate gyrus exhibits pairedpulse depression and, in slices from wild-type mice, test fEPSPs recorded in the dentate gyrus 200 ms after a burst conditioning stimulus consisting of seven stimuli at 100Hz were depressed relative to the first fEPSP elicited in the conditioning stimulus (fEPSPtest / fEPSP1 ⫽ 0.57 ± 0.02, n ⫽ 4). Application of LY341495 (1
µM) resulted in a significant increase in the amplitude of the test fEPSP following a 20 min wash in period (Fig. 3A,C; P ⬍ 0.01, ANOVA) with no effect on that of the first stimulus in the conditioning burst, resulting in an significantly increased relative amplitude of the test pulse (fEPSPtest / fEPSP1 ⫽ 0.62 ± 0.01, P ⬍ 0.05, ANOVA). In slices from mGlu2 -/- mice, test fEPSPs were also depressed relative to the first fEPSP elicited in the conditioning stimulus (fEPSPtest / fEPSP1 ⫽ 0.58 ± 0.01, n ⫽ 8). However, application of LY341495 did not change the amplitude of either the test pulse or that of the first stimulus in the conditioning burst (Fig. 3B,D; P ⬎ 0.1, ANOVA). Consequently, there was no change in the relative amplitude of the test pulse (fEPSPtest / fEPSP1 ⫽ 0.58 ± 0.02). The perforant path input to the CA1 stratum lacunosum moleculare exhibits paired-pulse facilitation and, in slices from wild-type mice, test fEPSPs recorded 200 ms after a burst conditioning stimulus consisting of five stimuli at 100 Hz were facilitated relative to the first fEPSP elicited in the conditioning stimulus (fEPSPtest / fEPSP1 ⫽ 1.56 ± 0.14, n ⫽ 4). Application of LY341495 (1 µM) resulted in a significant
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Fig. 2. DCG-IV-mediated inhibition in hippocampal slices from mGlu2 -/- mice is accompanied by an enhanced paired-pulse facilitation. (A) Application of DCG-IV (1 µM) resulted in an inhibition of fEPSP amplitude [amplitude fEPSP1(open circles), data points are mean ± SE, expressed as a percentage of the pre-drug control value (n ⫽ 5)], accompanied by an enhanced paired-pulse facilitation [EPSP2/EPSP1 (closed circles), data points: mean±SE]. (B) Representative paired fEPSPs (interstimulus interval: 50 ms) are shown under control conditions (top) and in the presence of DCG-IV (1 µM) (bottom). Scale bar, 0.2 mV, 5 ms.
Fig. 3. Activation of presynaptic group II mGlu receptors by synaptically released glutamate following burst stimulation in the dentate gyrus midmoleculare of wild-type, but not mGlu2 -/- mice. A burst stimulation protocol was used consisting of a seven pulse (100 Hz) conditioning burst followed by a test pulse after 200 ms. (A) In slices from wild-type mice (n ⫽ 4), LY341495 (1 µM) application resulted in the facilitation of a test fEPSP (closed circles, P ⬍ 0.01, ANOVA) but not the first fEPSP of the conditioning burst (open circles). Data shown are the last four control (pre-drug) fEPSPs and the last four fEPSPs evoked in the presence of LY341495 following a 20 min wash in period, expressed as mean±SE amplitude relative to respective mean pre-drug control. (B) In slices from mGlu2 -/- mice (n ⫽ 8), LY341495 (1 µM) application did not change the amplitudes of either the test fEPSP (closed circles) or the first fEPSP of the conditioning burst (open circles). Representative recordings are shown from wild-type (C) and mGlu2 -/- (D) slices under control conditions (top) and in the presence of 1 µM LY341495 (bottom). Scale bar, 0.2 mV, 50 ms.
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increase in the amplitude of the test fEPSP following a 20 min wash in period with no effect on that of the first stimulus in the conditioning burst (Fig. 4A,C; P ⬍ 0.001, ANOVA), resulting in a significantly increased relative amplitude of the test pulse (fEPSPtest /fEPSP1 ⫽ 2.00 ± 0.12, P ⬍ 0.05, ANOVA). In slices from mGlu2 -/- mice, test fEPSPs were also facilitated relative to the first fEPSP elicited in the conditioning stimulus (fEPSPtest / fEPSP1 ⫽ 1.80 ± 0.12, n ⫽ 7). Application of LY341495 resulted in significant increases in the amplitude of both the test stimulus and that of the first stimulus in the conditioning burst (Fig. 4B,D: P ⬍ 0.01, ANOVA) resulting in a small, non-significant, increase in the relative amplitude of the test pulse (fEPSPtest / fEPSP1 ⫽ 1.90 ± 0.14, P ⬎ 0.5, ANOVA). Whilst LY341495 is selective for group II mGluRs it also exhibits significant activity at mGlu 7 and 8 (Schoepp et al., 1999). Therefore, to attempt to dissociate effects mediated by the group II and group III mGlu receptors, we repeated the experimental paradigm using 100 nM LY341495. In slices from wild-type mice, application of LY341495 resulted in a slow onset significant facilitation of the test fEPSP reaching a
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maximum after 80 min (Fig. 5A; P ⬍ 0.005, ANOVA), resulting in an significantly increased relative amplitude of the test pulse (fEPSPtest /fEPSP1, control ⫽ 1.52 ± 0.04, ⫹ LY341495 ⫽ 1.86 ± 0.13, n ⫽ 7, P ⬍ 0.05, ANOVA). In slices from mGlu2 -/- mice, application of LY341495 over the same timecourse did not significantly effect amplitude of either the test fEPSP or that of the first stimulus in the conditioning burst (Fig. 5B). There was no significant change in the relative amplitude of the test pulse (fEPSPtest /fEPSP1, control ⫽1.79 ± 0.17, ⫹LY341495 ⫽ 1.83 ± 0.15, n ⫽ 5,P ⬎ 0.5, ANOVA). 4. Discussion In this study we have utilised mGlu2 receptor -/- mice to investigate the relative contributions of the two group II mGlu receptors, mGlu2 and mGlu3, to the inhibition of synaptic transmission at the perforant path inputs to the dentate gyrus mid-molecular layer and the CA1 stratum lacunosum moleculare mediated by both the selective group II agonist, DCG-IV, and by synaptically released glutamate. Importantly, the expression level and
Fig. 4. mGlu2 is the major presynaptic autoreceptor activated by burst stimulation of the perforant path input to the CA1 stratum lacunosum moleculare. A burst stimulation protocol was used consisting of a five pulse (100 Hz) conditioning burst followed by a test pulse after 200 ms. (A) In slices from wild-type mice (n ⫽ 4), LY341495 (1 µM) application resulted in the facilitation of a test fEPSP (closed circles, P ⬍ 0.001, ANOVA) but not the first fEPSP of the conditioning burst (open circles). Data shown are the last four control (pre-drug) fEPSPs and the last four fEPSPs evoked in the presence of LY341495 following a 20 min wash in period, expressed as mean±SE amplitude relative to respective mean pre-drug control. (B) In slices from mGlu2 -/- mice (n ⫽ 7), LY341495 (1 µM) application resulted in significant increases in the amplitudes of both the test fEPSP (closed circles) and the first fEPSP of the conditioning burst (open circles, P ⬍ 0.01, ANOVA). Representative recordings are shown from wild-type (C) and mGlu2 -/- (D) slices under control conditions (top) and in the presence of 1 µM LY341495 (bottom). Stimulus artefacts have been truncated. Scale bar, 0.2 mV, 20 ms.
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Fig. 5. Selective antagonism of group II mGlu receptors with 100 nM LY341495 does not increase baseline synaptic transmission in the stratum lacunosum moleculare of mGlu2 -/- mice. A burst stimulation protocol was used consisting of a five pulse (100 Hz) conditioning burst followed by a test pulse after 200 ms. (A) In slices from wild-type mice (n ⫽ 7), LY341495 (100 nM) application resulted in the facilitation of a test fEPSP (closed circles, P ⬍ 0.005, ANOVA) but not the first fEPSP of the conditioning burst (open circles). Data shown are the last four control (predrug) fEPSPs and the last four fEPSPs evoked in the presence of LY341495 following an 80 min wash in period, expressed as mean±SE amplitude relative to respective mean pre-drug control. (B) In slices from mGlu2 -/- mice (n ⫽ 5), LY341495 (100 nM) application did not significantly change the amplitudes of either the test fEPSP (closed circles) or the first fEPSP of the conditioning burst (open circles, P ⬎ 0.1, ANOVA).
distribution of mGlu3 appears to be unaltered in mGlu2 /- mice (Yokoi et al., 1996; Tamaru et al., 2001). Whilst we have observed a DCG-IV-sensitive inhibition of synaptic transmission likely to be mediated via mGlu3 in both pathways, it is mGlu2 that appears to act as the major presynaptic group II mGlu autoreceptor activated by synaptically released glutamate. DCG-IV exhibits similar affinities for recombinant mGlu2 and mGlu3 receptors. In rat hippocampal slices we have previously determined IC50 values for DCG-IV of 150 and 140 nM in the dentate gyrus and stratum lacunosum moleculare, respectively (Kew et al., 2001). In slices from wild-type mice DCG-IV potently inhibited fEPSPs recorded in both pathways with similar IC50 values, although with a somewhat higher potency in the stratum lacunosum moleculare relative to the dentate gyrus, perhaps reflecting the seemingly higher receptor density in this pathway. In mGlu2 -/- slices, the extent of the DCG-IV-induced inhibition of dentate gyrus fEPSPs was markedly reduced, demonstrating that in wild-type slices inhibition of synaptic transmission by group II mGlu receptor agonists is mediated predominantly via mGlu2. In the stratum lacunosum moleculare of mGlu2 -/- mice, the DCG-IV-mediated inhibition was also attenuated, although to a lesser extent, illustrating a more significant role for mGlu3 in the regulation of synaptic transmission in response to exogenous group II agonists. In both pathways the potency of the DCG-IVmediated inhibition in mGlu2 -/- slices was similar to that of wild-type, which together with the functional selectivity of DCG-IV for group II mGlu receptors (Brabet et al., 1998) suggests that the effects are mediated via mGlu3. In agreement, the DCG-IV-mediated inhibition in both pathways was blocked by the group II selective antagonist LY341495. In agreement with our previous observations in rat hippocampal slices (Kew et al., 2001), application of
LY341495 resulted in an increase in the amplitude of a test fEPSP evoked in the dentate gyrus mid-molecular layer of wild-type mice after a high frequency conditioning burst whilst the amplitude of the first fEPSP of the conditioning burst was unchanged, resulting in an increased relative amplitude of the test pulse (fEPSPtest/fEPSP1). In contrast, LY341495 application exerted no effect in mGlu2 -/- slices. Thus, in line with the observed reduced sensitivity to DCG-IV, the burstinduced activation of presumptive presynaptic group II mGlu autoreceptors by synaptically released glutamate is absent in mGlu2 -/- mice. Therefore, in wild-type animals, mGlu2 is the group II mGlu presynaptic autoreceptor which is activated after burst stimulation, consistent with its likely preterminal localisation (Shigemoto et al., 1997). Under these stimulation conditions we found no evidence for the activation of the presumptive mGlu3, DCG-IV-sensitive receptors in mGlu2 -/- mice suggesting a localisation distant to the synapses, inaccessible to released glutamate. In agreement, ultrastructural analysis has demonstrated both pre- and postsynaptic localisations for neuronal mGlu3 in the dentate gyrus molecular layer (Tamaru et al., 2001), with immunolabelling of axon terminals mostly remote from synapses. In the stratum lacunosum moleculare of wild-type mice, again in agreement with our previous observations in rat hippocampal slices (Kew et al., 2001), application of LY341495 produced an increase in the amplitude of the test fEPSP, whilst the amplitude of the first fEPSP of the conditioning burst was unchanged, resulting in an increased relative amplitude of the test pulse (fEPSPtest/fEPSP1). In mGlu2 -/- slices, application of 1µM LY341495 resulted in increases in both the test fEPSP and the first fEPSP in the conditioning burst, with no significant change in the relative amplitude of the test pulse. Notably, the absolute increase in amplitude of the test pulse was markedly less than that observed in wild-
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type slices. Whilst LY341495 is selective for group II mGlu receptors it also exhibits significant affinity at both mGlu7 and mGlu8 receptors (Schoepp et al., 1999). The increase in baseline transmission in mGlu2 -/- slices, which may reflect an increase in postsynaptic excitability, was absent in experiments using 100 nM LY341495, whilst the facilitation of the test pulse in wild-type slices persisted, suggesting that it may be mediated via blockade of group III mGlu receptors. It is unclear why such an effect should be apparent in slices from mGlu2 -/- and not wild-type mice but this may result from some form of developmental compensation such that their physiology may differ. Nevertheless, these observations demonstrate that in the CA1 stratum lacunosum moleculare of wild-type slices it is mGlu2 that acts as the major presynaptic group II mGlu autoreceptor activated by synaptically released glutamate. Whilst the DCG-IV effects on paired-pulse facilitation clearly suggest a presynaptic localisation for mGlu3, it seems likely that, as in the dentate, presynaptic mGlu3 is distant from the synaptic glutamate release sites. In conclusion, we have utilised mGlu2 -/- mice to probe the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission. Whilst activation of mGlu3 can inhibit synaptic transmission, it is mGlu2 that appears to play the major presynaptic autoreceptor role.
Acknowledgements We thank Professor Shigetada Nakanishi for the provision of mGlu2 -/- mice and Dr. Laurence Ozmen and Patrick Biry for their breeding and genotyping. We thank Drs. Geo Adam and Thomas Woltering for the synthesis and provision of LY341495.
References Anwyl, R., 1999. Metabotropic glutamate receptors: electrophysiological properties and role in plasticity. Brain Research Reviews 29, 83–120. Brabet, I., Parmentier, M.-L., De Colle, C., Bockaert, J., Acher, F., Pin, J.-P., 1998. Comparative effect of L-CCG-I, DCG-IV and gcarboxy-L-glutamate on all cloned metabotropic glutamate receptor subtypes. Neuropharmacology 37, 1043–1051. Cartmell, J., Schoepp, D.D., 2000. Regulation of neurotransmitter release by metabotropic glutamate receptors. Journal of Neurochemistry 75, 889–907. Conn, P.J., Pin, J.P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annual Review of Pharmacology and Toxicology 37, 205–237. Kew, J.N.C., Ducarre, J.-M., Pflimlin, M.-C., Mutel, V., Kemp, J.A., 2001. Activity-dependent presynaptic autoinhibition by group II
221
metabotropic receptors at perforant path inputs to the dentate gyrus and CA1. Neuropharmacology 40, 20–27. Kilbride, J., Huang, L., Rowan, M.J., Anwyl, R., 1998. Presynaptic inhibitory action of the group II metabotropic glutamate receptor agonists, LY354740 and DCG-IV. European Journal of Pharmacology 356, 149–157. Macek, T.A., Winder, D.G., Gereau, R.W., Ladd, C.O., Conn, P.J., 1996. Differential involvement of group II and proup III mGluRs as autoreceptors at lateral and medial perforant path synapses. Journal of Neurophysiology 76, 3798–3806. Mutel, V., Adam, G., Chaboz, S., Kemp, J.A., Klingelschmidt, A., Messer, J., Wichmann, J., Woltering, T., Richards, J.G., 1998. Characterization of (2S,2⬘R,3⬘R)-2-(2⬘,3⬘-[3H]-dicarboxycyclopropyl)glycine binding in rat brain. Journal of Neurochemistry 71, 2558–2564. Neki, A., Ohishi, H., Kaneko, T., Shigemoto, R., Nakanishi, S., Mizuno, N., 1996. Pre- and postsynaptic localization of a metabotropic glutamate receptor, mGluR2, in the rat brain: An immunohistochemical study with a monoclonal antibody. Neuroscience Letters 202, 197–200. Ohishi, H., Neki, A., Mizuno, N., 1998. Distribution of a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat and mouse: an immunohistochemical study with a monoclonal antibody. Neuroscience Research 30, 65–82. Ohishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N., 1993a. Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat. Neuroscience 53, 1009–1018. Ohishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N., 1993b. Distribution of the mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ hybridization study. Journal of Comparative Neurology 335, 252–266. Otmakhova, N.A., Lisman, J.E., 1998. Dopamine selectively inhibits the direct cortical pathway to the CA1 hippocampal region. Journal of Neuroscience 19, 1437–1445. Petralia, R.S., Wang, Y.X., Niedzielski, A.S., Wenthold, R.J., 1996. The metabotropic glutamate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynaptic and glial localizations. Neuroscience 71, 949–976. Schaffhauser, H., Richards, J.G., Cartmell, J., Chaboz, S., Kemp, J.A., Klingelschmidt, A., Messer, J., Stadler, H., Woltering, T., Mutel, V., 1998. In vitro binding characteristics of a new selective group II metabotropic glutamate receptor radioligand, [3H] LY354740, in rat brain. Molecular Pharmacology 53, 228–233. Schoepp, D.D., Jane, D.E., Monn, J.A., 1999. Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38, 1431–1476. Shigemoto, R., Kinoshita, A., Wada, E., Nomura, S., Ohishi, H., Takada, M., Flor, P.J., Neki, A., Abe, T., Nakanishi, S., Mizuno, N., 1997. Differential presynaptic localization of metabotropic glutamate receptor subtypes in the rat hippocampus. Journal of Neuroscience 17, 7503–7522. Tamaru, Y., Nomura, S., Mizuno, N., Shigemoto, R., 2001. Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre- and postsynaptic sites. Neuroscience 106, 481–503. Ugolini, A., Bordi, F., 1995. Metabotropic glutamate group II receptors are responsible for the depression of synaptic transmission induced by ACPD in the dentate gyrus. European Journal of Pharmacology 294, 403–410. Yokoi, M., Kobayashi, K., Manabe, T., Takahashi, T., Sakaguchi, I., Katsuura, G., Shigemoto, R., Ohishi, H., Nomura, S., Nakamura, K., Nakao, K., Katsuki, M., Nakanishi, S., 1996. Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2. Science 273, 645–647.
Differential regulation of synaptic transmission by mGlu2 and mGlu3 at the perforant path inputs to the dentate gyrus and CA1 revealed in mGlu2 -/- mice James N.C. Kew ∗, Marie-Claire Pflimlin, John A. Kemp, Vincent Mutel F. Hoffmann-La Roche Ltd, Preclinical CNS Research, CH-4070 Basel, Switzerland Received 8 April 2002; received in revised form 31 May 2002; accepted 5 June 2002
Abstract Group II metabotropic glutamate (mGlu) receptors can act as presynaptic autoinhibitory receptors at perforant path inputs to the hippocampus under conditions of high frequency synaptic activation. We have used mGlu2 -/- mice to examine the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, DCG-IV, and by synaptically released glutamate. Field excitatory postsynaptic potentials evoked by stimulation of either the perforant path inputs to the dentate gyrus mid-moleculare or the CA1 stratum lacunosum moleculare were inhibited by DCGIV with IC50 values and maximum percentage inhibition of: 169 nM (60%) and 41 nM (72%) in wild-type mice and 273 nM (19%) and 116 nM (49%) in mGlu2 -/- mice, respectively. Activation of presynaptic group II mGlu autoreceptors by synaptically released glutamate, as revealed by a LY341495-mediated increase in the relative amplitude of a test fEPSP evoked after a conditioning burst, was observed in both the dentate gyrus and the stratum lacunosum of wild-type, but not mGlu2 -/- mice. These observations demonstrate that activation of mGlu3 receptors can regulate synaptic transmission at perforant path synapses but suggest that mGlu2 is the major presynaptic group II autoreceptor activated by synaptically released glutamate. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Metabotropic glutamate receptor; Autoreceptor; Synaptic transmission
1. Introduction Whilst fast excitatory glutamatergic transmission is mediated via the ionotropic AMPA and NMDA receptors, glutamate also activates a family of G-proteincoupled receptors, termed metabotropic glutamate (mGlu) receptors, which can modulate neuronal excitability and synaptic transmission (Anwyl, 1999). To date, eight mGlu receptors have been identified (mGlu1– 8) and these have been divided into three groups according to their sequence homology: group I receptors, mGlu1 and 5; group II receptors, mGlu2 and 3 and group III receptors, mGlu4, 6, 7 and 8 (Conn and Pin, 1997). Whereas group I mGlu receptors are primarily localised Corresponding author. Tel.: +44-1279-622150; fax: +44-1279875389. Present address: Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. E-mail address: [email protected] (J.N.C. Kew). ∗
postsynaptically, group II and III receptors are typically presynaptic and can regulate neurotransmitter release (Cartmell and Schoepp, 2000). Group II mGlu receptors are highly expressed in the terminal fields of the perforant path input from the enthorinal cortex in the mid-molecular layer of the dentate gyrus and the CA1 stratum lacunosum moleculare as revealed by both immunohistochemistry (Neki et al., 1996; Petralia et al., 1996; Shigemoto et al., 1997; Ohishi et al., 1998) and radioautography (Mutel et al., 1998; Schaffhauser et al., 1998) studies. Application of group II mGlu receptor selective agonists inhibits synaptic transmission in these pathways (Ugolini and Bordi, 1995; Macek et al., 1996; Kilbride et al., 1998; Kew et al., 2001) and activation of these receptors by synaptically released glutamate has been observed following high frequency burst stimulation (Kew et al., 2001). In situ hybridisation studies have indicated that mGlu2 is largely neuronal whilst mGlu3 is expressed in both neurones and glia (Ohishi et al., 1993a; b) and immunocyto-
0028-3908/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 8 4 - 9
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chemistry studies have confirmed the neuronal localisation of mGlu2 (Neki et al., 1996; Ohishi et al., 1998). A recent study using an mGlu3-selective antibody, that cross reacts weakly with mGlu2, in both wild-type and mGlu2 -/- mice has confirmed the mixed neuronal / glial distribution of the receptor (Tamaru et al., 2001). Moderate labelling was observed in the CA1 stratum lacunosum moleculare, with strong immunoreactivity evident throughout the dentate gyrus molecular layer, where ultrastructural analysis identified labelled glial processes, dendritic spines and axons / axon terminals (Tamaru et al., 2001). In this study, we have utilised mGlu2 -/- mice (Yokoi et al., 1996) to investigate the relative contributions of mGlu2 and mGlu3 to the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, (2S,2⬘R,3⬘R)-2-(2⬘3⬘dicarboxycyclopropyl)glycine (DCG-IV) (Brabet et al., 1998) and by synaptically released glutamate.
2. Methods Mice were anaesthetised in a 3.5% halothane/96.5% oxygen mixture and decapitated, as approved by the local institutional animal welfare committee. Hippocampal slices (400 µm) were cut from 4–5 month old mGlu2 -/- mice, or wild-type littermates derived from heterozygote matings, with a Sorvall tissue chopper. Slices were maintained in a submerged chamber and perfused at 35 °C with a simple salt solution containing (mM): NaCl, 124; KCl, 2.5; MgSO4, 2; CaCl2, 2.5; KH2PO4, 1.25; NaHCO3, 26; glucose, 10; sucrose, 4; gassed with 95% O2/5% CO2 (pH 7.4, 307 mOsm). The perforant path inputs to either the mid-molecular layer of the dentate gyrus or the CA1 stratum lacunosum moleculare were stimulated (0.033 Hz, 100 µs) at a strength adjusted to evoke field excitatory post synaptic potentials (fEPSPs) equal to 40 or 60% of the relative maximum amplitudes without superimposed population spike, respectively. For stimulation of the medial perforant path input to the dentate gyrus, stimulating and recording electrodes were placed in the middle third of the molecular layer. For stimulation of the perforant path input to the CA1 stratum lacunosum moleculare, stimulation and recording electrodes were placed in the distal region of the CA1 stratum lacunosum moleculare. Field EPSPs evoked in this region are characterised by a high fibre volley:fEPSP ratio which we used as an exclusion criteria for each slice (see Otmakhova and Lisman (1998). Responses were recorded with a glass micropipette (1–3 M⍀) containing 2M NaCl and were averaged over a 2 min period (i.e. each data point is the mean of four stimuli). fEPSP amplitudes are expressed as the percent of mean stable baseline values recorded for 10 min before the start of the experiment. Paired stimulation
or burst conditioning was initiated after stable baseline recordings at 0.033 Hz. For paired stimulation (50 ms interstimulus interval), paired-pulse facilitation data is expressed as a ratio of amplitudes fEPSP2/fEPSP1 for each stimulus pair. Burst conditioning stimulation consisted of a conditioning train (5 or 7 stimuli at 100Hz) followed after a 200 ms interval by a test stimulus. Amplitudes of the first fEPSP evoked by the conditioning train or the test fEPSP are expressed as the percent of mean stable baseline values recorded for 10 min before application of drug. The relative amplitude of the test fEPSP is expressed as a percentage of that of the first fEPSP evoked by the respective conditioning train (fEPSPtest/fEPSP1) and was calculated as the mean of the last four observations recorded pre- or post- drug application. All data are expressed as mean±standard error (SE). Drugs were applied by bath perfusion. LY341495 (2-amino-2-(2-carboxycyclopropan-1-yl)-3(dibenzopyran-4-yl)propanoic acid) was synthesised at F. Hoffmann-La Roche (Basel, Switzerland). DCG-IV ((2S,2⬘R,3⬘R)-2-(2⬘3⬘-dicarboxycyclopropyl)glycine) was purchased from Tocris Cookson (Bristol, UK). Statistical significance was determined by two-way ANOVA for repeated measures at a significance level of 0.05. Inhibition curves were fitted according to the Hill equation with baseline: Y ⫽ ( 100 ⫺ Ybase ) / ( 1 ⫹ ( x / IC50 )n) ⫹ Ybase where Ybase is maximum inhibition level and n is the slope factor.
3. Results To investigate the relative modulation of synaptic transmission by mGlu2 and mGlu3 receptor activation, we assayed the effects of the selective group II mGlu receptor agonist, DCG-IV, on fEPSPs elicited via stimulation of the perforant path inputs to the dentate gyrus mid-molecular layer and the CA1 stratum lacunosum moleculare in slices from wild-type and mGlu2 -/- mice. In slices from wild-type mice, fEPSPs elicited in the dentate gyrus and stratum lacunosum moleculare were inhibited by DCG-IV in a concentration-dependent manner. IC50s and maximum percentage inhibitions were 169 nM, 60% and 41 nM, 72%, respectively (Fig. 1). In slices from mGlu2 -/- mice a relatively small inhibition of dentate gyrus fEPSPs was observed, IC50 ⫽ 273 nM, 19%, whilst a much larger inhibition of stratum lacunosum moleculare fEPSPs, IC50 ⫽ 116 nM, 49%, was evident. DCG-IV (1 µM)-mediated inhibition in mGlu2 -/mice was blocked by 20 min pre-application of the group II selective mGlu receptor antagonist, LY341495 (1 µM), in both the dentate gyrus and stratum lacunosum moleculare (data not shown). To determine whether the action of DCG-IV in the stratum lacunosum moleculare
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Fig. 1. DCG-IV inhibits perforant path synaptic transmission in wild-type and mGlu2 -/- mice. Concentration-dependent inhibition of fEPSPs evoked by stimulation of the perforant path and recorded in either the dentate gyrus mid-moleculare (A), or the CA1 stratum lacunosum moleculare (C) in hippocampal slices from wild-type (filled circles) and mGlu2 -/- (open circles) mice. Data points are mean±SE fEPSP amplitudes expressed as a percentage of the pre-drug control value (n ⫽ 4–5). Fitted curves yielded IC50 values of 169 nM (slope: 1.8, maximum percentage inhibition: 60%) and 273 nM (slope: 1, 19%) for wild-type and mGlu2 -/- slices, respectively, in (A), and values of 41 nM (slope: 1.4, 72%) and 116 nM (slope: 1.1, 49%), respectively, in (C). Representative fEPSPs recorded in the dentate gyrus mid-moleculare (B) and CA1 stratum lacunosum moleculare (D) from wild-type (left) and mGlu2 -/- (right) slices are shown under control conditions and in the presence of 1 µM DCG-IV. Scale bars, 0.1 mV, 5 ms.
of mGlu2 -/- mice was mediated pre- or postsynaptically, we examined its effects on paired-pulse facilitation. Paired pulses (50ms interstimulus interval) were elicited at 0.033 Hz. Application of DCG-IV (1 µM) inhibited evoked fEPSPs and this inhibition was accompanied by an enhanced paired-pulse facilitation (Fig. 2), consistent with a presynaptic mode of action. To compare the effects of synaptically released glutamate on transmission in both pathways we employed a protocol consisting of a burst conditioning stimulus followed after 200 ms by a test stimulus. We have previously demonstrated that application of LY341495 to rat hippocampal slices resulted in an increase in the relative amplitude of test fEPSPs evoked 200 ms after a conditioning burst, but not after a single conditioning stimulus, in both pathways (Kew et al., 2001). The medial perforant path input to the dentate gyrus exhibits pairedpulse depression and, in slices from wild-type mice, test fEPSPs recorded in the dentate gyrus 200 ms after a burst conditioning stimulus consisting of seven stimuli at 100Hz were depressed relative to the first fEPSP elicited in the conditioning stimulus (fEPSPtest / fEPSP1 ⫽ 0.57 ± 0.02, n ⫽ 4). Application of LY341495 (1
µM) resulted in a significant increase in the amplitude of the test fEPSP following a 20 min wash in period (Fig. 3A,C; P ⬍ 0.01, ANOVA) with no effect on that of the first stimulus in the conditioning burst, resulting in an significantly increased relative amplitude of the test pulse (fEPSPtest / fEPSP1 ⫽ 0.62 ± 0.01, P ⬍ 0.05, ANOVA). In slices from mGlu2 -/- mice, test fEPSPs were also depressed relative to the first fEPSP elicited in the conditioning stimulus (fEPSPtest / fEPSP1 ⫽ 0.58 ± 0.01, n ⫽ 8). However, application of LY341495 did not change the amplitude of either the test pulse or that of the first stimulus in the conditioning burst (Fig. 3B,D; P ⬎ 0.1, ANOVA). Consequently, there was no change in the relative amplitude of the test pulse (fEPSPtest / fEPSP1 ⫽ 0.58 ± 0.02). The perforant path input to the CA1 stratum lacunosum moleculare exhibits paired-pulse facilitation and, in slices from wild-type mice, test fEPSPs recorded 200 ms after a burst conditioning stimulus consisting of five stimuli at 100 Hz were facilitated relative to the first fEPSP elicited in the conditioning stimulus (fEPSPtest / fEPSP1 ⫽ 1.56 ± 0.14, n ⫽ 4). Application of LY341495 (1 µM) resulted in a significant
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Fig. 2. DCG-IV-mediated inhibition in hippocampal slices from mGlu2 -/- mice is accompanied by an enhanced paired-pulse facilitation. (A) Application of DCG-IV (1 µM) resulted in an inhibition of fEPSP amplitude [amplitude fEPSP1(open circles), data points are mean ± SE, expressed as a percentage of the pre-drug control value (n ⫽ 5)], accompanied by an enhanced paired-pulse facilitation [EPSP2/EPSP1 (closed circles), data points: mean±SE]. (B) Representative paired fEPSPs (interstimulus interval: 50 ms) are shown under control conditions (top) and in the presence of DCG-IV (1 µM) (bottom). Scale bar, 0.2 mV, 5 ms.
Fig. 3. Activation of presynaptic group II mGlu receptors by synaptically released glutamate following burst stimulation in the dentate gyrus midmoleculare of wild-type, but not mGlu2 -/- mice. A burst stimulation protocol was used consisting of a seven pulse (100 Hz) conditioning burst followed by a test pulse after 200 ms. (A) In slices from wild-type mice (n ⫽ 4), LY341495 (1 µM) application resulted in the facilitation of a test fEPSP (closed circles, P ⬍ 0.01, ANOVA) but not the first fEPSP of the conditioning burst (open circles). Data shown are the last four control (pre-drug) fEPSPs and the last four fEPSPs evoked in the presence of LY341495 following a 20 min wash in period, expressed as mean±SE amplitude relative to respective mean pre-drug control. (B) In slices from mGlu2 -/- mice (n ⫽ 8), LY341495 (1 µM) application did not change the amplitudes of either the test fEPSP (closed circles) or the first fEPSP of the conditioning burst (open circles). Representative recordings are shown from wild-type (C) and mGlu2 -/- (D) slices under control conditions (top) and in the presence of 1 µM LY341495 (bottom). Scale bar, 0.2 mV, 50 ms.
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increase in the amplitude of the test fEPSP following a 20 min wash in period with no effect on that of the first stimulus in the conditioning burst (Fig. 4A,C; P ⬍ 0.001, ANOVA), resulting in a significantly increased relative amplitude of the test pulse (fEPSPtest /fEPSP1 ⫽ 2.00 ± 0.12, P ⬍ 0.05, ANOVA). In slices from mGlu2 -/- mice, test fEPSPs were also facilitated relative to the first fEPSP elicited in the conditioning stimulus (fEPSPtest / fEPSP1 ⫽ 1.80 ± 0.12, n ⫽ 7). Application of LY341495 resulted in significant increases in the amplitude of both the test stimulus and that of the first stimulus in the conditioning burst (Fig. 4B,D: P ⬍ 0.01, ANOVA) resulting in a small, non-significant, increase in the relative amplitude of the test pulse (fEPSPtest / fEPSP1 ⫽ 1.90 ± 0.14, P ⬎ 0.5, ANOVA). Whilst LY341495 is selective for group II mGluRs it also exhibits significant activity at mGlu 7 and 8 (Schoepp et al., 1999). Therefore, to attempt to dissociate effects mediated by the group II and group III mGlu receptors, we repeated the experimental paradigm using 100 nM LY341495. In slices from wild-type mice, application of LY341495 resulted in a slow onset significant facilitation of the test fEPSP reaching a
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maximum after 80 min (Fig. 5A; P ⬍ 0.005, ANOVA), resulting in an significantly increased relative amplitude of the test pulse (fEPSPtest /fEPSP1, control ⫽ 1.52 ± 0.04, ⫹ LY341495 ⫽ 1.86 ± 0.13, n ⫽ 7, P ⬍ 0.05, ANOVA). In slices from mGlu2 -/- mice, application of LY341495 over the same timecourse did not significantly effect amplitude of either the test fEPSP or that of the first stimulus in the conditioning burst (Fig. 5B). There was no significant change in the relative amplitude of the test pulse (fEPSPtest /fEPSP1, control ⫽1.79 ± 0.17, ⫹LY341495 ⫽ 1.83 ± 0.15, n ⫽ 5,P ⬎ 0.5, ANOVA). 4. Discussion In this study we have utilised mGlu2 receptor -/- mice to investigate the relative contributions of the two group II mGlu receptors, mGlu2 and mGlu3, to the inhibition of synaptic transmission at the perforant path inputs to the dentate gyrus mid-molecular layer and the CA1 stratum lacunosum moleculare mediated by both the selective group II agonist, DCG-IV, and by synaptically released glutamate. Importantly, the expression level and
Fig. 4. mGlu2 is the major presynaptic autoreceptor activated by burst stimulation of the perforant path input to the CA1 stratum lacunosum moleculare. A burst stimulation protocol was used consisting of a five pulse (100 Hz) conditioning burst followed by a test pulse after 200 ms. (A) In slices from wild-type mice (n ⫽ 4), LY341495 (1 µM) application resulted in the facilitation of a test fEPSP (closed circles, P ⬍ 0.001, ANOVA) but not the first fEPSP of the conditioning burst (open circles). Data shown are the last four control (pre-drug) fEPSPs and the last four fEPSPs evoked in the presence of LY341495 following a 20 min wash in period, expressed as mean±SE amplitude relative to respective mean pre-drug control. (B) In slices from mGlu2 -/- mice (n ⫽ 7), LY341495 (1 µM) application resulted in significant increases in the amplitudes of both the test fEPSP (closed circles) and the first fEPSP of the conditioning burst (open circles, P ⬍ 0.01, ANOVA). Representative recordings are shown from wild-type (C) and mGlu2 -/- (D) slices under control conditions (top) and in the presence of 1 µM LY341495 (bottom). Stimulus artefacts have been truncated. Scale bar, 0.2 mV, 20 ms.
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Fig. 5. Selective antagonism of group II mGlu receptors with 100 nM LY341495 does not increase baseline synaptic transmission in the stratum lacunosum moleculare of mGlu2 -/- mice. A burst stimulation protocol was used consisting of a five pulse (100 Hz) conditioning burst followed by a test pulse after 200 ms. (A) In slices from wild-type mice (n ⫽ 7), LY341495 (100 nM) application resulted in the facilitation of a test fEPSP (closed circles, P ⬍ 0.005, ANOVA) but not the first fEPSP of the conditioning burst (open circles). Data shown are the last four control (predrug) fEPSPs and the last four fEPSPs evoked in the presence of LY341495 following an 80 min wash in period, expressed as mean±SE amplitude relative to respective mean pre-drug control. (B) In slices from mGlu2 -/- mice (n ⫽ 5), LY341495 (100 nM) application did not significantly change the amplitudes of either the test fEPSP (closed circles) or the first fEPSP of the conditioning burst (open circles, P ⬎ 0.1, ANOVA).
distribution of mGlu3 appears to be unaltered in mGlu2 /- mice (Yokoi et al., 1996; Tamaru et al., 2001). Whilst we have observed a DCG-IV-sensitive inhibition of synaptic transmission likely to be mediated via mGlu3 in both pathways, it is mGlu2 that appears to act as the major presynaptic group II mGlu autoreceptor activated by synaptically released glutamate. DCG-IV exhibits similar affinities for recombinant mGlu2 and mGlu3 receptors. In rat hippocampal slices we have previously determined IC50 values for DCG-IV of 150 and 140 nM in the dentate gyrus and stratum lacunosum moleculare, respectively (Kew et al., 2001). In slices from wild-type mice DCG-IV potently inhibited fEPSPs recorded in both pathways with similar IC50 values, although with a somewhat higher potency in the stratum lacunosum moleculare relative to the dentate gyrus, perhaps reflecting the seemingly higher receptor density in this pathway. In mGlu2 -/- slices, the extent of the DCG-IV-induced inhibition of dentate gyrus fEPSPs was markedly reduced, demonstrating that in wild-type slices inhibition of synaptic transmission by group II mGlu receptor agonists is mediated predominantly via mGlu2. In the stratum lacunosum moleculare of mGlu2 -/- mice, the DCG-IV-mediated inhibition was also attenuated, although to a lesser extent, illustrating a more significant role for mGlu3 in the regulation of synaptic transmission in response to exogenous group II agonists. In both pathways the potency of the DCG-IVmediated inhibition in mGlu2 -/- slices was similar to that of wild-type, which together with the functional selectivity of DCG-IV for group II mGlu receptors (Brabet et al., 1998) suggests that the effects are mediated via mGlu3. In agreement, the DCG-IV-mediated inhibition in both pathways was blocked by the group II selective antagonist LY341495. In agreement with our previous observations in rat hippocampal slices (Kew et al., 2001), application of
LY341495 resulted in an increase in the amplitude of a test fEPSP evoked in the dentate gyrus mid-molecular layer of wild-type mice after a high frequency conditioning burst whilst the amplitude of the first fEPSP of the conditioning burst was unchanged, resulting in an increased relative amplitude of the test pulse (fEPSPtest/fEPSP1). In contrast, LY341495 application exerted no effect in mGlu2 -/- slices. Thus, in line with the observed reduced sensitivity to DCG-IV, the burstinduced activation of presumptive presynaptic group II mGlu autoreceptors by synaptically released glutamate is absent in mGlu2 -/- mice. Therefore, in wild-type animals, mGlu2 is the group II mGlu presynaptic autoreceptor which is activated after burst stimulation, consistent with its likely preterminal localisation (Shigemoto et al., 1997). Under these stimulation conditions we found no evidence for the activation of the presumptive mGlu3, DCG-IV-sensitive receptors in mGlu2 -/- mice suggesting a localisation distant to the synapses, inaccessible to released glutamate. In agreement, ultrastructural analysis has demonstrated both pre- and postsynaptic localisations for neuronal mGlu3 in the dentate gyrus molecular layer (Tamaru et al., 2001), with immunolabelling of axon terminals mostly remote from synapses. In the stratum lacunosum moleculare of wild-type mice, again in agreement with our previous observations in rat hippocampal slices (Kew et al., 2001), application of LY341495 produced an increase in the amplitude of the test fEPSP, whilst the amplitude of the first fEPSP of the conditioning burst was unchanged, resulting in an increased relative amplitude of the test pulse (fEPSPtest/fEPSP1). In mGlu2 -/- slices, application of 1µM LY341495 resulted in increases in both the test fEPSP and the first fEPSP in the conditioning burst, with no significant change in the relative amplitude of the test pulse. Notably, the absolute increase in amplitude of the test pulse was markedly less than that observed in wild-
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type slices. Whilst LY341495 is selective for group II mGlu receptors it also exhibits significant affinity at both mGlu7 and mGlu8 receptors (Schoepp et al., 1999). The increase in baseline transmission in mGlu2 -/- slices, which may reflect an increase in postsynaptic excitability, was absent in experiments using 100 nM LY341495, whilst the facilitation of the test pulse in wild-type slices persisted, suggesting that it may be mediated via blockade of group III mGlu receptors. It is unclear why such an effect should be apparent in slices from mGlu2 -/- and not wild-type mice but this may result from some form of developmental compensation such that their physiology may differ. Nevertheless, these observations demonstrate that in the CA1 stratum lacunosum moleculare of wild-type slices it is mGlu2 that acts as the major presynaptic group II mGlu autoreceptor activated by synaptically released glutamate. Whilst the DCG-IV effects on paired-pulse facilitation clearly suggest a presynaptic localisation for mGlu3, it seems likely that, as in the dentate, presynaptic mGlu3 is distant from the synaptic glutamate release sites. In conclusion, we have utilised mGlu2 -/- mice to probe the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission. Whilst activation of mGlu3 can inhibit synaptic transmission, it is mGlu2 that appears to play the major presynaptic autoreceptor role.
Acknowledgements We thank Professor Shigetada Nakanishi for the provision of mGlu2 -/- mice and Dr. Laurence Ozmen and Patrick Biry for their breeding and genotyping. We thank Drs. Geo Adam and Thomas Woltering for the synthesis and provision of LY341495.
References Anwyl, R., 1999. Metabotropic glutamate receptors: electrophysiological properties and role in plasticity. Brain Research Reviews 29, 83–120. Brabet, I., Parmentier, M.-L., De Colle, C., Bockaert, J., Acher, F., Pin, J.-P., 1998. Comparative effect of L-CCG-I, DCG-IV and gcarboxy-L-glutamate on all cloned metabotropic glutamate receptor subtypes. Neuropharmacology 37, 1043–1051. Cartmell, J., Schoepp, D.D., 2000. Regulation of neurotransmitter release by metabotropic glutamate receptors. Journal of Neurochemistry 75, 889–907. Conn, P.J., Pin, J.P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annual Review of Pharmacology and Toxicology 37, 205–237. Kew, J.N.C., Ducarre, J.-M., Pflimlin, M.-C., Mutel, V., Kemp, J.A., 2001. Activity-dependent presynaptic autoinhibition by group II
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metabotropic receptors at perforant path inputs to the dentate gyrus and CA1. Neuropharmacology 40, 20–27. Kilbride, J., Huang, L., Rowan, M.J., Anwyl, R., 1998. Presynaptic inhibitory action of the group II metabotropic glutamate receptor agonists, LY354740 and DCG-IV. European Journal of Pharmacology 356, 149–157. Macek, T.A., Winder, D.G., Gereau, R.W., Ladd, C.O., Conn, P.J., 1996. Differential involvement of group II and proup III mGluRs as autoreceptors at lateral and medial perforant path synapses. Journal of Neurophysiology 76, 3798–3806. Mutel, V., Adam, G., Chaboz, S., Kemp, J.A., Klingelschmidt, A., Messer, J., Wichmann, J., Woltering, T., Richards, J.G., 1998. Characterization of (2S,2⬘R,3⬘R)-2-(2⬘,3⬘-[3H]-dicarboxycyclopropyl)glycine binding in rat brain. Journal of Neurochemistry 71, 2558–2564. Neki, A., Ohishi, H., Kaneko, T., Shigemoto, R., Nakanishi, S., Mizuno, N., 1996. Pre- and postsynaptic localization of a metabotropic glutamate receptor, mGluR2, in the rat brain: An immunohistochemical study with a monoclonal antibody. Neuroscience Letters 202, 197–200. Ohishi, H., Neki, A., Mizuno, N., 1998. Distribution of a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat and mouse: an immunohistochemical study with a monoclonal antibody. Neuroscience Research 30, 65–82. Ohishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N., 1993a. Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat. Neuroscience 53, 1009–1018. Ohishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N., 1993b. Distribution of the mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ hybridization study. Journal of Comparative Neurology 335, 252–266. Otmakhova, N.A., Lisman, J.E., 1998. Dopamine selectively inhibits the direct cortical pathway to the CA1 hippocampal region. Journal of Neuroscience 19, 1437–1445. Petralia, R.S., Wang, Y.X., Niedzielski, A.S., Wenthold, R.J., 1996. The metabotropic glutamate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynaptic and glial localizations. Neuroscience 71, 949–976. Schaffhauser, H., Richards, J.G., Cartmell, J., Chaboz, S., Kemp, J.A., Klingelschmidt, A., Messer, J., Stadler, H., Woltering, T., Mutel, V., 1998. In vitro binding characteristics of a new selective group II metabotropic glutamate receptor radioligand, [3H] LY354740, in rat brain. Molecular Pharmacology 53, 228–233. Schoepp, D.D., Jane, D.E., Monn, J.A., 1999. Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38, 1431–1476. Shigemoto, R., Kinoshita, A., Wada, E., Nomura, S., Ohishi, H., Takada, M., Flor, P.J., Neki, A., Abe, T., Nakanishi, S., Mizuno, N., 1997. Differential presynaptic localization of metabotropic glutamate receptor subtypes in the rat hippocampus. Journal of Neuroscience 17, 7503–7522. Tamaru, Y., Nomura, S., Mizuno, N., Shigemoto, R., 2001. Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre- and postsynaptic sites. Neuroscience 106, 481–503. Ugolini, A., Bordi, F., 1995. Metabotropic glutamate group II receptors are responsible for the depression of synaptic transmission induced by ACPD in the dentate gyrus. European Journal of Pharmacology 294, 403–410. Yokoi, M., Kobayashi, K., Manabe, T., Takahashi, T., Sakaguchi, I., Katsuura, G., Shigemoto, R., Ohishi, H., Nomura, S., Nakamura, K., Nakao, K., Katsuki, M., Nakanishi, S., 1996. Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2. Science 273, 645–647.