- Email: [email protected]
Selective activation of either mGlu2 or mGlu3 receptors can induce LTD in the amygdala
Neuropharmacology 66 (2013) 196e201
Contents lists available at SciVerse ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Selective activation of either mGlu2 or mGlu3 receptors can induce LTD in the amygdala Sarah J. Lucas a, *, Zuner A. Bortolotto a, Graham L. Collingridge a, b, David Lodge a a b
MRC Centre for Synaptic Plasticity, University of Bristol, Bristol BS8 1TD, UK Department of Brain and Cognitive Sciences, College of Natural Sciences, Seoul National University, Gwanak-gu, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 December 2011 Received in revised form 15 March 2012 Accepted 2 April 2012
Group II metabotropic glutamate (mGlu) receptors are known to induce a long-term depression (LTD) of synaptic transmission in many brain regions including the amygdala. However the roles of the individual receptor subtypes, mGlu2 and mGlu3, in LTD are not well understood. In particular, it is unclear whether activation of mGlu3 receptors is sufficient to induce LTD at synapses in the CNS. In the present study, advantage was taken of a Wistar rat strain not expressing mGlu2 receptors (Ceolin et al., 2011) to investigate the function of mGlu3 receptors in the amygdala. In this preparation, the group II agonist, DCG-IV induced an LTD of the cortical, but not the intra-nuclear, synaptic input to the lateral amygdala. This LTD was concentration dependent and was blocked by the group II mGlu receptor antagonist, LY341495. To investigate further the role of mGlu3 receptors, we used LY395756 (an mGlu2 agonist and mGlu3 antagonist), which acts as a pure mGlu3 receptor antagonist in this rat strain. This compound alone had no effect on basal synaptic transmission, but blocked the LTD induced by DCG-IV. Furthermore, we found that DCG-IV also induces LTD in mGlu2 receptor knock-out (KO) mice to a similar extent as in wild-type mice. This confirms that the activation of mGlu3 receptors alone is sufficient to induce LTD at this amygdala synapse. To address whether mGlu2 activation alone is also sufficient to induce LTD at this synapse we used LY541850 (the active enantiomer of LY395756) in wild-type mice. LY541850 induced a substantial LTD showing that either receptor alone is capable of inducing LTD in this pathway. This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Metabotropic glutamate receptors mGlu3 mGlu2 Lateral amygdala Long-term depression
1. Introduction The eight, G-protein coupled, metabotropic glutamate receptors are divided into 3 groups; group I comprises mGlu1 and mGlu5 receptors, group II comprises mGlu2 and mGlu3 receptors, and group III comprises mGlu4, mGlu6, mGlu7 and mGlu8 receptors (Pin and Duvoisin, 1995). Group II mGlu receptors are believed to act primarily as presynaptic autoreceptors, modulating synaptic transmission (Conn and Pin, 1997; Anwyl, 1999). Group II mGlu receptor agonists have been shown to have therapeutic potential. In initial clinical studies these agonists have been found to have anxiolytic properties in patients with anxiety disorders (Levine et al., 2002; Dunayevich et al., 2008), as well as antipsychotic properties, reducing both the positive and negative symptoms in schizophrenics (Patil et al., 2007). It appears that the * Corresponding author. Present address: Laboratory of Neurobiology, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA. Tel.: þ1 919 316 4819. E-mail address: [email protected] (S.J. Lucas). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2012.04.006
anxiolytic effect of group II mGlu receptor agonists may be mediated in the basolateral amygdala (BLA) complex, as infusion of group II agonists bilaterally into the BLA complex blocks the expression of fear potentiated startle in rats and this effect is blocked by the group II antagonist, LY341495 (Walker et al., 2002; Lin et al., 2005). Group II agonists also disrupt the acquisition of fear conditioning, indicating mGlu2/3 receptor activation can inhibit amygdala dependent learning (Walker et al., 2002; Lin et al., 2005). The activation of group II mGlu receptors has been shown to induce a long-term depression (LTD) of synaptic transmission in various brain regions, including in the thalamic input to the lateral amygdala (Heinbockel and Pape, 2000) and within the BLA complex (Lin et al., 2000; Kaschel et al., 2004). It is therefore suggested that an LTD of synaptic transmission in the amygdala induced by group II mGlu receptor activation may underlie the anxiolytic effects of these receptors (Walker et al., 2002). Due to a relative lack of agonists and antagonists that can differentiate between mGlu2 and mGlu3 receptors, the roles of the individual subtypes are not well understood. Many studies, some using transgenic mice, have indicated a role for mGlu2 and/or mGlu3
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
197
receptors as mediators of reversible depression of synaptic transmission at synapses throughout the CNS (Higgins et al., 2004; Kew et al., 2002; Yokoi et al., 1996; Ceolin et al., 2011; Alexander and Godwin, 2006) but there have been very few studies of their roles in LTD. Activity dependent LTD in the mossy fibre pathway has been shown to involve mGlu2 receptor activation (Yokoi et al.,1996). In the perforant path, however, the mGlu3 receptor agonist, NAAG, gave a slow onset LTD in hippocampal slices (Huang et al., 1999b) and in vivo a high concentration of, NAAG was sufficient to induce an LTD (Poschel and Manahan-Vaughan, 2005). It should be noted, however, that doubt has been cast on the reliability of NAAG as an mGlu3 agonist (Chopra et al., 2009; Fricker et al., 2009). In the BLA, the group II mGlu receptor agonist, LY354740, increased c-fos in mGlu3 receptor knock-out (KO) mice, but not in wild-type or mGlu2 receptor KO mice; indicating that the activation of mGlu3 receptors in the BLA may normally counteract the effects of mGlu2 receptor activation that lead to induction of c-fos (Linden et al., 2006). Muly et al. (2007) proposed, however, that both mGlu2 and mGlu3 receptors may contribute to the reversible depression of synaptic transmission in the BLA but the roles of the individual receptors in LTD in the amygdala has not been investigated. It remains, therefore, unclear whether mGlu2 and mGlu3 receptors play similar roles in long-term depression of synaptic transmission in the amygdala or whether the two receptor subtypes may even have opposing roles. Here we have used LY395756, or its active enantiomer LY541850, which we have recently shown is a selective mGlu2 agonist and mGlu3 antagonist at native receptors in the hippocampus (Ceolin et al., 2011; Hanna et al., 2013), to investigate the roles of the individual group II mGlu receptor subtypes in the amygdala. Using a Han strain of Wistar rats that lacks mGlu2 receptors, as assessed pharmacologically and using western blotting (Ceolin et al., 2011) or mGlu2 KO mice we observed an LTD induced by DCG-IV that was prevented by LY395756. In wild-type mice, LY541850 was able to induce an LTD similar to that induced by DCG-IV. Therefore activation of either the mGlu3 or mGlu2 receptor subtype alone can be sufficient to induce an LTD of synaptic transmission in the cortical input to the lateral amygdala (LA). 2. Methods 2.1. Slice preparation Horizontal amygdala slices (500 mm) were prepared from 5e6 week old Wistar rats obtained from B&K Universal Ltd for field electrophysiology recordings and coronal amygdala slices (400 mm) from 5e6 week old CD-1 mice from Charles River, or 10e13 week old mGlu2 receptor / mice and their wild-type littermates were prepared for whole-cell patch-clamp electrophysiology recordings. The knock-out mice were generated in a CD-1 background by Eli Lilly and Co. Ltd (Linden et al., 2005), and bred at Charles River, UK. All experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986. The animals were killed by cervical dislocation and the brain was removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) which was composed of (in mM); NaCl (124), NaHCO3 (26), KCl (3), NaH2PO4 (1.4), MgSO4 (1), D-glucose (10) and CaCl2 (2) saturated with 95% O2 and 5% CO2. Amygdala slices were cut using a Vibroslicer and placed into a Petri dish of oxygenated aCSF, and allowed to recover for at least 1 hour at room temperature. 2.2. Field recordings Slices were transferred to a humidified interface recording chamber where they were perfused at 2 ml/min with oxygenated aCSF and maintained at 28 1 C. Glass microelectrodes, used to record the extracellular field potentials generated in the LA, were filled with 3 M NaCl and had a resistance of 3e6 MU. A bipolar stimulating electrode, made from twisted chromium/nickel wire (0.05 mm diameter), was placed outside of the external capsule in the entorhinal cortex (see Fig. 1A). Afferents from the entorhinal cortex to the LA have been shown to be preserved in horizontal slice preparations (von Bohlen und Halbach and Albrecht, 2002). In some experiments a second stimulating electrode was placed within the LA to stimulate intranuclearly. Stimuli of 100 ms duration were delivered at 0.033 Hz through each electrode; stimulus strength was adjusted to about 80% of the maximal response and
Fig. 1. DCG-IV, a selective group II agonist, depresses synaptic transmission, in the lateral amygdala. (A) A schematic showing the horizontal slice preparation used including the electrode placement. Example traces are shown for each input, and the addition of Ca2þ free aCSF confirms the measured response is synaptic. (B) Application of 1 mM DCG-IV for 10 min induces an LTD of synaptic transmission in the entorhinal cortex input (5 slices from 4 rats (6 slices from 5 rats to 90 min)) (filled circles), while the fibre volley amplitude remains unchanged (open circles). (C) Application of 1 mM DCG-IV for 10 min induces a reversible depression of synaptic transmission in the intra-nuclear input (5 slices from 4 rats) (filled circles), while the fibre volley amplitude remains unchanged (open circles). The example traces show the mean field potential, for a single experiment, at the time points ‘a’, ‘b’, ‘c’ and ‘d’ indicated on the graph.
was less than 400 mA. When two electrodes were used there was a 15 s separation between the stimulation of each input. Recordings were made using an AxoClamp 2B amplifier (Axon Instruments), and data were acquired and re-analysed using WinLTP software (www.winltp.com; Anderson and Collingridge, 2007). The recordings were digitised at 20 kHz and filtered at 10 kHz. In initial experiments to verify the synaptic nature of the potential being measured, Ca2þ-free aCSF was superfused for 20 min (Fig. 1A). 2.3. Whole-cell recordings For whole-cell patch-clamp recordings slices were held in a recording chamber at room temperature (w21 C) and perfused at 2 ml/min with oxygenated aCSF containing 50 mM picrotoxin. A twisted chromium/nickel wire, bipolar stimulating electrode was placed in the external capsule dorsal to the LA (Inset Fig. 4). Glass
198
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
recording pipettes with a resistance of 2e4 MU were filled with a whole cell solution composed of (in mM) cesium methane sulfonate (130), HEPES (10), EGTA (0.5), Mg-ATP (4), Na-GTP (0.3), QX-314 (5) and NaCl (8). The preparation was visualized using a Nikon Eclipse E600FN microscope and a 40X DIC objective was used to visually identify cells as pyramidal on the basis of having a pyriform cell soma. Recordings were made using a Multiclamp 700B amplifier (Axon instruments) and WinLTP software for data acquisition and re-analysis. Responses were digitised at 20 kHz and filtered at 4 kHz. Cells were used if their resting membrane potential was between 50 and 75 mV. Recordings were made in voltage-clamp mode with cells held at 70 mV. The holding current, input resistance and series resistance were monitored throughout the recording, and experiments were discarded if the series resistance changed by more than 20% during the recording. The stimulation frequency was set to 0.033 Hz, the stimulus duration to 100 ms and an intensity of less than 400 mA was used. All KO mice experiments were performed blind to genotype. 2.4. Analysis
(Fig. 2A). Following each application the peak depression was measured, which showed a significant effect of concentration (one-way repeated measures ANOVA, n ¼ 3, p < 0.01). There was no significant depression with either 100 nM (0 4%, Bonferroni post hoc, p > 0.05) or with 300 nM (9 1%, Bonferroni post hoc, p > 0.05), but as stated previously 1 mM DCG-IV gave a significant depression of the field potential (27 5%, Bonferroni post hoc, p < 0.01). In order to confirm that the observed effect of DCG-IV was due to its selective activation of group II mGlu receptors, LY341495, an orthosteric antagonist selective for group II mGlu receptors at 300 nM, was applied for 30 min before, during and for 30 min after DCG-IV application (Fig. 2B and C). In the entorhinal cortex input there was no significant effect of LY341495 alone after a 30 min
For each individual experiment the responses were averaged every 2 min (i.e. 4 field potentials/EPSCs) and these averaged responses were used to measure the peak amplitude of the response. The responses were measured in WinLTP by taking the average amplitude of the DC baseline (measured in the 2e8 ms prior to stimulation) and comparing it to the peak data point between two post-pulse time points (which were 5 ms apart). The data were normalised relative to the baseline period (30 min for field recordings and 10 min for whole-cell recordings). All graphs show the mean normalised field potential/EPSC amplitude with the error bars giving the standard error of the mean, Fig. 1B and C also shows the mean normalised fibre volley amplitude. Values given are the mean change in amplitude standard error of the mean. For statistical analysis 2 min averages at the points indicated on the graphs (‘a’, ‘b’, ‘c’, ‘d’) were compared using paired t-tests, one- or two-way repeated measures ANOVA, followed by Bonferroni post hoc tests, as appropriate. SigmaStat was used for statistical analysis. Sample size (n) is the number of animals used. 2.5. Compounds 2S,20 R,30 R)-2-(20 ,30 -dicarboxycyclopropyl)glycine (DCG-IV) and (2S)-2-amino-2[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495) were obtained from Tocris Bioscience (Bristol, UK). (1SR,2SR,4RS,5RS,6SR)-2-amino-4methylbicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY395756), and its (1S,2S,4R,5R,6S)active enantiomer (LY541850), were gifts from James Monn, Eli Lilly & Co. Ltd. DCG-IV was dissolved in water, LY341495, LY395756 and LY541850 were dissolved in equivalent NaOH, and all drugs were stored as frozen stock solutions. The drugs were then diluted at least 1:1000 in the aCSF and bath applied in the perfusing aCSF.
3. Results After obtaining at least 30 min of stable recording of field potential amplitude in both the entorhinal cortex input and the intranuclear input, the competitive group II mGlu receptor agonist, DCGIV, was applied at 1 mM for 10 min (Fig. 1B and C). In both inputs, after an initial temporary and variable increase in amplitude, there was a significant inhibitory effect of DCG-IV on the entorhinal cortex input (one-way repeated measures ANOVA, n ¼ 5, p < 0.01) and the intra-nuclear input (one-way repeated measures ANOVA, n ¼ 4, p < 0.01). Thus in the entorhinal input there was a significant depression of synaptic transmission measured 20 min after the end of the DCG-IV application (56 4%, Bonferroni post hoc, p < 0.01, Fig. 1Bc), which was maintained following 60 min of washout of DCG-IV (47 6%, Bonferroni post hoc, p < 0.01, Fig. 1Bd). Similarly, there was a significant inhibition of synaptic transmission by DCG-IV in the intra-nuclear input 20 min after the end of the DCG-IV application (52 10%, Bonferroni post hoc. p < 0.01, Fig. 1Cc). However, in contrast to the entorhinal cortex input, in the intra-nuclear input following 60 min of washout of DCG-IV, there was no significant long-lasting depression (11 8%, Bonferroni post hoc, p > 0.05, Fig. 1Cd). Throughout these DCG-IV application experiments, the amplitude of the fibre volley did not significantly change in either the entorhinal cortex input (paired t-test, n ¼ 4, p > 0.05, Fig.1B) or in the intra-nuclear input (paired t-test, n ¼ 4, p > 0.05, Fig. 1C). To determine the concentration response to DCG-IV, 10 min applications of 100 nM, 300 nM, and 1 mM DCG-IV were applied while recording the entorhinal cortex evoked field potential
Fig. 2. (A) The concentration dependent effects of DCG-IV in the entorhinal cortex input; DCG-IV is applied for 10 min at 100 nM, 300 nM and then 1 mM (n ¼ 3). (B,C) 300 nM LY341495, a selective group II mGlu receptor antagonist, has no effect on synaptic transmission alone. However it does block the DCG-IV induced depression in the entorhinal cortex input (B; n ¼ 6) and in the intra-nuclear input (C; n ¼ 3).
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
application (8 7%), nor was there any depression 20 min after DCG-IV application in the presence of LY341495 (0 10%, one-way repeated measures ANOVA, n ¼ 6, p > 0.05). Similarly, in the intranuclear input there was no significant effect of LY341495 alone (6 6%) or of DCG-IV in the presence of LY341495 (3 4%, one-way repeated measures ANOVA, n ¼ 3, p > 0.05). Because the Wistar rats used in this study are known to lack mGlu2 receptors (Ceolin et al., 2011), the DCG-IV induced LTD is most likely mediated by mGlu3 receptors. To test this conclusion, we used LY395756, a selective mGlu2 receptor agonist and mGlu3 receptor antagonist. LY395756 (10 mM) was applied for 30 min before, during and for 30 min after DCG-IV (1 mM) application (Fig. 3A); these experiments were interleaved with control experiments where DCG-IV was applied alone (Fig. 3B). A two-way repeated measures ANOVA revealed a significant effect of time (p < 0.01), of group (p < 0.05) and a significant interaction between the two conditions (p < 0.01). Firstly, consistent with the absence of mGlu2 receptors, LY395756 applied alone had no significant effect on synaptic transmission (5 3%, n ¼ 5, Fig. 3Ab) compared to the interleaved control experiments (3 4%, n ¼ 5, Fig. 3Bb) (Bonferroni post hoc, p > 0.05). Secondly, in the presence of LY395756, the field potential was unchanged by DCG-IV (1 5%, n ¼ 5, Fig. 3Ac) or slightly increased (13 7%, n ¼ 5, Fig. 3Ad) compared to baseline. These effects were significantly less than in the interleaved control experiments where DCG-IV application in the absence of LY395756 gave an acute depression of 28 5% (Bonferroni post hoc, n ¼ 5, p < 0.01, Fig. 3Bc) and an LTD of 15 9% (Bonferroni post hoc, n ¼ 5, p < 0.01, Fig. 3Bd). Thus, LY395756 was able to antagonize the
Fig. 3. The effect of the mGlu2 receptor agonist and mGlu3 receptor antagonist, LY395756. (A) 10 mM LY395756 has no effect on the field potential amplitude, but does block the DCG-IV induced depression in the entorhinal cortex input (n ¼ 5). (B) Interleaved control experiments show DCG-IV applied alone (n ¼ 5).
199
effects of DCG-IV, presumably due to its action as an mGlu3 receptor antagonist. To further confirm the ability of mGlu3 receptor activation alone to induce LTD, 1 mM DCG-IV was applied for 20 min while stimulating the cortical input and performing whole-cell recordings from pyramidal cells in the LA of mGlu2 receptor KO mice, and wild-type littermates (Fig. 4). In these mice, there was a significant depression induced by DCG-IV (two-way repeated measures ANOVA, p < 0.01). There was a significant acute depression at the end of DCG-IV application in the mGlu2 receptor KO mice (34 5%, n ¼ 9) and in their wild-type littermates (46 8%, n ¼ 6) (Bonferroni post hoc, p < 0.01). Following 50 min of washout in the mGlu2 receptor KO mice there was a significant LTD of 35 12% (n ¼ 9), and in their wild-type littermates an LTD of 30 11% (n ¼ 6) (Bonferroni post hoc, p < 0.01). The magnitude of the acute depression and LTD were not significantly different between the mGlu2 receptor KO mice and their wild-type littermates (two-way repeated measures ANOVA, p > 0.05). In conclusion, these studies using mice have confirmed that it is possible at this synapse to induce LTD via the selective activation of mGlu3 receptors. To examine the role of mGlu2 receptors alone in the induction of LTD in this pathway, we used the active enantiomer of LY395756, LY541850 which we have recently shown using KO mice to be an mGlu2 agonist and mGlu3 antagonist (Hanna et al., 2013). In CD-1 wild-type mice, 1 mM LY541850 for 20 min induced a significant depression which lasted a further 50 min without decay (one-way repeated measures ANOVA, n ¼ 5, p < 0.01; 53 4% during LY541850 versus 57 11% 50 min later Bonferroni post hocs, p < 0.01; Fig. 5A). To confirm that this action was via mGlu2 receptors, we carried out similar experiments on mGlu2 receptor KO mice and their wild-type litter mates. NaOH (1 mM), which did not change the pH of the aCSF, was applied as a vehicle control in mGlu2 receptor KO mice. A two-way repeated measures ANOVA showed there was a significant effect of LY541850 (p < 0.01) and that there was a significant difference between the groups (p < 0.01) (Fig. 5B). In the mGlu2 receptor KO mice while LY541850 gave a very slight depression of synaptic transmission 12 3% (n ¼ 7) this was not significantly different from the vehicle control group (9 6%, n ¼ 5)
Fig. 4. DCG-IV induces an LTD in wild-type and mGlu2 knock-out mice. Inset shows schematic of the coronal slice preparation and electrode placements. Application of 1 mM DCG-IV for 20 min induces a LTD in the cortical input to the lateral amygdala in coronal slices from mGlu2 receptor knock-out mice (n ¼ 9) and their wild-type littermates (n ¼ 6).
200
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
Fig. 5. Selective activation of mGlu2 receptors induces an LTD. (A) 1 mM LY541850, an mGlu2 receptor agonist and mGlu3 receptor antagonist, gives an LTD in wild-type CD-1 mice (n ¼ 5). (B) In mGlu2 receptor knock-out mice the effect of 1 mM LY541850 (n ¼ 7) was no different from vehicle controls (n ¼ 5), while in wild-type littermates LY541850 still induces an LTD (n ¼ 6).
(Bonferroni post hoc, p > 0.05). In the wild-type littermates LY541850 gave a significant depression of 42 7% following 20 min of washout (Bonferroni post hoc, p < 0.01, n ¼ 6). 4. Discussion The primary finding of the present study is that it is possible to induce a form of LTD in the amygdala via the selective activation of either mGlu2 or mGlu3 receptors. In horizontal slices from Wistar rats, our data show that DCG-IV produces an acute depression of synaptic transmission through the selective activation of group II mGlu receptors in both entorhinal cortex and intra-nuclear inputs to the lateral amygdala, and induces a form of LTD in the entorhinal cortex input but not in the intra-nuclear input. Given the full recovery of the DCG-IV depression within 60 min in the intranuclear input, it can be concluded that DCG-IV is being washed out from the tissue, suggesting that there is a bona fide LTD in the entorhinal cortex input rather than DCG-IV remaining bound to the receptors. The reduction of both acute and LTD effects of DCG-IV by LY341495 (300 nM) a selective group II mGlu receptor antagonist (Kingston et al., 1998) identified these effects as mediated by group II mGlu receptors, and confirmed that group II mGlu receptors are not tonically activated in the amygdala (Li et al., 1998; Lin et al., 2000; Heinbockel and Pape, 2000; Neugebauer et al., 1997). In some studies, group II mGlu receptor agonists have shown only a reversible depression of transmission in the hippocampus (Macek et al., 1996; Yokoi et al., 1996), striatum (Lovinger and McCool, 1995) and subthalamic nucleus (Shen and Johnson,
2003). Previous studies of this acute depression have indicated a major role for mGlu2 receptors as inhibitors of hippocampal synaptic transmission (Ceolin et al., 2011; Higgins et al., 2004; Kew et al., 2002; Yokoi et al., 1996). In the present study, we show that mGlu3 receptors are able to exert a profound acute inhibition as well as inducing an LTD. LTD, induced by group II mGlu receptors, has been previously reported within the BLA complex and in the thalamic input to the LA (Heinbockel and Pape, 2000; Lin et al., 2000; Kaschel et al., 2004) and also in the hippocampus (Huang et al., 1999a), prefrontal cortex (Otani et al., 2002), striatum (Kahn et al., 2001) and nucleus accumbens (Robbe et al., 2002). The lack of LTD in the intra-nuclear input is most likely due to different receptor distributions or coupling in the two pathways. Previously it has been shown that there are differences between the intra-nuclear pathway and the external capsule input in terms of the magnitude of activity dependent LTD (Kaschel et al., 2004). Furthermore, the thalamic and cortical inputs to the LA have been shown to respond differently to theta burst stimulation and to group II mGlu receptor agonists (Heinbockel and Pape, 2000). It can therefore be concluded that group II mGlu receptors are capable of mediating LTD at many, but not all, central synapses; there is, however, little evidence identifying the role of the individual group II mGlu receptor subtypes in this form of plasticity. In the present study we specifically used the Han Wistar rat strain, supplied by B&K, in order to rule out mGlu2 receptor involvement. These rats have been shown to lack mGlu2 receptors throughout the brain (Ceolin et al., 2011) and hence it can be asserted that mGlu2 receptors cannot mediate the DCG-IV induced LTD in the present study. In order to test this hypothesis further, we utilized the properties of LY395756, which has been shown, using human recombinant receptors, to be an agonist at mGlu2 receptors and antagonist at mGlu3 receptors (Dominguez et al., 2005). The mGlu2 receptor agonist and mGlu3 receptor antagonist actions of LY395756 have been confirmed in studies using mGlu2 and mGlu3 receptor KO mice (Ceolin et al., 2011). LY395756 has an EC50 of 0.40 mM at mGlu2 receptors and an IC50 of 2.94 mM versus 30 mM 1S,3R-ACPD, at recombinant human receptors (Dominguez et al., 2005), and in rat hippocampal slices has an EC50 of 0.63 mM and an IC50 of 0.66 mM versus 100 nM DCG-IV (Ceolin et al., 2011). In the present study, LY395756 alone did not affect the amplitude of the field potential, which is consistent with the lack of mGlu2 receptors in the B&K Wistar rats used. However, LY395756 blocked both the acute depression and the LTD induced by DCG-IV, providing further evidence that mGlu3 receptor activation alone is sufficient to induce an LTD. Additionally we further confirmed the ability of mGlu3 activation alone to induce LTD, by showing that DCG-IV induces an LTD in the cortical input to the lateral amygdala in mGlu2 receptor KO mice. Previous work has shown that activation of mGlu2 receptors is sufficient to induce LTD at mossy fibre synapses in the hippocampus (Yokoi et al., 1996). However, it has been less clear whether activation of mGlu3 receptors alone can induce LTD. A putative mGlu3 receptor agonist, NAAG, has been found to induce a slowly developing LTD in the dentate gyrus (Huang et al., 1999b; Poschel and Manahan-Vaughan, 2005). In the study by Huang et al. (1999b) the effect of NAAG was mimicked by group II mGlu receptor antagonists (MCPG and EGLU), and doubts have been raised as to whether NAAG acts as a selective mGlu3 receptor agonist (Chopra et al., 2009; Fricker et al., 2009). By using a rat strain and transgenic mice deficient in mGlu2 receptors, we have confirmed that mGlu3 receptor activation alone can be sufficient to induce LTD. It is possible in these mGlu2 receptor deficient rats and mice that mGlu3 receptors are in some way upregulated to play a greater contribution than is normal, although this was not observed in
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
either the Han Wistar rats (Ceolin et al., 2011) or these transgenic mice (Wright et al., 2013). Assessing the contribution of mGlu3 receptors in normal animals requires more selective pharmacological tools. We were, however, able to investigate whether mGlu2 receptors can induce LTD at these synapses in normal animals. The observation that LY541850 was able to induce LTD, comparable to that induced by DCG-IV, in wild-type mice but not in mGlu2 KO mice demonstrates that activation of mGlu2 receptors alone is sufficient to induce LTD at these synapses. In conclusion, using rodents that lack expression of mGlu2 receptors, in conjunction with a compound that acts as a pure mGlu3 receptor antagonist in these preparations, we demonstrate that activation of mGlu3 receptors alone is sufficient to induce LTD at a central synapse. Acknowledgements Funded by the MRC and the Centre for Cognitive Neuroscience (Eli Lilly). SL was a MRC scholar. LY395756, LY541850 and the KO mice were kind gifts from Eli Lilly & Co. Ltd. GLC is a WCU International Scholar supported by the WCU program through the KOSEF funded by the MEST (R31-10089). References Alexander, G.M., Godwin, D.W., 2006. Unique presynaptic and postsynaptic roles of group II metabotropic glutamate receptors in the modulation of thalamic network activity. Neuroscience 141, 501e513. Anderson, W.W., Collingridge, G.L., 2007. Capabilities of the WinLTP data acquisition program extending beyond basic LTP experimental functions. J. Neurosci. Methods 162, 346e356. Anwyl, R., 1999. Metabotropic glutamate receptors: electrophysiological properties and role in plasticity. Brain Res. Rev. 29, 83e120. Ceolin, L., Kantamneni, S., Barker, G.R., Hanna, L., Murray, L., Warburton, E.C., Robinson, E.S., Monn, J.A., Fitzjohn, S.M., Collingridge, G.L., Bortolotto, Z.A., Lodge, D., 2011. Study of novel selective mGlu2 agonist in the temporoammonic input to CA1 neurons reveals reduced mGlu2 receptor expression in a Wistar substrain with an Anxiety-like phenotype. J. Neurosci. 31, 6721e6731. Chopra, M., Yao, Y., Blake, T.J., Hampson, D.R., Johnson, E.C., 2009. The neuroactive peptide N-acetylaspartylglutamate is not an agonist at the metabotropic glutamate receptor subtype 3 of metabotropic glutamate receptor. J. Pharmacol. Exp. Ther. 330, 212e219. Conn, P.J., Pin, J.-P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205e237. Dominguez, C., Prieto, L., Valli, M.J., Massey, S.M., Bures, M., Wright, R.A., Johnson, B.G., Andis, S.L., Kingston, A., Schoepp, D.D., Monn, J.A., 2005. Methyl substitution of 2-aminobicyclo[3.1.0]hexane 2,6-dicarboxylate (LY354740) determines functional activity at metabotropic glutamate receptors: identification of a subtype selective mGlu2 receptor agonist. J. Med. Chem. 48, 3605e3612. Dunayevich, E., Erickson, J., Levine, L., Landbloom, R., Schoepp, D.D., Tollefson, G.D., 2008. Efficacy and tolerability of an mGlu2/3 agonist in the treatment of generalised anxiety disorder. Neuropsychopharmacology 33, 1603e1610. Fricker, A.C., Mok, M.H., de la Flor, R., Shah, A.J., Woolley, M., Dawson, L.A., Kew, J.N., 2009. Effects of N-acetylaspartylglutamate (NAAG) at group II mGluRs and NMDAR. Neuropharmacology 56, 1060e1067. Hanna, L., Ceolin, L., Lucas, S., Monn, J., Johnson, B., Collingridge, G., Bortolotto, Z., Lodge, D., 2013. Differentiating the roles of mGlu2 and mGlu3 receptors using LY541850, an mGlu2 agonist / mGlu3 antagonist. Neuropharmacology 66, 114e121. Heinbockel, T., Pape, H.-C., 2000. Input-specific long-term depression in the lateral amygdala evoked by theta frequency stimulation. J. Neurosci. 20, 1e5. Higgins, G.A., Ballard, T.M., Kew, J.N.C., Richards, J.G., Kemp, J.A., Adam, G., Woltering, T., Nakanishi, S., Mutel, V., 2004. Pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent. Neuropharmacology 46, 907e917. Huang, L., Killbride, J., Rowan, M.J., Anwyl, R., 1999a. Activation of mGluRII induces LTD via activation of protein kinase A and protein kinase C in the dentate gyrus of the hippocampus in vitro. Neuropharmacology 38, 73e83. Huang, L., Rowan, M.J., Anwyl, R., 1999b. Induction of long-lasting depression by (þ)-alpha-methyl-4-carboxyphenylglycine and other group II mGlu receptor ligands in the dentate gyrus of the hippocampus in vitro. Eur. J. Pharmacol. 366, 151e158.
201
Kahn, L., Alonso, G., Robbe, D., Bockaert, J., Manzoni, O.J., 2001. Group 2 metabotropic glutamate receptors induced long term depression in mouse striatal slices. Neurosci. Lett. 316, 178e182. Kaschel, T., Schubert, M., Albrecht, D., 2004. Long-term depression in horizontal slices of the rat lateral amygdala. Synapse 53, 141e150. Kew, J.N.C., Pflimlin, M.-C., Kemp, J.A., Mutel, V., 2002. Differential regulation of synaptic transmission by mGlu2 and mGlu3 at the perforant path inputs to the dentate gyrus and CA1 revealed in mGlu2 / mice. Neuropharmacology 43, 215e221. Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors. Neuropharmacology 37, 1e12. Levine, L., Gaydos, B., Sheehan, D., Goddard, A., Feighner, J., Potter, W.Z., 2002. The mGlu2/3 receptor agonist, LY354740, reduces panic anxiety induced by a CO2 challenge in patients diagnosed with panic disorder. Neuropharmacology 43, 294e295. Li, H., Weiss, S.R.B., Chuang, D.-C., Post, R.M., Rogawski, M.A., 1998. Bidirectional synaptic plasticity in the rat basolateral amygdala: characterization of an activity-dependent switch sensitive to the presynaptic metabotropic glutamate receptor antagonist 2S-a-ethylglutamic acid. J. Neurosci. 18, 1662e1670. Lin, H.-C., Lee, C.-C., Huang, Y.-C., Wang, S.-J., Gean, P.-W., 2005. Activation of group II metabotropic glutamate receptors induces depotentiation in amygdala slices and reduces fear potentiated startle in rats. Learn Mem. 12, 130e137. Lin, H.-C., Wang, S.-J., Luo, M.-Z., Gean, P.-W., 2000. Activation of group II metabotropic glutamate receptors induces long-term depression of synaptic transmission in the rat amygdala. J. Neurosci. 20, 9017e9024. Linden, A.-M., Shannon, H., Baez, M., Yu, J.L., Koester, A., Schoepp, D.D., 2005. Anxiolytic-like activity of the mGLU2/3 receptor agonist LY354740 in the elevated plus maze test is disrupted in metabotropic glutamate receptor 2 and 3 knock-out mice. Psychopharmacology 179, 284e291. Linden, A.-M., Baez, M., Bergeron, M., Schoepp, D.D., 2006. Effects of mGlu2 and mGlu3 receptor deletions on mGlu2/3 receptor agonist (LY354740)-induced brain c-Fos expression: specific roles for mGlu2 in the amygdala and subcortical nuclei, and mGlu3 in the hippocampus. Neuropharmacology 51, 213e228. Lovinger, D.M., McCool, B.A., 1995. Metabotropic glutamate receptor-mediated presynaptic depression at corticostriatal synapses involves mGluR2 or 3. J. Neurophysiol. 73, 1076e1083. Macek, T.A., Winder, D.G., Gereau, R.W.T., Ladd, C.O., Conn, P.J., 1996. Differential involvement of group II and group III mGluRs as autoreceptors at lateral and medial perforant path synapses. J. Neurophysiol. 76, 3798e3806. Muly, E.C., Mania, I., Guo, J.-D., Rainnie, D.G., 2007. Group II metabotropic glutamate receptors in anxiety circuitry: correspondence of physiological response and subcellular distribution. J. Comp. Neurol. 505, 682e700. Neugebauer, V., Keele, N.B., Shinnick-Gallagher, P., 1997. Epileptogenesis in vivo enhances the sensitivity of inhibitory presynaptic metabotropic glutamate receptors in basolateral amygdala neurons in vitro. J. Neurosci. 17, 983e995. Otani, S., Daniel, H., Takita, M., Crepel, F., 2002. Long-term depression induced by postsynaptic group II metabotropic glutamate receptors linked to phospholipase C and intracellular calcium rises in rat prefrontal cortex. J. Neurosci. 22, 3434e3444. Patil, S.T., Zhang, L., Martenyi, F., Lowe, S.L., Jackson, K.A., Andreev, B.V., Avedisova, A.S., Bardenstein, L.M., Gurovich, I.Y., Morozova, M.A., Mosolov, S.N., Neznanov, N.G., Reznik, A.M., Smulevich, A.B., Tochilov, V.A., Johnson, B.G., Monn, J.A., Schoepp, D.D., 2007. Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized Phase 2 clinical trial. Nat. Med. 13, 1102e1107. Pin, J.-P., Duvoisin, R., 1995. Review: neurotransmitter receptors I. Neuropharmacology 34, 1e26. Poschel, B., Manahan-Vaughan, D., 2005. Group II mGluR-induced long term depression in the dentate gyrus in vivo is NMDA receptor-independent and does not require protein synthesis. Neuropharmacology 49, 1e12. Robbe, D., Alonso, G., Chaumont, S., Bockhaert, J., Manzoni, O.J., 2002. Role of p/q-Ca2þ channels in metabotropic glutamate receptor 2/3-dependent presynaptic longterm depression at nucleus accumbens synapses. J. Neurosci. 22, 4346e4356. Shen, K.Z., Johnson, S.W., 2003. Group II metabotropic glutamate receptor modulation of excitatory transmission in rat subthalamic nucleus. J. Physiol. 553, 489e496. von Bohlen und Halbach, O., Albrecht, D., 2002. Reciprocal connections of the hippocampal area CA1, the lateral nucleus of the amygdala and cortical areas in a combined horizontal slice preparation. Neurosci. Res. 44, 91e100. Walker, D.L., Rattiner, L.M., Davis, M., 2002. Group II metabotropic glutamate receptors within the amygdala regulate fear as assessed with potentiated startle in rats. Behav. Neurosci. 116, 1075e1083. Wright, R.A., Johnson, B.G., Zhang, C., Salhoff, C., Kingston, A.E., Calligaro, D.O., Monn, J.A., Schoepp, D.D., Marek, G.J., 2013. CNS distribution of metabotropic glutamate 2 and 3 receptors: transgenic mice and [(3)H]LY459477 autoradiography. Neuropharmacology 66, 89e98. 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, 645e647.
Contents lists available at SciVerse ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Selective activation of either mGlu2 or mGlu3 receptors can induce LTD in the amygdala Sarah J. Lucas a, *, Zuner A. Bortolotto a, Graham L. Collingridge a, b, David Lodge a a b
MRC Centre for Synaptic Plasticity, University of Bristol, Bristol BS8 1TD, UK Department of Brain and Cognitive Sciences, College of Natural Sciences, Seoul National University, Gwanak-gu, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 December 2011 Received in revised form 15 March 2012 Accepted 2 April 2012
Group II metabotropic glutamate (mGlu) receptors are known to induce a long-term depression (LTD) of synaptic transmission in many brain regions including the amygdala. However the roles of the individual receptor subtypes, mGlu2 and mGlu3, in LTD are not well understood. In particular, it is unclear whether activation of mGlu3 receptors is sufficient to induce LTD at synapses in the CNS. In the present study, advantage was taken of a Wistar rat strain not expressing mGlu2 receptors (Ceolin et al., 2011) to investigate the function of mGlu3 receptors in the amygdala. In this preparation, the group II agonist, DCG-IV induced an LTD of the cortical, but not the intra-nuclear, synaptic input to the lateral amygdala. This LTD was concentration dependent and was blocked by the group II mGlu receptor antagonist, LY341495. To investigate further the role of mGlu3 receptors, we used LY395756 (an mGlu2 agonist and mGlu3 antagonist), which acts as a pure mGlu3 receptor antagonist in this rat strain. This compound alone had no effect on basal synaptic transmission, but blocked the LTD induced by DCG-IV. Furthermore, we found that DCG-IV also induces LTD in mGlu2 receptor knock-out (KO) mice to a similar extent as in wild-type mice. This confirms that the activation of mGlu3 receptors alone is sufficient to induce LTD at this amygdala synapse. To address whether mGlu2 activation alone is also sufficient to induce LTD at this synapse we used LY541850 (the active enantiomer of LY395756) in wild-type mice. LY541850 induced a substantial LTD showing that either receptor alone is capable of inducing LTD in this pathway. This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Metabotropic glutamate receptors mGlu3 mGlu2 Lateral amygdala Long-term depression
1. Introduction The eight, G-protein coupled, metabotropic glutamate receptors are divided into 3 groups; group I comprises mGlu1 and mGlu5 receptors, group II comprises mGlu2 and mGlu3 receptors, and group III comprises mGlu4, mGlu6, mGlu7 and mGlu8 receptors (Pin and Duvoisin, 1995). Group II mGlu receptors are believed to act primarily as presynaptic autoreceptors, modulating synaptic transmission (Conn and Pin, 1997; Anwyl, 1999). Group II mGlu receptor agonists have been shown to have therapeutic potential. In initial clinical studies these agonists have been found to have anxiolytic properties in patients with anxiety disorders (Levine et al., 2002; Dunayevich et al., 2008), as well as antipsychotic properties, reducing both the positive and negative symptoms in schizophrenics (Patil et al., 2007). It appears that the * Corresponding author. Present address: Laboratory of Neurobiology, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA. Tel.: þ1 919 316 4819. E-mail address: [email protected] (S.J. Lucas). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2012.04.006
anxiolytic effect of group II mGlu receptor agonists may be mediated in the basolateral amygdala (BLA) complex, as infusion of group II agonists bilaterally into the BLA complex blocks the expression of fear potentiated startle in rats and this effect is blocked by the group II antagonist, LY341495 (Walker et al., 2002; Lin et al., 2005). Group II agonists also disrupt the acquisition of fear conditioning, indicating mGlu2/3 receptor activation can inhibit amygdala dependent learning (Walker et al., 2002; Lin et al., 2005). The activation of group II mGlu receptors has been shown to induce a long-term depression (LTD) of synaptic transmission in various brain regions, including in the thalamic input to the lateral amygdala (Heinbockel and Pape, 2000) and within the BLA complex (Lin et al., 2000; Kaschel et al., 2004). It is therefore suggested that an LTD of synaptic transmission in the amygdala induced by group II mGlu receptor activation may underlie the anxiolytic effects of these receptors (Walker et al., 2002). Due to a relative lack of agonists and antagonists that can differentiate between mGlu2 and mGlu3 receptors, the roles of the individual subtypes are not well understood. Many studies, some using transgenic mice, have indicated a role for mGlu2 and/or mGlu3
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
197
receptors as mediators of reversible depression of synaptic transmission at synapses throughout the CNS (Higgins et al., 2004; Kew et al., 2002; Yokoi et al., 1996; Ceolin et al., 2011; Alexander and Godwin, 2006) but there have been very few studies of their roles in LTD. Activity dependent LTD in the mossy fibre pathway has been shown to involve mGlu2 receptor activation (Yokoi et al.,1996). In the perforant path, however, the mGlu3 receptor agonist, NAAG, gave a slow onset LTD in hippocampal slices (Huang et al., 1999b) and in vivo a high concentration of, NAAG was sufficient to induce an LTD (Poschel and Manahan-Vaughan, 2005). It should be noted, however, that doubt has been cast on the reliability of NAAG as an mGlu3 agonist (Chopra et al., 2009; Fricker et al., 2009). In the BLA, the group II mGlu receptor agonist, LY354740, increased c-fos in mGlu3 receptor knock-out (KO) mice, but not in wild-type or mGlu2 receptor KO mice; indicating that the activation of mGlu3 receptors in the BLA may normally counteract the effects of mGlu2 receptor activation that lead to induction of c-fos (Linden et al., 2006). Muly et al. (2007) proposed, however, that both mGlu2 and mGlu3 receptors may contribute to the reversible depression of synaptic transmission in the BLA but the roles of the individual receptors in LTD in the amygdala has not been investigated. It remains, therefore, unclear whether mGlu2 and mGlu3 receptors play similar roles in long-term depression of synaptic transmission in the amygdala or whether the two receptor subtypes may even have opposing roles. Here we have used LY395756, or its active enantiomer LY541850, which we have recently shown is a selective mGlu2 agonist and mGlu3 antagonist at native receptors in the hippocampus (Ceolin et al., 2011; Hanna et al., 2013), to investigate the roles of the individual group II mGlu receptor subtypes in the amygdala. Using a Han strain of Wistar rats that lacks mGlu2 receptors, as assessed pharmacologically and using western blotting (Ceolin et al., 2011) or mGlu2 KO mice we observed an LTD induced by DCG-IV that was prevented by LY395756. In wild-type mice, LY541850 was able to induce an LTD similar to that induced by DCG-IV. Therefore activation of either the mGlu3 or mGlu2 receptor subtype alone can be sufficient to induce an LTD of synaptic transmission in the cortical input to the lateral amygdala (LA). 2. Methods 2.1. Slice preparation Horizontal amygdala slices (500 mm) were prepared from 5e6 week old Wistar rats obtained from B&K Universal Ltd for field electrophysiology recordings and coronal amygdala slices (400 mm) from 5e6 week old CD-1 mice from Charles River, or 10e13 week old mGlu2 receptor / mice and their wild-type littermates were prepared for whole-cell patch-clamp electrophysiology recordings. The knock-out mice were generated in a CD-1 background by Eli Lilly and Co. Ltd (Linden et al., 2005), and bred at Charles River, UK. All experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986. The animals were killed by cervical dislocation and the brain was removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) which was composed of (in mM); NaCl (124), NaHCO3 (26), KCl (3), NaH2PO4 (1.4), MgSO4 (1), D-glucose (10) and CaCl2 (2) saturated with 95% O2 and 5% CO2. Amygdala slices were cut using a Vibroslicer and placed into a Petri dish of oxygenated aCSF, and allowed to recover for at least 1 hour at room temperature. 2.2. Field recordings Slices were transferred to a humidified interface recording chamber where they were perfused at 2 ml/min with oxygenated aCSF and maintained at 28 1 C. Glass microelectrodes, used to record the extracellular field potentials generated in the LA, were filled with 3 M NaCl and had a resistance of 3e6 MU. A bipolar stimulating electrode, made from twisted chromium/nickel wire (0.05 mm diameter), was placed outside of the external capsule in the entorhinal cortex (see Fig. 1A). Afferents from the entorhinal cortex to the LA have been shown to be preserved in horizontal slice preparations (von Bohlen und Halbach and Albrecht, 2002). In some experiments a second stimulating electrode was placed within the LA to stimulate intranuclearly. Stimuli of 100 ms duration were delivered at 0.033 Hz through each electrode; stimulus strength was adjusted to about 80% of the maximal response and
Fig. 1. DCG-IV, a selective group II agonist, depresses synaptic transmission, in the lateral amygdala. (A) A schematic showing the horizontal slice preparation used including the electrode placement. Example traces are shown for each input, and the addition of Ca2þ free aCSF confirms the measured response is synaptic. (B) Application of 1 mM DCG-IV for 10 min induces an LTD of synaptic transmission in the entorhinal cortex input (5 slices from 4 rats (6 slices from 5 rats to 90 min)) (filled circles), while the fibre volley amplitude remains unchanged (open circles). (C) Application of 1 mM DCG-IV for 10 min induces a reversible depression of synaptic transmission in the intra-nuclear input (5 slices from 4 rats) (filled circles), while the fibre volley amplitude remains unchanged (open circles). The example traces show the mean field potential, for a single experiment, at the time points ‘a’, ‘b’, ‘c’ and ‘d’ indicated on the graph.
was less than 400 mA. When two electrodes were used there was a 15 s separation between the stimulation of each input. Recordings were made using an AxoClamp 2B amplifier (Axon Instruments), and data were acquired and re-analysed using WinLTP software (www.winltp.com; Anderson and Collingridge, 2007). The recordings were digitised at 20 kHz and filtered at 10 kHz. In initial experiments to verify the synaptic nature of the potential being measured, Ca2þ-free aCSF was superfused for 20 min (Fig. 1A). 2.3. Whole-cell recordings For whole-cell patch-clamp recordings slices were held in a recording chamber at room temperature (w21 C) and perfused at 2 ml/min with oxygenated aCSF containing 50 mM picrotoxin. A twisted chromium/nickel wire, bipolar stimulating electrode was placed in the external capsule dorsal to the LA (Inset Fig. 4). Glass
198
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
recording pipettes with a resistance of 2e4 MU were filled with a whole cell solution composed of (in mM) cesium methane sulfonate (130), HEPES (10), EGTA (0.5), Mg-ATP (4), Na-GTP (0.3), QX-314 (5) and NaCl (8). The preparation was visualized using a Nikon Eclipse E600FN microscope and a 40X DIC objective was used to visually identify cells as pyramidal on the basis of having a pyriform cell soma. Recordings were made using a Multiclamp 700B amplifier (Axon instruments) and WinLTP software for data acquisition and re-analysis. Responses were digitised at 20 kHz and filtered at 4 kHz. Cells were used if their resting membrane potential was between 50 and 75 mV. Recordings were made in voltage-clamp mode with cells held at 70 mV. The holding current, input resistance and series resistance were monitored throughout the recording, and experiments were discarded if the series resistance changed by more than 20% during the recording. The stimulation frequency was set to 0.033 Hz, the stimulus duration to 100 ms and an intensity of less than 400 mA was used. All KO mice experiments were performed blind to genotype. 2.4. Analysis
(Fig. 2A). Following each application the peak depression was measured, which showed a significant effect of concentration (one-way repeated measures ANOVA, n ¼ 3, p < 0.01). There was no significant depression with either 100 nM (0 4%, Bonferroni post hoc, p > 0.05) or with 300 nM (9 1%, Bonferroni post hoc, p > 0.05), but as stated previously 1 mM DCG-IV gave a significant depression of the field potential (27 5%, Bonferroni post hoc, p < 0.01). In order to confirm that the observed effect of DCG-IV was due to its selective activation of group II mGlu receptors, LY341495, an orthosteric antagonist selective for group II mGlu receptors at 300 nM, was applied for 30 min before, during and for 30 min after DCG-IV application (Fig. 2B and C). In the entorhinal cortex input there was no significant effect of LY341495 alone after a 30 min
For each individual experiment the responses were averaged every 2 min (i.e. 4 field potentials/EPSCs) and these averaged responses were used to measure the peak amplitude of the response. The responses were measured in WinLTP by taking the average amplitude of the DC baseline (measured in the 2e8 ms prior to stimulation) and comparing it to the peak data point between two post-pulse time points (which were 5 ms apart). The data were normalised relative to the baseline period (30 min for field recordings and 10 min for whole-cell recordings). All graphs show the mean normalised field potential/EPSC amplitude with the error bars giving the standard error of the mean, Fig. 1B and C also shows the mean normalised fibre volley amplitude. Values given are the mean change in amplitude standard error of the mean. For statistical analysis 2 min averages at the points indicated on the graphs (‘a’, ‘b’, ‘c’, ‘d’) were compared using paired t-tests, one- or two-way repeated measures ANOVA, followed by Bonferroni post hoc tests, as appropriate. SigmaStat was used for statistical analysis. Sample size (n) is the number of animals used. 2.5. Compounds 2S,20 R,30 R)-2-(20 ,30 -dicarboxycyclopropyl)glycine (DCG-IV) and (2S)-2-amino-2[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495) were obtained from Tocris Bioscience (Bristol, UK). (1SR,2SR,4RS,5RS,6SR)-2-amino-4methylbicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY395756), and its (1S,2S,4R,5R,6S)active enantiomer (LY541850), were gifts from James Monn, Eli Lilly & Co. Ltd. DCG-IV was dissolved in water, LY341495, LY395756 and LY541850 were dissolved in equivalent NaOH, and all drugs were stored as frozen stock solutions. The drugs were then diluted at least 1:1000 in the aCSF and bath applied in the perfusing aCSF.
3. Results After obtaining at least 30 min of stable recording of field potential amplitude in both the entorhinal cortex input and the intranuclear input, the competitive group II mGlu receptor agonist, DCGIV, was applied at 1 mM for 10 min (Fig. 1B and C). In both inputs, after an initial temporary and variable increase in amplitude, there was a significant inhibitory effect of DCG-IV on the entorhinal cortex input (one-way repeated measures ANOVA, n ¼ 5, p < 0.01) and the intra-nuclear input (one-way repeated measures ANOVA, n ¼ 4, p < 0.01). Thus in the entorhinal input there was a significant depression of synaptic transmission measured 20 min after the end of the DCG-IV application (56 4%, Bonferroni post hoc, p < 0.01, Fig. 1Bc), which was maintained following 60 min of washout of DCG-IV (47 6%, Bonferroni post hoc, p < 0.01, Fig. 1Bd). Similarly, there was a significant inhibition of synaptic transmission by DCG-IV in the intra-nuclear input 20 min after the end of the DCG-IV application (52 10%, Bonferroni post hoc. p < 0.01, Fig. 1Cc). However, in contrast to the entorhinal cortex input, in the intra-nuclear input following 60 min of washout of DCG-IV, there was no significant long-lasting depression (11 8%, Bonferroni post hoc, p > 0.05, Fig. 1Cd). Throughout these DCG-IV application experiments, the amplitude of the fibre volley did not significantly change in either the entorhinal cortex input (paired t-test, n ¼ 4, p > 0.05, Fig.1B) or in the intra-nuclear input (paired t-test, n ¼ 4, p > 0.05, Fig. 1C). To determine the concentration response to DCG-IV, 10 min applications of 100 nM, 300 nM, and 1 mM DCG-IV were applied while recording the entorhinal cortex evoked field potential
Fig. 2. (A) The concentration dependent effects of DCG-IV in the entorhinal cortex input; DCG-IV is applied for 10 min at 100 nM, 300 nM and then 1 mM (n ¼ 3). (B,C) 300 nM LY341495, a selective group II mGlu receptor antagonist, has no effect on synaptic transmission alone. However it does block the DCG-IV induced depression in the entorhinal cortex input (B; n ¼ 6) and in the intra-nuclear input (C; n ¼ 3).
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
application (8 7%), nor was there any depression 20 min after DCG-IV application in the presence of LY341495 (0 10%, one-way repeated measures ANOVA, n ¼ 6, p > 0.05). Similarly, in the intranuclear input there was no significant effect of LY341495 alone (6 6%) or of DCG-IV in the presence of LY341495 (3 4%, one-way repeated measures ANOVA, n ¼ 3, p > 0.05). Because the Wistar rats used in this study are known to lack mGlu2 receptors (Ceolin et al., 2011), the DCG-IV induced LTD is most likely mediated by mGlu3 receptors. To test this conclusion, we used LY395756, a selective mGlu2 receptor agonist and mGlu3 receptor antagonist. LY395756 (10 mM) was applied for 30 min before, during and for 30 min after DCG-IV (1 mM) application (Fig. 3A); these experiments were interleaved with control experiments where DCG-IV was applied alone (Fig. 3B). A two-way repeated measures ANOVA revealed a significant effect of time (p < 0.01), of group (p < 0.05) and a significant interaction between the two conditions (p < 0.01). Firstly, consistent with the absence of mGlu2 receptors, LY395756 applied alone had no significant effect on synaptic transmission (5 3%, n ¼ 5, Fig. 3Ab) compared to the interleaved control experiments (3 4%, n ¼ 5, Fig. 3Bb) (Bonferroni post hoc, p > 0.05). Secondly, in the presence of LY395756, the field potential was unchanged by DCG-IV (1 5%, n ¼ 5, Fig. 3Ac) or slightly increased (13 7%, n ¼ 5, Fig. 3Ad) compared to baseline. These effects were significantly less than in the interleaved control experiments where DCG-IV application in the absence of LY395756 gave an acute depression of 28 5% (Bonferroni post hoc, n ¼ 5, p < 0.01, Fig. 3Bc) and an LTD of 15 9% (Bonferroni post hoc, n ¼ 5, p < 0.01, Fig. 3Bd). Thus, LY395756 was able to antagonize the
Fig. 3. The effect of the mGlu2 receptor agonist and mGlu3 receptor antagonist, LY395756. (A) 10 mM LY395756 has no effect on the field potential amplitude, but does block the DCG-IV induced depression in the entorhinal cortex input (n ¼ 5). (B) Interleaved control experiments show DCG-IV applied alone (n ¼ 5).
199
effects of DCG-IV, presumably due to its action as an mGlu3 receptor antagonist. To further confirm the ability of mGlu3 receptor activation alone to induce LTD, 1 mM DCG-IV was applied for 20 min while stimulating the cortical input and performing whole-cell recordings from pyramidal cells in the LA of mGlu2 receptor KO mice, and wild-type littermates (Fig. 4). In these mice, there was a significant depression induced by DCG-IV (two-way repeated measures ANOVA, p < 0.01). There was a significant acute depression at the end of DCG-IV application in the mGlu2 receptor KO mice (34 5%, n ¼ 9) and in their wild-type littermates (46 8%, n ¼ 6) (Bonferroni post hoc, p < 0.01). Following 50 min of washout in the mGlu2 receptor KO mice there was a significant LTD of 35 12% (n ¼ 9), and in their wild-type littermates an LTD of 30 11% (n ¼ 6) (Bonferroni post hoc, p < 0.01). The magnitude of the acute depression and LTD were not significantly different between the mGlu2 receptor KO mice and their wild-type littermates (two-way repeated measures ANOVA, p > 0.05). In conclusion, these studies using mice have confirmed that it is possible at this synapse to induce LTD via the selective activation of mGlu3 receptors. To examine the role of mGlu2 receptors alone in the induction of LTD in this pathway, we used the active enantiomer of LY395756, LY541850 which we have recently shown using KO mice to be an mGlu2 agonist and mGlu3 antagonist (Hanna et al., 2013). In CD-1 wild-type mice, 1 mM LY541850 for 20 min induced a significant depression which lasted a further 50 min without decay (one-way repeated measures ANOVA, n ¼ 5, p < 0.01; 53 4% during LY541850 versus 57 11% 50 min later Bonferroni post hocs, p < 0.01; Fig. 5A). To confirm that this action was via mGlu2 receptors, we carried out similar experiments on mGlu2 receptor KO mice and their wild-type litter mates. NaOH (1 mM), which did not change the pH of the aCSF, was applied as a vehicle control in mGlu2 receptor KO mice. A two-way repeated measures ANOVA showed there was a significant effect of LY541850 (p < 0.01) and that there was a significant difference between the groups (p < 0.01) (Fig. 5B). In the mGlu2 receptor KO mice while LY541850 gave a very slight depression of synaptic transmission 12 3% (n ¼ 7) this was not significantly different from the vehicle control group (9 6%, n ¼ 5)
Fig. 4. DCG-IV induces an LTD in wild-type and mGlu2 knock-out mice. Inset shows schematic of the coronal slice preparation and electrode placements. Application of 1 mM DCG-IV for 20 min induces a LTD in the cortical input to the lateral amygdala in coronal slices from mGlu2 receptor knock-out mice (n ¼ 9) and their wild-type littermates (n ¼ 6).
200
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
Fig. 5. Selective activation of mGlu2 receptors induces an LTD. (A) 1 mM LY541850, an mGlu2 receptor agonist and mGlu3 receptor antagonist, gives an LTD in wild-type CD-1 mice (n ¼ 5). (B) In mGlu2 receptor knock-out mice the effect of 1 mM LY541850 (n ¼ 7) was no different from vehicle controls (n ¼ 5), while in wild-type littermates LY541850 still induces an LTD (n ¼ 6).
(Bonferroni post hoc, p > 0.05). In the wild-type littermates LY541850 gave a significant depression of 42 7% following 20 min of washout (Bonferroni post hoc, p < 0.01, n ¼ 6). 4. Discussion The primary finding of the present study is that it is possible to induce a form of LTD in the amygdala via the selective activation of either mGlu2 or mGlu3 receptors. In horizontal slices from Wistar rats, our data show that DCG-IV produces an acute depression of synaptic transmission through the selective activation of group II mGlu receptors in both entorhinal cortex and intra-nuclear inputs to the lateral amygdala, and induces a form of LTD in the entorhinal cortex input but not in the intra-nuclear input. Given the full recovery of the DCG-IV depression within 60 min in the intranuclear input, it can be concluded that DCG-IV is being washed out from the tissue, suggesting that there is a bona fide LTD in the entorhinal cortex input rather than DCG-IV remaining bound to the receptors. The reduction of both acute and LTD effects of DCG-IV by LY341495 (300 nM) a selective group II mGlu receptor antagonist (Kingston et al., 1998) identified these effects as mediated by group II mGlu receptors, and confirmed that group II mGlu receptors are not tonically activated in the amygdala (Li et al., 1998; Lin et al., 2000; Heinbockel and Pape, 2000; Neugebauer et al., 1997). In some studies, group II mGlu receptor agonists have shown only a reversible depression of transmission in the hippocampus (Macek et al., 1996; Yokoi et al., 1996), striatum (Lovinger and McCool, 1995) and subthalamic nucleus (Shen and Johnson,
2003). Previous studies of this acute depression have indicated a major role for mGlu2 receptors as inhibitors of hippocampal synaptic transmission (Ceolin et al., 2011; Higgins et al., 2004; Kew et al., 2002; Yokoi et al., 1996). In the present study, we show that mGlu3 receptors are able to exert a profound acute inhibition as well as inducing an LTD. LTD, induced by group II mGlu receptors, has been previously reported within the BLA complex and in the thalamic input to the LA (Heinbockel and Pape, 2000; Lin et al., 2000; Kaschel et al., 2004) and also in the hippocampus (Huang et al., 1999a), prefrontal cortex (Otani et al., 2002), striatum (Kahn et al., 2001) and nucleus accumbens (Robbe et al., 2002). The lack of LTD in the intra-nuclear input is most likely due to different receptor distributions or coupling in the two pathways. Previously it has been shown that there are differences between the intra-nuclear pathway and the external capsule input in terms of the magnitude of activity dependent LTD (Kaschel et al., 2004). Furthermore, the thalamic and cortical inputs to the LA have been shown to respond differently to theta burst stimulation and to group II mGlu receptor agonists (Heinbockel and Pape, 2000). It can therefore be concluded that group II mGlu receptors are capable of mediating LTD at many, but not all, central synapses; there is, however, little evidence identifying the role of the individual group II mGlu receptor subtypes in this form of plasticity. In the present study we specifically used the Han Wistar rat strain, supplied by B&K, in order to rule out mGlu2 receptor involvement. These rats have been shown to lack mGlu2 receptors throughout the brain (Ceolin et al., 2011) and hence it can be asserted that mGlu2 receptors cannot mediate the DCG-IV induced LTD in the present study. In order to test this hypothesis further, we utilized the properties of LY395756, which has been shown, using human recombinant receptors, to be an agonist at mGlu2 receptors and antagonist at mGlu3 receptors (Dominguez et al., 2005). The mGlu2 receptor agonist and mGlu3 receptor antagonist actions of LY395756 have been confirmed in studies using mGlu2 and mGlu3 receptor KO mice (Ceolin et al., 2011). LY395756 has an EC50 of 0.40 mM at mGlu2 receptors and an IC50 of 2.94 mM versus 30 mM 1S,3R-ACPD, at recombinant human receptors (Dominguez et al., 2005), and in rat hippocampal slices has an EC50 of 0.63 mM and an IC50 of 0.66 mM versus 100 nM DCG-IV (Ceolin et al., 2011). In the present study, LY395756 alone did not affect the amplitude of the field potential, which is consistent with the lack of mGlu2 receptors in the B&K Wistar rats used. However, LY395756 blocked both the acute depression and the LTD induced by DCG-IV, providing further evidence that mGlu3 receptor activation alone is sufficient to induce an LTD. Additionally we further confirmed the ability of mGlu3 activation alone to induce LTD, by showing that DCG-IV induces an LTD in the cortical input to the lateral amygdala in mGlu2 receptor KO mice. Previous work has shown that activation of mGlu2 receptors is sufficient to induce LTD at mossy fibre synapses in the hippocampus (Yokoi et al., 1996). However, it has been less clear whether activation of mGlu3 receptors alone can induce LTD. A putative mGlu3 receptor agonist, NAAG, has been found to induce a slowly developing LTD in the dentate gyrus (Huang et al., 1999b; Poschel and Manahan-Vaughan, 2005). In the study by Huang et al. (1999b) the effect of NAAG was mimicked by group II mGlu receptor antagonists (MCPG and EGLU), and doubts have been raised as to whether NAAG acts as a selective mGlu3 receptor agonist (Chopra et al., 2009; Fricker et al., 2009). By using a rat strain and transgenic mice deficient in mGlu2 receptors, we have confirmed that mGlu3 receptor activation alone can be sufficient to induce LTD. It is possible in these mGlu2 receptor deficient rats and mice that mGlu3 receptors are in some way upregulated to play a greater contribution than is normal, although this was not observed in
S.J. Lucas et al. / Neuropharmacology 66 (2013) 196e201
either the Han Wistar rats (Ceolin et al., 2011) or these transgenic mice (Wright et al., 2013). Assessing the contribution of mGlu3 receptors in normal animals requires more selective pharmacological tools. We were, however, able to investigate whether mGlu2 receptors can induce LTD at these synapses in normal animals. The observation that LY541850 was able to induce LTD, comparable to that induced by DCG-IV, in wild-type mice but not in mGlu2 KO mice demonstrates that activation of mGlu2 receptors alone is sufficient to induce LTD at these synapses. In conclusion, using rodents that lack expression of mGlu2 receptors, in conjunction with a compound that acts as a pure mGlu3 receptor antagonist in these preparations, we demonstrate that activation of mGlu3 receptors alone is sufficient to induce LTD at a central synapse. Acknowledgements Funded by the MRC and the Centre for Cognitive Neuroscience (Eli Lilly). SL was a MRC scholar. LY395756, LY541850 and the KO mice were kind gifts from Eli Lilly & Co. Ltd. GLC is a WCU International Scholar supported by the WCU program through the KOSEF funded by the MEST (R31-10089). References Alexander, G.M., Godwin, D.W., 2006. Unique presynaptic and postsynaptic roles of group II metabotropic glutamate receptors in the modulation of thalamic network activity. Neuroscience 141, 501e513. Anderson, W.W., Collingridge, G.L., 2007. Capabilities of the WinLTP data acquisition program extending beyond basic LTP experimental functions. J. Neurosci. Methods 162, 346e356. Anwyl, R., 1999. Metabotropic glutamate receptors: electrophysiological properties and role in plasticity. Brain Res. Rev. 29, 83e120. Ceolin, L., Kantamneni, S., Barker, G.R., Hanna, L., Murray, L., Warburton, E.C., Robinson, E.S., Monn, J.A., Fitzjohn, S.M., Collingridge, G.L., Bortolotto, Z.A., Lodge, D., 2011. Study of novel selective mGlu2 agonist in the temporoammonic input to CA1 neurons reveals reduced mGlu2 receptor expression in a Wistar substrain with an Anxiety-like phenotype. J. Neurosci. 31, 6721e6731. Chopra, M., Yao, Y., Blake, T.J., Hampson, D.R., Johnson, E.C., 2009. The neuroactive peptide N-acetylaspartylglutamate is not an agonist at the metabotropic glutamate receptor subtype 3 of metabotropic glutamate receptor. J. Pharmacol. Exp. Ther. 330, 212e219. Conn, P.J., Pin, J.-P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205e237. Dominguez, C., Prieto, L., Valli, M.J., Massey, S.M., Bures, M., Wright, R.A., Johnson, B.G., Andis, S.L., Kingston, A., Schoepp, D.D., Monn, J.A., 2005. Methyl substitution of 2-aminobicyclo[3.1.0]hexane 2,6-dicarboxylate (LY354740) determines functional activity at metabotropic glutamate receptors: identification of a subtype selective mGlu2 receptor agonist. J. Med. Chem. 48, 3605e3612. Dunayevich, E., Erickson, J., Levine, L., Landbloom, R., Schoepp, D.D., Tollefson, G.D., 2008. Efficacy and tolerability of an mGlu2/3 agonist in the treatment of generalised anxiety disorder. Neuropsychopharmacology 33, 1603e1610. Fricker, A.C., Mok, M.H., de la Flor, R., Shah, A.J., Woolley, M., Dawson, L.A., Kew, J.N., 2009. Effects of N-acetylaspartylglutamate (NAAG) at group II mGluRs and NMDAR. Neuropharmacology 56, 1060e1067. Hanna, L., Ceolin, L., Lucas, S., Monn, J., Johnson, B., Collingridge, G., Bortolotto, Z., Lodge, D., 2013. Differentiating the roles of mGlu2 and mGlu3 receptors using LY541850, an mGlu2 agonist / mGlu3 antagonist. Neuropharmacology 66, 114e121. Heinbockel, T., Pape, H.-C., 2000. Input-specific long-term depression in the lateral amygdala evoked by theta frequency stimulation. J. Neurosci. 20, 1e5. Higgins, G.A., Ballard, T.M., Kew, J.N.C., Richards, J.G., Kemp, J.A., Adam, G., Woltering, T., Nakanishi, S., Mutel, V., 2004. Pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent. Neuropharmacology 46, 907e917. Huang, L., Killbride, J., Rowan, M.J., Anwyl, R., 1999a. Activation of mGluRII induces LTD via activation of protein kinase A and protein kinase C in the dentate gyrus of the hippocampus in vitro. Neuropharmacology 38, 73e83. Huang, L., Rowan, M.J., Anwyl, R., 1999b. Induction of long-lasting depression by (þ)-alpha-methyl-4-carboxyphenylglycine and other group II mGlu receptor ligands in the dentate gyrus of the hippocampus in vitro. Eur. J. Pharmacol. 366, 151e158.
201
Kahn, L., Alonso, G., Robbe, D., Bockaert, J., Manzoni, O.J., 2001. Group 2 metabotropic glutamate receptors induced long term depression in mouse striatal slices. Neurosci. Lett. 316, 178e182. Kaschel, T., Schubert, M., Albrecht, D., 2004. Long-term depression in horizontal slices of the rat lateral amygdala. Synapse 53, 141e150. Kew, J.N.C., Pflimlin, M.-C., Kemp, J.A., Mutel, V., 2002. Differential regulation of synaptic transmission by mGlu2 and mGlu3 at the perforant path inputs to the dentate gyrus and CA1 revealed in mGlu2 / mice. Neuropharmacology 43, 215e221. Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors. Neuropharmacology 37, 1e12. Levine, L., Gaydos, B., Sheehan, D., Goddard, A., Feighner, J., Potter, W.Z., 2002. The mGlu2/3 receptor agonist, LY354740, reduces panic anxiety induced by a CO2 challenge in patients diagnosed with panic disorder. Neuropharmacology 43, 294e295. Li, H., Weiss, S.R.B., Chuang, D.-C., Post, R.M., Rogawski, M.A., 1998. Bidirectional synaptic plasticity in the rat basolateral amygdala: characterization of an activity-dependent switch sensitive to the presynaptic metabotropic glutamate receptor antagonist 2S-a-ethylglutamic acid. J. Neurosci. 18, 1662e1670. Lin, H.-C., Lee, C.-C., Huang, Y.-C., Wang, S.-J., Gean, P.-W., 2005. Activation of group II metabotropic glutamate receptors induces depotentiation in amygdala slices and reduces fear potentiated startle in rats. Learn Mem. 12, 130e137. Lin, H.-C., Wang, S.-J., Luo, M.-Z., Gean, P.-W., 2000. Activation of group II metabotropic glutamate receptors induces long-term depression of synaptic transmission in the rat amygdala. J. Neurosci. 20, 9017e9024. Linden, A.-M., Shannon, H., Baez, M., Yu, J.L., Koester, A., Schoepp, D.D., 2005. Anxiolytic-like activity of the mGLU2/3 receptor agonist LY354740 in the elevated plus maze test is disrupted in metabotropic glutamate receptor 2 and 3 knock-out mice. Psychopharmacology 179, 284e291. Linden, A.-M., Baez, M., Bergeron, M., Schoepp, D.D., 2006. Effects of mGlu2 and mGlu3 receptor deletions on mGlu2/3 receptor agonist (LY354740)-induced brain c-Fos expression: specific roles for mGlu2 in the amygdala and subcortical nuclei, and mGlu3 in the hippocampus. Neuropharmacology 51, 213e228. Lovinger, D.M., McCool, B.A., 1995. Metabotropic glutamate receptor-mediated presynaptic depression at corticostriatal synapses involves mGluR2 or 3. J. Neurophysiol. 73, 1076e1083. Macek, T.A., Winder, D.G., Gereau, R.W.T., Ladd, C.O., Conn, P.J., 1996. Differential involvement of group II and group III mGluRs as autoreceptors at lateral and medial perforant path synapses. J. Neurophysiol. 76, 3798e3806. Muly, E.C., Mania, I., Guo, J.-D., Rainnie, D.G., 2007. Group II metabotropic glutamate receptors in anxiety circuitry: correspondence of physiological response and subcellular distribution. J. Comp. Neurol. 505, 682e700. Neugebauer, V., Keele, N.B., Shinnick-Gallagher, P., 1997. Epileptogenesis in vivo enhances the sensitivity of inhibitory presynaptic metabotropic glutamate receptors in basolateral amygdala neurons in vitro. J. Neurosci. 17, 983e995. Otani, S., Daniel, H., Takita, M., Crepel, F., 2002. Long-term depression induced by postsynaptic group II metabotropic glutamate receptors linked to phospholipase C and intracellular calcium rises in rat prefrontal cortex. J. Neurosci. 22, 3434e3444. Patil, S.T., Zhang, L., Martenyi, F., Lowe, S.L., Jackson, K.A., Andreev, B.V., Avedisova, A.S., Bardenstein, L.M., Gurovich, I.Y., Morozova, M.A., Mosolov, S.N., Neznanov, N.G., Reznik, A.M., Smulevich, A.B., Tochilov, V.A., Johnson, B.G., Monn, J.A., Schoepp, D.D., 2007. Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized Phase 2 clinical trial. Nat. Med. 13, 1102e1107. Pin, J.-P., Duvoisin, R., 1995. Review: neurotransmitter receptors I. Neuropharmacology 34, 1e26. Poschel, B., Manahan-Vaughan, D., 2005. Group II mGluR-induced long term depression in the dentate gyrus in vivo is NMDA receptor-independent and does not require protein synthesis. Neuropharmacology 49, 1e12. Robbe, D., Alonso, G., Chaumont, S., Bockhaert, J., Manzoni, O.J., 2002. Role of p/q-Ca2þ channels in metabotropic glutamate receptor 2/3-dependent presynaptic longterm depression at nucleus accumbens synapses. J. Neurosci. 22, 4346e4356. Shen, K.Z., Johnson, S.W., 2003. Group II metabotropic glutamate receptor modulation of excitatory transmission in rat subthalamic nucleus. J. Physiol. 553, 489e496. von Bohlen und Halbach, O., Albrecht, D., 2002. Reciprocal connections of the hippocampal area CA1, the lateral nucleus of the amygdala and cortical areas in a combined horizontal slice preparation. Neurosci. Res. 44, 91e100. Walker, D.L., Rattiner, L.M., Davis, M., 2002. Group II metabotropic glutamate receptors within the amygdala regulate fear as assessed with potentiated startle in rats. Behav. Neurosci. 116, 1075e1083. Wright, R.A., Johnson, B.G., Zhang, C., Salhoff, C., Kingston, A.E., Calligaro, D.O., Monn, J.A., Schoepp, D.D., Marek, G.J., 2013. CNS distribution of metabotropic glutamate 2 and 3 receptors: transgenic mice and [(3)H]LY459477 autoradiography. Neuropharmacology 66, 89e98. 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, 645e647.