Antagonists reversibly reverse chemical LTD induced by group I, group II and group III metabotropic glutamate receptors

Neuropharmacology 74 (2013) 135e146

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Antagonists reversibly reverse chemical LTD induced by group I, group II and group III metabotropic glutamate receptors David Lodge a, *, Patrick Tidball a, Marion S. Mercier a, Sarah J. Lucas a, b, Lydia Hanna a, Laura Ceolin a, c, Minos Kritikos a, Stephen M. Fitzjohn a, d, John L. Sherwood a, d, Neil Bannister a, Arturas Volianskis a, David E. Jane a, Zuner A. Bortolotto a, Graham L. Collingridge a, e a

Centre for Synaptic Plasticity, School of Physiology and Pharmacology, Dorothy Hodgkin Building, University of Bristol, Bristol BS1 3NY, UK Laboratory of Neurobiology, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA c Institute of Functional Genomics, CNRS-UMR5203, INSERM-U661, Université Montpellier 1, Université Montpellier 2, 141 Rue de la Cardonille, 34000 Montpellier, France d Neuroscience Research Division, Lilly Research Centre, Eli Lilly & Co. Ltd., Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK e Department of Brain and Cognitive Sciences, College of Natural Sciences, Seoul National University, Shilim, Gwanak, Seoul 151-746, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 December 2012 Received in revised form 25 February 2013 Accepted 7 March 2013

Metabotropic glutamate (mGlu) receptors are implicated in many neurological and psychiatric diseases and are the targets of therapeutic agents currently in clinical development. Their activation has diverse effects in the central nervous system (CNS) that includes an involvement in synaptic plasticity. We previously reported that the brief exposure of hippocampal slices to dihydroxyphenylglycine (DHPG) can result in a long-term depression (LTD) of excitatory synaptic transmission. Surprisingly, this LTD could be fully reversed by mGlu receptor antagonists in a manner that was itself fully reversible upon washout of the antagonist. Here, 15 years after the discovery of DHPG-LTD and its reversible reversibility, we summarise these initial findings. We then present new data on DHPG-LTD, which demonstrates that evoked epileptiform activity triggered by activation of group I mGlu receptors can also be reversibly reversed by mGlu receptor antagonists. Furthermore, we show that the phenomenon of reversible reversibility is not specific to group I mGlu receptors. We report that activation of group II mGlu receptors in the temporo-ammonic pathway (TAP) and mossy fibre pathway within the hippocampus and in the cortical input to neurons of the lateral amygdala induces an LTD that is reversed by LY341495, a group II mGlu receptor antagonist. We also show that activation of group III mGlu8 receptors induces an LTD at lateral perforant path inputs to the dentate gyrus and that this LTD is reversed by MDCPG, an mGlu8 receptor antagonist. In conclusion, we have shown that activation of representative members of each of the three groups of mGlu receptors can induce forms of LTD than can be reversed by antagonists, and that in each case washout of the antagonist is associated with the re-establishment of the LTD. This article is part of the Special Issue entitled ‘Glutamate Receptor-Dependent Synaptic Plasticity’. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Metabotropic glutamate receptors mGlu1 mGlu2 mGlu5 mGlu8 DHPG DCG-IV Long-term depression

1. Introduction Glutamate receptors comprise three main ionotropic subtypes, AMPA, NMDA and kainate (Collingridge et al., 2009), and a family of G-protein coupled metabotropic (mGlu) receptors (Pin and Duvoisin, 1995). The eight known mGlu receptors are further subdivided into three groups based on structural and functional criteria: group I (mGlu1 and mGlu5), group II (mGlu2 and mGlu3) and group III (mGlu4, mGlu6, mGlu7 and mGlu8). There is currently * Corresponding author. Tel.: þ44 1173313148; fax: þ44 1173313029. E-mail address: [email protected] (D. Lodge). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.03.011

considerable interest in mGlu receptors as targets for therapeutic agents (Nicoletti et al., 2011; Niswender and Conn, 2010). For example, negative allosteric modulators (NAMs) acting at mGlu5 receptors are being explored as potential treatments for epileptogenesis, Fragile-X syndrome, tardive dyskinesias and autism spectrum disorders (Bianchi et al., 2012; Gürkan and Hagerman, 2012; Michalon et al., 2012; Rylander et al., 2009). In addition, agonists acting at group II mGlu receptors have shown efficacy in man as therapies for anxiety and schizophrenia (Dunayevich et al., 2008; Grillon et al., 2003; Patil et al., 2007). Furthermore, mGlu5 receptors have been linked to the aetiology of Alzheimer’s disease (Hu et al., 2012). Therefore understanding both the functions of

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Abbreviations 1S,3R-ACPD (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid DCG-IV (2S,20 R,30 R)-2-(20 ,30 -dicarboxycyclopropyl)glycine DCPG (S)-3,4-dicarboxyphenylglycine DHPG (RS)-3,5-dihydroxyphenylglycine L-AP4 L-(þ)-2-amino-4-phosphonobutyric acid L-689,560 trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline LY341495 (2S,10 S,20 S)-2-(9-xanthylmethyl)-2-(20 -carboxycyclopropyl)glycine LY367385 (S)-(þ)-a-amino-4-carboxy-2-methylbenzeneacetic acid LY379268 (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid LY395756 (1SR,2SR,4RS,5RS,6SR)-2-amino-4-methylbicyclo[3.1.0]-hexane-2,6-dicarboxylic acid a-Methyl-4-carboxyphenylglycine MCPG MDCPG (RS)-a-methyl-3,4-dicarboxyphenylglycine MPEP 2-methyl-6-(phenylethynyl)-pyridine hydrochloride

mGlu receptors and the actions of ligands that affect these receptors is of relevance to the development of therapeutic agents active against these, and other, disorders. Fifteen years ago we described a form of LTD following the selective activation of group I mGlu receptors using the agonist, DHPG (Fitzjohn et al., 1998; Palmer et al., 1997). This form of synaptic plasticity, referred to hereafter as DHPG-LTD, has been extensively studied by many groups (e.g. Huang and Hsu, 2006; Huber et al., 2000; Massey and Bashir, 2007; Watabe et al., 2002; Xiao et al., 2001) A similar form of LTD has also been seen using the less selective mGlu receptor agonist, 1S,3R-ACPD, (O’Mara et al., 1995; Overstreet et al., 1997) although this is not invariably the case (Palmer et al., 1997). DHPG-LTD is mechanistically different from the other major form of chemical LTD that can be induced by activation of NMDA receptors (NMDA-LTD) (see Collingridge et al., 2010). However, there is still disagreement over some aspects of the pharmacology and biochemistry of the induction and expression mechanisms. For example, which group I receptor mediates the DHPG-LTD (Faas et al., 2002; Gladding et al., 2009; Kumar and Foster, 2007; Moult et al., 2006; Volk et al., 2006), which kinases and phosphatases are involved (Gallagher et al., 2004; Hou and Klann, 2004; Mockett et al., 2011; Moult et al., 2008; Schnabel et al., 1999) and the relative contributions of pre- and post-synaptic mechanisms (Fitzjohn et al., 2001; Moult et al., 2006; Qian and Noebels, 2006; Tan et al., 2003; Upreti et al., 2013; Watabe et al., 2002; Waung et al., 2008; Xiao et al., 2001) are all subject to controversy. These aspects of LTD are, however, not the subject of the present article but rather we concentrate on the pharmacology of the maintenance phase of LTD induced by the pharmacological activation of mGlu receptors. More specifically, at the same time as reporting DHPG-LTD, we noted the curious phenomenon that mGlu receptor antagonists, such as a-methyl-4-carboxyphenylglycine (MCPG), can reverse this plasticity long after it has been induced and that the plasticity is restored after washout of the antagonist (Fitzjohn et al., 1998; Palmer et al., 1997). Here we summarise these historical findings and present a series of new observations concerning the antagonist reversal of the maintained phase of group I mGlu receptortriggered LTD (mGlu receptor-LTD). We also demonstrate similar phenomena for other mGlu receptor subtypes in groups II (mGlu2 and mGlu3) and III (mGlu8). 2. Materials and methods 2.1. Animals and slice preparation Experiments were performed according UK Scientific Procedures Act, 1986 and EU Guidelines for Animal Care and conducted as described in previous publications (Ceolin

et al., 2011; Fitzjohn et al., 1999; Hanna et al., 2012; Lucas et al., 2012; Mercier et al., 2013; Sherwood et al., 2012). Briefly, electrophysiological recordings were obtained from slices prepared from rats and mice. Extracellular and whole cell experiments were performed as described in the text. Stimulating electrodes were placed in the Schaffer collaterals, the temporo-ammonic pathway, mossy fibre pathway, the external capsule or the lateral perforant path and recordings were made in the CA1 stratum radiatum, the CA1 stratum lacunosum moleculare, the CA3 stratum lucidum, the lateral amygdala or the outer third of the dentate gyrus stratum moleculare, respectively. 2.2. Analyses of electrophysiological recordings Data were captured and analysed using the WinLTP program (Anderson and Collingridge, 2007). In extracellular experiments, the effects of compounds on AMPA receptormediated synaptic transmission were quantified by measuring either initial slopes or peak amplitudes of AMPA receptor-mediated field excitatory post-synaptic potentials (fEPSPs) and normalised to baseline. Similarly, peak excitatory postsynaptic currents (EPSCs) were quantified in whole cell experiments. Changes in excitability due to DHPG application were quantified using coastline analyses, which are built into WinLTP (www.winltp.com). Data are presented both as single experiments and as mean values of experimental groups (S.E.M). 2.3. Chemicals Compounds were purchased from Tocris Cookson, Bristol, U.K.:-(2S,20 R,30 R)-2(20 ,30 -dicarboxycyclopropyl)glycine (DCG-IV) and trans-2-carboxy-5,7-dichloro-4phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline (L-689,560), or from Abcam, Cambridge, U.K.:- picrotoxin (PTX), (S)-3,4-dicarboxyphenylglycine (DCPG), (2S,1’S,20 S)-2-(9-xanthylmethyl)-2-(20 -carboxycyclopropylglycine (LY341495), (RS)3,5-dihydroxyphenylglycine (DHPG), D-2-amino-5-phosphono-pentanoate (D-AP5), (S)-(þ)-a-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), a-methyl-4carboxyphenylglycine (MCPG) and 2-methyl-6-(phenylethynyl)-pyridine hydrochloride (MPEP). (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) and (1SR,2SR,4RS,5RS,6SR)-2-amino-4-methylbicyclo[3.1.0]-hexane-2,6-dicarboxylic acid (LY395756) were kind gifts of Dr James Monn, Eli Lilly & Co. and (RS)-a-methyl-3,4-dicarboxyphenylglycine (MDCPG) was synthesized in house. All other fine chemicals were purchased from Fisher Scientific or Sigma.

3. Results 3.1. Group I mGlu receptor-mediated LTD The original observation that MCPG (Palmer et al., 1997), and other mGlu receptor antagonists (Fitzjohn et al., 1998, 1999; Palmer et al., 1997), can reverse DHPG-LTD in a reversible manner is illustrated in Fig. 1. Fig. 1A shows a single example time-course plot. MCPG alone had little effect on the synaptic response when applied during baseline recording but, when applied after the induction of DHPG-LTD, it was able to fully reverse the synaptic depression. Significantly, DHPG-LTD was fully restored upon washout of MCPG. The consistency of this observation can be seen in the pooled timecourse data plot (Fig. 1B). The effect is extremely unlikely to be due

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Fig. 1. Transient reversal of the expression of group I mGlu receptor mediated LTD by antagonists. A. Single example showing the lack of effect of (S)-MCPG (1 mM) on baseline but complete reversal of LTD induced by DHPG (100 mM). Note that upon washout of MCPG, LTD was re-instated. B. Pooled data (n ¼ 5). C. The ability of DHPG (100 mM) to potentiate depolarisations induced by NMDA (20 mM, 2 min applied at times indicated by arrowheads) is rapidly reversible, demonstrating rapid washout of DHPG. D. A single example to show the ability of a range of mGlu receptor antagonists (1 mM (S)-4CPG, 0.5 mM MTPG, 0.5 mM MPPG and 1 mM (S)-MCPG) to reverse the expression of DHPG-LTD. E. Reversal of LTD induced by three applications of DHPG (30 mM), by LY341495 (100 mM). F. Reversal of DHPG-LTD by LY393053 (n ¼ 5). Data shown in panels AeE are from hippocampal slices obtained from adult rats (4e10 week old), bathed in Mg2þ-free medium (AeD; (Palmer et al., 1997) and in 1 mM Mg2þ-containing medium (E; Fitzjohn et al., 1998). Data shown in panel F are from hippocampal slices obtained from juvenile (12e18 day old) rats, bathed in 1 mM Mg2þ-containing medium (Fitzjohn et al., 1999).

to a slow washout of DHPG for two reasons. First, in interleaved experiments the ability of DHPG to acutely potentiate depolarisations induced by NMDA was rapidly reversible (Fig. 1C). Second, as shown in Fig. 1A and B, the synaptic plasticity was restored following washout of MCPG. The reversal of DHPG-LTD was not unique to MCPG. In Fig. 1D, a range of phenylglycine antagonists, which have a varying degree of activity at the various mGlu receptor subtypes (Schoepp et al., 1999; Watkins and Collingridge, 1994), were compared. The spectrum of activity was consistent with the reversal effect being due to persistent activity of group I mGlu receptors. Note that, even when applied approximately 3 h after the induction of DHPG-LTD, MCPG fully reversed the LTD in a completely reversible manner. Fig. 1E shows an example of full reversal of a large DHPG-LTD, induced by three applications of the agonist, by LY341495 applied at a concentration (100 mM) that blocks all mGlu receptor subtypes (Fitzjohn et al., 1999). Fig. 1F shows pooled data of the effects of LY393053, an antagonist that has a degree of selectively towards group I mGlu receptors (Fitzjohn et al., 1998). The data illustrated in Fig. 1AeE were obtained from slices prepared from 4 to 10 week old rats whilst those in Fig. 1F were obtained from slices obtained from 12 to 18 day old rats. Therefore this phenomenon appears not to be unique to a particular developmental time point. The above observations with antagonists have been largely replicated in several other laboratories. Thus, there is general

agreement that persistent activation of group I mGlu receptors is involved in the maintenance of DHPG-LTD (Huang and Hsu, 2006; Palmer et al., 1997; Watabe et al., 2002), rather than other mGlu receptors (Palmer et al., 1997) or NMDA receptors (Huang and Hsu, 2006). Most studies, however, do not differentiate between the contribution of mGlu1 and mGlu5 receptor subtypes to the maintenance of this form of plasticity. By comparing the actions of LY367385 and MPEP, selective antagonists for mGlu1 and mGlu5 receptors, respectively (Schoepp et al., 1999), it was shown that either mGlu1 (Volk et al., 2006) or mGlu5 (Huang and Hsu, 2006; Ronesi et al., 2012) receptors provided the greater role in the maintenance of DHPG-LTD. We recently investigated this issue in parasagittal hippocampal slices with the CA3 field removed from 8 to 12 week old rats, using methods described previously, which include addition of GABAA (picrotoxin) and NMDA (L-689,560) receptor antagonists in the perfusate (Palmer et al., 1997). Bath application of DHPG 100 mM for 10 min reduced the fEPSP recorded in stratum radiatum of CA1 following submaximal stimulation of the Schaffer collateral/commissural input (Fig. 2). The acute phase was followed by a sustained reduction (i.e., DHPG-LTD), which lasted for as long as recordings were maintained (typically up to 2 h after washout of DHPG). During this sustained phase application of the mGlu1 antagonist, LY367385 (100 mM; applied 70 min after stopping the perfusion with DHPG) produced a partial reversal of the LTD, and following washout of LY367385, the DHPG-LTD was re-

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Fig. 2. The effects of the subtype-selective antagonists, LY367385 and MPEP, on the maintenance of DHPG-LTD and DHPG-induced epileptiform bursting. A. Representative example, and B. pooled data (n ¼ 6) showing that chemical LTD of Schaffer collateral-CA1 fEPSPs induced by DHPG (100 mM, 10 min), was transiently reversed by the mGlu1 selective antagonist LY367385 (100 mM, 20 min). Sample traces are taken from the time points indicated in the figure (in this and subsequent figures, traces are an average of 4 consecutive sweeps). LTD was 69  5%, 82  3% and 73  6% of baseline levels respectively before, during and after LY367385. C. Pooled data (n ¼ 4) illustrating equivalent experiment with the mGlu5 selective antagonist, MPEP (10 mM; 20 min). LTD was 81  3%, 85  3% and 80  4% of baseline before, during and after MPEP, respectively. D. Single example (same experiment as panel A) and E. pooled data (n ¼ 6) of ‘coastline’ measurement of fEPSP waveforms showing persistent enhancement of evoked epileptiform bursting by DHPG and its reversal by LY367385. Coastline values were 171  14%, 140  8% and 182  18% of baseline before, during and after LY367385, respectively. F. Pooled coastline plot (n ¼ 4) showing no effect of MPEP on the DHPG-induced enhancement of evoked epileptiform bursting with coastline values of 146  14%, 144  22% and 137  15% before, during and after application of MPEP, respectively.

established (Fig. 2A and B). Bath application of the mGlu5 antagonist, MPEP (10 mM), had a much smaller effect (Fig. 2C). The present data, supporting a major role for mGlu1 receptors in the maintenance of DHPG-LTD, are in agreement with those of Volk et al. (2006) but not with those of Huang and Hsu (2006) and Ronesi et al. (2012); the reason for this discrepancy is presently unknown. It should, however, be noted that heterodimers between mGlu1 and mGlu5 receptors (Doumazane et al., 2011) and variability in proportions of heterodimers and homodimers could potentially lead to experimental differences. During these experiments, it became clear that, in addition to inducing LTD of synaptic transmission, exposure to DHPG-induced a persistent enhancement of evoked epileptiform activity that followed the initial fEPSPs in the presence of picrotoxin (Fig. 2DeF). Increases in excitability following DHPG administration are well documented (Davies et al., 1995). For example, an enhancement of spontaneous epileptiform activity following DHPG administration in the CA3 region of disinhibited slices has been extensively described (e.g. Merlin and Wong, 1997 and reviewed by Bianchi

et al., 2012), but overt epileptiform activity was not observed in our earlier experiments (Fitzjohn et al., 1998, 1999; Palmer et al., 1997), possibly due to methodological differences. Persistent, non-synaptic bursting network activity has also been observed following DHPG application to hippocampal slices, in the absence of Ca2þ-dependent synaptic transmission (Piccinin et al., 2008). Our study of synaptically-evoked epileptiform activity in the CA1 region of the hippocampus showed that, similar to DHPG-LTD, the increase in population spike activity was maintained for up to 2 h after DHPG application, and was reduced by LY367385, but not by MPEP, in a fully reversible manner (Fig. 2DeF). The major role played by mGlu1 receptors in this form of plasticity parallels that for the maintenance of spontaneous DHPG-induced epileptiform activity in CA3 (Merlin, 2002). 3.2. Group II mGlu receptor-mediated LTD In addition to group I mGlu receptor-mediated LTD, there are several reports of both synaptic (e.g. Altinbilek and Manahan-

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Vaughan, 2009; Huang et al., 1997; Manahan-Vaughan, 1997; Yokoi et al., 1996) and chemical (e.g. Huang et al., 1999; Pöschel and Manahan-Vaughan, 2005; Tzounopoulos et al., 1998; Wostrack and Dietrich, 2009) LTD induced by group II mGlu receptor activation in various hippocampal pathways. Group II mGlu receptormediated LTD has also been described in several other central pathways, including the prefrontal cortex (Otani et al., 1999, 2002), the striatum (Kahn et al., 2001), the nucleus accumbens (Robbe et al., 2002) and the amygdala (Lin et al., 2000; Lucas et al., 2012). In the majority of cases, the ability of mGlu receptor antagonists to affect the maintained phase of group II mGlu receptormediated LTD has not been explored, or not been observed. We have reported that the fEPSP in CA1 evoked by stimulation of the temporo-ammonic pathway (TAP) is inhibited by DCG-IV (0.3e1 mM) in a fully reversible manner (Ceolin et al., 2011; Hanna et al., 2012). By contrast, using these same methods in parasagittal slices from 12 to 16 day old rats, inhibition induced by bath application of a more potent mGlu2/3 agonist, LY379268 (0.3 mM; Fig. 3A and B) was maintained for several hours. Interestingly, LY341495 (0.3 mM), a concentration likely to act preferentially as an antagonist of group II mGlu receptors (Kingston et al., 1998), fully restored this LTD of the fEPSP back to baseline values (Fig. 3A and B). This antagonism was, however, at least partially reversible on washout of LY341495, suggesting a maintained chemical LTD mediated by group II mGlu receptors in this pathway in rat slices (Fig. 3A and B). We extended these observations to TAP stimulation in coronal slices from 10 to 16 week old CD-1 mice (Fig. 3C) and investigated the role of mGlu2 and mGlu3 receptors by comparing the effects of both LY379268 (0.3 mM) and LY341495 (0.3 mM) in mGlu3/ or mGlu2/ mice with those in their wild-type (WT) littermates (Linden et al., 2005). Fig. 3D illustrates the data showing

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that specific activation of mGlu2 receptors is able to induce an LTD of the CA1 fEPSP in these knock-out mice comparable to that seen in WT animals (Fig. 3D) whereas activation of mGlu3 receptors alone induces a smaller LTD. In all three cases, LY341495 induced a reversible reversal of the LTD (Fig. 3D). To further investigate the role of mGlu2 receptors in chemical LTD, the effects of DCG-IV and of LY395756, a selective mGlu2 receptor agonist but an antagonist at mGlu3 receptors (Ceolin et al., 2011; Dominguez et al., 2005) were examined in the mossy fibre pathway to CA3 (Fig. 4). In a previous study we demonstrated that sensitivity to DCG-IV in the mossy fibre pathway was dependent on experimental conditions and in particular on slice orientation (Sherwood et al., 2012). We therefore confined this investigation of group II mGlu receptor-mediated LTD of mossy fibre transmission to transverse slices, in which sensitivity to DCG-IV (Fig. 4A) and facilitation to increased stimulation frequency (Fig. 4B) were used to identify the mossy fibre input to CA3 (Sherwood et al., 2012; Weisskopf et al., 1994). The reduction in fEPSP amplitude by DCG-IV (0.1 mM) outlasted the drug application (Fig. 4A), as reported previously with 1 mM DCG-IV (Wostrack and Dietrich, 2009). This chemical LTD was also elicited by the mGlu2 receptor agonist, LY395756, (10e30 mM; Fig. 4C), which in turn was reversibly reversed on application of LY341495 (0.3 mM; Fig. 4C). We have also extended our observations from hippocampal fields to amygdala neurons by studying the effect of the group II mGlu receptor antagonist, LY341495 (0.3 mM) on the LTD evoked by DCG-IV (1 mM) using patch-clamp recordings from neurons within the lateral amygdala of 5e6 week old mice (Lucas et al., 2012). The evoked EPSC remained depressed for at least 1 h after removal of DCG-IV (Fig. 5A and C) but LY341495 fully reversed the LTD in 6 out of 8 neurons examined (Fig. 5B and C). With respect to

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Fig. 3. The group II mGlu receptor antagonist, LY341495, reverses LY379268 induced LTD in the temporo-ammonic pathway of the hippocampus. A. Representative example, and B. pooled data (n ¼ 4) showing that chemical LTD of TAP-CA1 fEPSPs induced in rat hippocampal slices by LY379268 (0.03 and 0.3 mM, 40 min each) was reversed by the group II mGlu receptor antagonist, LY341495 (0.3 mM; 60 min). Sample traces are taken from the time points indicated in the figure. LTD was 23  9%, 102  23% and 70  21% of baseline levels before, during and after LY341495, respectively. C. Representative experiment from a CD-1 wild-type (WT) mouse and D. pooled data from transgenic and WT littermate mice showing that LTD induced by LY379268 (0.3 mM, 30 min) in hippocampal slices was transiently reversed by LY341495 (0.3 mM, 60 min). Specifically, the results from mGlu3/ mice (B; n ¼ 6) and WT mice (C; n ¼ 12) were very similar, whereas in mGlu2/ mice ( ; n ¼ 6) the LTD was significantly smaller. Thus the values were 29  3%, 35  5% and 74  5% of baseline before, 103  10%, 126  9% and 95  9% of baseline during and 52  7%, 58  5% and 76  8% of baseline after LY341495 application for mGlu3/, WT and mGlu2/ CD1 mice, respectively.

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receptor subtype, we have previously reported, using knock-out mice, that both mGlu2 and mGlu3 receptors are separately capable of inducing this DCG-IV-LTD in amygdala neurons (Lucas et al., 2012).

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3.3. Group III mGlu receptor-mediated LTD Having demonstrated antagonist reversal of established chemical LTD in relation to group I and II mGlu receptors, it was of interest to see if this phenomenon extended to agonists of group III mGlu receptors. The paucity of subtype-selective and potent agonists for group III mGlu receptors has, thus far, limited the investigation of group III mGlu receptor subtypes in synaptic plasticity in general, and chemical LTD in particular. There is, however, some evidence for group III mGlu receptor-mediated LTD. For instance, the mGlu4 and mGlu8 preferring agonist, L-(þ)-2-amino-4phosphonobutyric acid (L-AP4) (Schoepp et al., 1999) has been

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4. Discussion In the present study we have extended our analysis of DHPGLTD in CA1 and its reversal by group I mGlu receptor antagonists. In particular, we have shown that antagonists are able to reverse both the LTD of stimulus-evoked synaptic activity and stimulusevoked epileptiform discharges. Significantly, we have demonstrated that this unusual phenomenon of reversible reversal of LTD by antagonists also applies to members of mGlu receptor groups II (mGlu2 and mGlu3) and III (mGlu8). 4.1. Chemical forms of LTD induced by group II and III mGlu receptors We have extended the number of pathways where a chemical form of group II or III mGlu receptor mediated LTD can be observed and demonstrated that appropriate antagonists reduce the maintenance phase of the LTD with subsequent reversal on washout of the antagonist. With respect to group II mGlu receptors, we have found that activation of mGlu2 can induce LTD in the TAP. Interestingly, this LTD was consistently observed with LY379268 but not with DCG-IV. LY379268 is a more potent mGlu2/3 agonist than DCG-IV (Schoepp et al., 1999) and so whether LTD is induced or not could relate to the level of intrinsic activity of the agonist. However, this cannot be the sole explanation, since DCG-IV was capable of inducing LTD in both the MF input to the CA3 (Fig. 4) and the cortical input to the lateral amygdala (Fig. 5 and Lucas et al., 2012). Perhaps, the ability of an agonist to induce LTD depends upon both its intrinsic activity for the receptor and the receptor density at the particular pathway under investigation. Our studies in the TAP-CA1 and MF-CA3 pathways identified a primary role for mGlu2

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shown to induce an LTD in ventral septal neurons in vitro (Tolchard et al., 2000) and in CA1 neurons in vivo (Naie et al., 2006). L-AP4 (200 mM) was also found to induce an LTD at mossy fibre synapses in interneurons but, significantly, not pyramidal neurons (Pelkey et al., 2005). The lack of effect with lower concentrations of L-AP4, selective for mGlu4 and mGlu8 receptors, indicated a role for mGlu7 receptors in this form of LTD. Although few studies have directly investigated plasticity following specific activation of mGlu8 receptors, incomplete recovery from the depressant actions of L-AP4 and DCPG, an mGlu8 receptor preferring agonist (Thomas et al., 2001), has been observed in the bed nucleus of stria terminalis (Gosnell et al., 2011; Grueter and Winder, 2005). During a recent investigation into the selectivity of DCPG as an mGlu8 receptor agonist (Mercier et al., 2013), we noticed that bath application of 100 mM DCPG induced a long lasting depression of the lateral perforant path (LPP) evoked fEPSP in the dentate gyrus. It was found that DCPG was a selective mGlu8 agonist at low concentrations but, at concentrations greater than 1 mM, DCPG also acted at other mGlu receptors, principally mGlu2 (Mercier et al., 2013). In order to avoid non-selective effects via mGlu2 receptors, experiments were conducted in a strain of Han Wistar rats (10 weeks old) known not to express mGlu2 receptors (Ceolin et al., 2011). We first demonstrated that the mGlu8 receptor antagonist, MDCPG (30 mM) (Mercier et al., 2013; Thomas et al., 2001), had no effect on baseline fEPSPs (Fig. 6A) and then that 30 min perfusion with DCPG (100 mM) induced a long lasting depression of fEPSPs (Fig. 6B and C), which we term DCPG-LTD. This novel form of chemical LTD was fully reversed to baseline levels by the application of 30 mM MDCPG and, as with the group I and II chemical LTD, washout of the antagonist re-instated the LTD (Fig. 6B and C). Again this indicates that activity of the mGlu receptor, in this case mGlu8, is required for the maintenance of DCPG-LTD, and extends this curious phenomenon to all three mGlu receptor subgroups.

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receptors in LTD, but also suggest a minor role for mGlu3 receptors. In the lateral amygdala, we have previously shown that both mGlu2 and mGlu3 receptors are capable of inducing LTD following application of DCG-IV (Lucas et al., 2012). Other reports (e.g. Johnson et al., 2011; Yokoi et al., 1996) emphasize the role of mGlu2 receptors as inducers of LTD in the CNS. Regarding group III mGlu receptors, we have shown that mGlu8 receptors can induce LTD, in this case in the LPP projection to the dentate gyrus. To achieve this effect it was necessary to apply a high concentration, relative to that needed to induce a transient synaptic

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depression (Mercier et al., 2013). However, we are confident that the LTD is due, primarily at least, to activation of mGlu8 receptors, since we performed our experiments in a rat strain that is devoid of mGlu2 receptors, which primarily mediate the non-specific effects of DCPG (Mercier et al., 2013). The maintained phase of the LTD was reduced by MDCPG, which is a selective antagonist for mGlu8 receptors (Mercier et al., 2013). However, the possibility that mGlu2 receptors contribute to DCPG-LTD in wild-type tissue cannot be ruled out. Indeed, lower concentrations of DCPG (e.g., 30 mM) induced a persistent depression in wild-type but not in mGlu2/ mice (Mercier et al., 2013). 4.2. Reversal of mGlu receptor-mediated LTD by antagonists The major focus of the present study has been to investigate the reversal of the maintenance of LTD by mGlu receptor antagonists. Our main conclusion is that the reversible reversal of LTD is observed across all three subgroups and so is likely to relate to a general property of these G-protein coupled receptors. So what are the possible explanations for the reversal of these chemical forms of LTD by the antagonists? Broadly speaking there are two primary explanations: 1) the exogenous agonist remains in the tissue and is available for activation of the receptor for long periods of time or 2) the receptor remains active in the absence of the exogenous agonist. 4.3. Exogenous agonist remains in the tissue A simple explanation of the antagonist reversal of LTD is that the exogenous agonist remains bound to the receptor and exposure to the antagonist displaces the agonist from the receptor and it is then removed from the slice. For example, Wostrack and Dietrich (2009) reported that LY341495 (3 mM) reversed the maintained long lasting inhibition of the mossy fibre input to dentate gyrus following DCG-IV (1 mM) but did not observe any recovery from the effects of this antagonist, unlike the present experiments where recovery followed washout of LY341495 (0.3 mM). Wostrack and Dietrich (2009) interpreted their data as indicating that the agonist remained bound to the group II receptor until displaced by LY341495. Although this is an attractively simple idea, it is difficult to reconcile with the fact that the LTD of each of the three mGlu receptor groups re-appears after washout of the antagonist, with the fact that the depression of synaptic activity by the same concentration of a group III agonist is long lasting (LTD) at one synapse but not at another synapse in the same preparation (Pelkey et al., 2005) and with the fact that long lasting inhibition of afterhyperpolarisations by DHPG remains intact after multiple applications of MCPG (Young et al., 2012). To retain this hypothesis, it is therefore necessary to conjecture that the exogenous agonist is sequestered in some form within the tissue such that it is able to reactivate the receptor and the transduction processes underlying the LTD once the antagonist is washed out. One such possibility is that the agonist is internalised along with the receptor and the receptor may continue to signal (Shenoy and Lefkowitz, 2011). This in itself seems unlikely to be the entire explanation since the extracellularly applied antagonists are able to reverse the LTD, unless this is also internalised and therefore prevents the transduction mechanisms internally. Alternatively, it is also possible to envisage a situation where a bound agonistereceptor complex is continuously recycled back to the membrane where its actions would be blocked during antagonist application. Thus, arrestins or other mediators (Hong et al., 2009; Mundell et al., 2001) are involved in resensitisation as well as desensitisation of G-protein coupled receptors (Bockaert et al., 2004; Ferguson, 2001), although whether this can occur with agonist bound during the recycling event is unclear. Such a long

lasting recycling of the receptoreagonist complex could be envisaged for a high affinity agonist, such as LY379268, but seems less likely an explanation for the LTD induced by relatively low affinity agonists such as DHPG and DCPG. A second type of sequestering of an agonist might be uptake into glia and/or presynaptic terminals. Subsequent hetero-exchange with, for example, synaptically released glutamate, or release from terminals would provide a continued background of the exogenous agonist and hence activation of the respective mGlu receptor. Group II mGlu receptor agonists inhibit the cysteine-glutamate antiporter via astrocytic mGlu3 receptors (Baker et al., 2002) and stimulate GLAST and GLT1 as well as the glutamate astrocycte transporters (Aronica et al., 2003; D’Antoni et al., 2008) all increasing uptake of glutamatelike substrates. Any subsequent release of such substrates is well positioned to activate presynaptic mGlu2/3 receptors (Kalivas, 2009). DHPG is also known to increase expression of the glutamate transporter, EAAC1 (Ross et al., 2011) and along with several other phenylglycine compounds may be a substrate for glutamate uptake (Gochenauer and Robinson, 2001). Nevertheless with all the above putative schemes, it seems likely that any recycling exogenous agonist would diffuse from the slice in a finite time and not result in LTD that is capable of lasting for hours. 4.4. Receptors remain active in the absence of exogenous agonist One possibility is that endogenous glutamate maintains activity of the mGlu receptors. For example, exposure to the exogenous agonist could lead to a chronic increase in the perisynaptic glutamate concentration, or to an increase in the affinity and/or efficacy of the mGlu receptors or to redistribution of receptors into a zone of higher glutamate exposure. Potentially, one or more of these mechanisms could result in an increase in glutamate receptor tone sufficient to maintain LTD. In the case of DHPG, the induced epileptiform activity reported here and previously (Merlin and Wong, 1997) may increase ambient glutamate levels and hence lead to continued mGlu receptor activation. This would constitute a form of “autopotentiation,” i.e., transient agonist-mediated selective activation results in long lasting enhanced responsiveness of group I mGlu receptors, allowing endogenous glutamate to now sustain the activation of the receptors (Bianchi et al., 2012). However, if ambient levels of glutamate do increase, one might then expect several of the mGlu receptor subtypes to be activated and contribute to the LTD. This appears not to be the case since we found that LTD induced by an agonist for a particular mGlu receptor subtype was essentially reversed fully by antagonists selective for that particular receptor (Figs. 1e6) and, where tested, not by antagonists selective for the other mGlu receptors (e.g. (Palmer et al., 1997)). Simplistically, one would not expect this hypothesis to extend to LTD evoked by agonists selective for group II and III mGlu receptors. These are largely presynaptic autoreceptors (Nicoletti et al., 2011; Niswender and Conn, 2010) which tend to reduce extracellular glutamate levels and hence would not be expected to contribute to their maintained activation. mGlu3 receptors located on glia could, however, contribute to changes in glutamate dynamics, possibly leading to agonist release onto presynaptic mGlu2 receptors (see 3.1). This is, however, not likely to be the major explanation for group II agonist-LTD since in the mGlu3/ mice, the extent of the LTD remained similar to that in wild-type mice (Fig. 3). The possibility that the induction of mGlu receptor mediated LTD involves an increase in receptor affinity could underlie each of the forms of LTD studied here. In each case, the acute affect of mGlu receptor activation is a depression of synaptic transmission and so an increase in the receptor affinity sufficient to permit its activation by ambient levels of L-glutamate would lead to a persistent

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reduction in synaptic strength. There is precedence for such a mechanism involving G-protein associated kainate receptors (Lauri et al., 2006). In neonatal tissue, an LTP stimulus leads to a rapid decrease in the affinity of kainate receptors such that they are no longer activated by ambient L-glutamate. This results in an increase in the probability of release. Conceivably, mGlu receptor mediated LTD could elicit the opposite effect; an increase in receptor affinity. This would need to be quite a substantial alteration, however, to account for the plasticity in tissue obtained from adult animals, where ambient L-glutamate concentrations are likely to be maintained at a very low level. Another possibility is that, after prolonged exposure to the exogenous agonist, the mGlu receptors become constitutively active, in a manner similar to that recently suggested for DHPGinduced epileptiform activity (Young et al., 2013). Prolonged or constitutive activity of G-proteins and downstream transduction processes per se is unlikely to be the explanation, since this would not be reversed by mGlu receptor antagonists. Constitutive activity of the receptor implies a conformational change such that even in the agonist-unbound state, the receptor continues to couple to intracellular G-proteins and the downstream transduction processes. Constitutive activity of G-protein coupled receptors is well

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documented (Cotecchia et al., 1990; Milligan et al., 1995; Seifert and Wenzel-Seifert, 2002; Tiberi and Caron, 1994, reviewed by Milligan, 2003) and has been proposed for mGlu receptors (Ango et al., 2001; Hermans and Challiss, 2001; Joly et al., 1995; Prézeau et al., 1996, and see Bockaert et al., 2004). Several conformational changes may induce such constitutive activity. Firstly, G-protein coupled receptors can interact with scaffolding and interacting proteins forming large macromolecular complexes. Such complexes have been termed ‘signalosomes’ and are capable of constitutive activity (Bockaert et al., 2004). One example of such a functional interaction with group I mGlu receptors occurs with the Homer class of scaffolding proteins. Normally Homer3 is present in the submembrane domains of these mGlu receptors and prevents constitutive signalling. This negative influence is not present in non-neuronal cells and hence constitutive activity is more common in heterologous systems especially at high levels of expression (e.g. Tiberi and Caron, 1994). Neuronal synaptic activity, however, can lead to expression of Homer1a which competes with Homer3 and allows G-protein activation by group I mGlu receptors to occur in the absence of agonist (Ango et al., 2001; Bockaert et al., 2004; Kammermeier and Worley, 2007). Interestingly, this form of constitutive activity is blocked by inverse agonists, e.g. MPEP, but

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Fig. 7. Hypothetical mechanism to explain mGlu receptor-LTD and its reversible reversal by antagonists. Baseline: mGlu receptors can bind synaptically released L-glutamate to become activated transiently or bind an antagonist that prevents their activation by L-glutamate (single monomers are shown for clarity). Under baseline conditions antagonists have little or no effect on synaptic strength and similarly the transient activation of mGlu receptors by L-glutamate has no discernible effect. Thus, the receptors display little or no constitutive activity (as indicated by the arrows favouring the inactive conformation of the receptor). Induction: Prolonged application of an exogenous agonist (such as DHPG) leads to a modification of the mGlu receptor such that there is an increased probability of it being in the constitutively active state. Expression: Upon washout of the exogenous agonist, the mGlu receptor remains in the constitutively active state, and actively depresses synaptic transmission. However, the constitutively active receptor is in equilibrium with the inactive conformation, which can bind the antagonist. Hence application of the antagonist leads to inhibition of the response but upon washout the equilibrium between inactive and constitutively active states is restored.

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not by competitive antagonists, e.g. LY367385 (Ango et al., 2001; Carroll et al., 2001). Therefore, it seems unlikely that this mechanism is the explanation for the LTDs induced by the agonists from each of the three mGlu receptor groups reported here, because all were sensitive to competitive antagonists. Secondly, in many G-protein coupled receptors, the agonist recognition site is within the heptahelical domain, although this is not the case for the transmitter glutamate with mGlu receptors. Nevertheless, class C receptors, to which mGlu receptors belong, have an equivalent domain which controls G-protein coupling (Malherbe et al., 2006). This site and other sites on the cytoplasmic and extracellular loops (Halls, 2012; Seifert and Wenzel-Seifert, 2002) can be modulated in the absence of a glutamate-like agonist to become constitutively active (Jensen and BräunerOsborne, 2007; Jensen et al., 2002; Yamashita et al., 2004; Yanagawa et al., 2009). Negative and positive allosteric modulators bind to this region or the equivalent region in other mGlu receptors (Carroll et al., 2001; Goudet et al., 2004; Malherbe et al., 2006; Pagano et al., 2000). In constitutive activity via this mechanism, both the orthosteric agonists and antagonists would be expected to be less effective (Yanagawa et al., 2009). A further possibility is shown schematically in Fig. 7. In the presence of agonist the clam shaped glutamate recognition domain adopts a closed (active) conformation whereas in the presence of the antagonist the clam is held open and hence inactive. However, in the absence of agonist or antagonist, these G-protein coupled receptors generally adopt a closed (active) conformation with a low probability relative to an open (active) conformation. It is therefore possible that prolonged administration of an agonist can induce a long lasting change in the receptor protein such that the closed (active) conformation becomes thermodynamically more probable. This scheme still allows an orthosteric antagonist to bind and hence block the response but upon washout the high probability of the closed state remains. Such a mechanism leading to constitutive activity has been induced in the presence of Gd3þ ions and possibly Ca2þ ions for the mGlu1 receptor (Jensen et al., 2002). Such constitutive activity, being sensitive to both orthosteric and allosteric antagonists, could underlie the observations in the present study. Presently, however, there is insufficient evidence to distinguish between the above possible mechanisms and to determine whether there are common features in the maintenance of LTD by all three groups of mGlu receptors. Further investigation is required. 4.5. Therapeutic relevance of the antagonist reversal phenomenon Does the ability of mGlu receptor antagonists to reverse the sustained effects of mGlu receptor ligands have any functional significance? With respect to group I mGlu receptors, the ability of antagonists to reverse epileptiform activity could be targeted therapeutically. It will be interesting to determine whether interictal, and/or ictal events present in epileptic human tissue are sensitive to mGlu receptor antagonists. Interestingly, mGlu5 receptor antagonists have been reported to have efficacy in animal models of various diseases, including autism spectrum disorders, fragile-X syndrome and tardive dyskinesia (Bianchi et al., 2012; Gürkan and Hagerman, 2012; Michalon et al., 2012; Rylander et al., 2009). The putative therapeutic effects could be due to antagonist prevention of the repeated induction of a pathological form of neuronal plasticity, or to antagonists reversing prolonged expression of the pathological plasticity. Conceivably, in various disease states, the affected mGlu receptor pathway may become constitutively active, in which case a therapeutic effect could be achieved by inhibition of the constitutive activity.

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