The potent mGlu receptor antagonist LY341495 identifies roles for both cloned and novel mGlu receptors in hippocampal synaptic plasticity

Abstract

Understanding the roles of metabotropic glutamate (mGlu) receptors has been severely hampered by the lack of potent antagonists. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid) has been shown to block group II mGlu receptors in low nanomolar concentrations (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 at group II metabotropic glutamate receptors. Neuropharmacology 37, 1–12) but can be used in higher concentrations to block all hippocampal mGlu receptors, identified so far by molecular cloning (mGlu_{1–5, 7,8}). Here we have further characterised the mGlu receptor antagonist activity of LY341495 and have used this compound to investigate roles of mGlu receptors in hippocampal long-term potentiation (LTP) and long-term depression (LTD). LY341495 competitively antagonised DHPG-stimulated PI hydrolysis in AV12-664 cells expressing either human mGlu₁ or mGlu₅ receptors with K_i-values of 7.0 and 7.6 μM, respectively. When tested against 10 μM l-glutamate-stimulated Ca²⁺ mobilisation in rat mGlu₅ expressing CHO cells, it produced substantial or complete block at a concentration of 100 μM. In rat hippocampal slices, LY341495 eliminated 30 μM DHPG-stimulated PI hydrolysis and 100 μM (1S,3R)-ACPD-inhibition of forskolin-stimulated cAMP formation at concentrations of 100 and 0.03 μM, respectively. In area CA1, it antagonised DHPG-mediated potentiation of NMDA-induced depolarisations and DHPG-induced long-lasting depression of AMPA receptor-mediated synaptic transmission. LY341495 also blocked NMDA receptor-independent depotentiation and setting of a molecular switch involved in the induction of LTP; effects which have previously been shown to be blocked by the mGlu receptor antagonist (S)-MCPG. These effects may therefore be due to activation of cloned mGlu receptors. In contrast, LY341495 did not affect NMDA receptor-dependent homosynaptic LTD; an effect which may therefore be independent of cloned mGlu receptors. Finally, LY341495 failed to antagonise NMDA receptor-dependent LTP and, in area CA3, NMDA receptor-independent, mossy fibre LTP. Since in the same inputs these forms of LTP were blocked by (S)-MCPG, a novel type of mGlu receptor may be involved in their induction.

Introduction

l-Glutamate, the principal excitatory neurotransmitter in the brain, activates a family of G-protein linked receptors, termed metabotropic glutamate (mGlu) receptors, of which eight subtypes have been cloned (Nakanishi and Masu, 1994, Conn and Pin, 1997). However, identification of the roles of these receptors has been hindered by the lack of potent antagonists (Watkins and Collingridge, 1994). This is typified in the hippocampus where pharmacological studies have investigated the potential roles of mGlu receptors in the induction of LTP and LTD (Bliss and Collingridge, 1993, Bear and Abraham, 1996). Of the cloned mGlu receptor subtypes, group I (i.e. mGlu₁ and mGlu₅ receptors) have been most often implicated in LTP for several reasons. Firstly, these receptors couple to PLC (Nakanishi and Masu, 1994, Conn and Pin, 1997) and can thereby affect Ca²⁺ signalling and kinases that have been implicated in the induction of LTP (Bliss and Collingridge, 1993). Secondly, antagonists that have actions at group I mGlu receptors, in particular (S)-MCPG (Eaton et al., 1993), can block the induction of LTP (Bashir et al., 1993a, Sergueeva et al., 1993, Aiba et al., 1994, Bortolotto et al., 1994, O’Connor et al., 1994, Richter-Levin et al., 1994, Little et al., 1995, Wang et al., 1995, Manahan-Vaughan, 1997, Breakwell et al., 1998). Thirdly, deficits in LTP have been described in mice lacking mGlu₁ receptors (Aiba et al., 1994, Conquet et al., 1994) and mGlu₅ receptors (Lu et al., 1997). However, these data are controversial, since (S)-MCPG does not invariably block the induction of LTP (Chinestra et al., 1993, Bortolotto et al., 1994, Manzoni et al., 1994, Selig et al., 1995, Thomas and O’Dell, 1995, Wang et al., 1995, Martin and Morris, 1997) and since deficits in LTP in area CA1 of the mGlu₁ receptor knockout were not observed in one of the two original reports (Conquet et al., 1994). A similar controversy surrounds studies on the roles of mGlu receptors in LTD with both positive (Bashir et al., 1993b, Bashir and Collingridge, 1994, Bolshakov and Siegelbaum, 1994, Yang et al., 1994, Manahan-Vaughan, 1997, Oliet et al., 1997) and negative (Selig et al., 1995, Oliet et al., 1997) reports on the effectiveness of (S)-MCPG.

Recently, LY341495 has been shown to inhibit all known mGlu receptor subtypes (except for retina-specific mGlu₆ which was not tested) with IC₅₀-values ranging from 14 nM (mGlu₃) to 22 μM (mGlu₄) (Kingston et al., 1998). Therefore, LY341495 is the first compound that can be used to block the activation of all the mGlu receptor subtypes in the brain that have been identified so far by cloning, and thereby determine to what extent the activation of one or more of these mGlu receptors is essential for specific functions.

In the present study we have further characterised LY341495 to determine the effective concentration required to antagonise activation of all mGlu receptors, so far identified by molecular cloning. We have then used 100 μM LY341495 to determine whether activation of any of these mGlu receptors is necessary for the induction of both NMDA receptor-dependent and NMDA receptor-independent forms of hippocampal LTP and LTD.

Some of these data have been presented as abstracts at the Forum of European Neuroscience, Berlin (Bortolotto et al., 1998, Fitzjohn et al., 1998).