Elsevier

Neuropharmacology

Volume 38, Issue 10, October 1999, Pages 1553-1567
Neuropharmacology

Modulation of synaptic transmission and differential localisation of mGlus in cultured hippocampal autapses

https://doi.org/10.1016/S0028-3908(99)00103-3Get rights and content

Abstract

Metabotropic glutamate receptors (mGlus) are known to modulate synaptic transmission in various pathways of the central nervous system, but the exact mechanisms by which this modulation occurs remain unclear. Here we utilise electrophysiological and immunocytochemical techniques on cultured autaptic hippocampal neurones to investigate the mechanism of action and distribution of mGlus. Agonists at all three groups of mGlus depressed glutamatergic transmission, whereas only agonists at group I mGlus depressed GABAergic transmission. Agonists at all mGlus failed to modulate Ca2+ and K+ channels in glutamatergic autapses whereas an agonist at group III mGlus did depress the frequency of miniature excitatory postsynaptic currents (mEPSCs). Agonists failed to modulate Ca2+ or K+ channels and miniature inhibitory postsynaptic currents (mIPSCs) in GABAergic autapses. Distribution studies using selective antibodies revealed punctate staining for group III mGlus that co-localised with the synaptic marker, synaptophysin. Staining for the remaining mGlus was more diffuse throughout the soma and processes with little co-localisation with synaptophysin. The distribution of the group III receptors is consistent with the direct ‘downstream’ modulation of mEPSCs, although the exact mechanism of action for the remaining receptors remains unclear.

Introduction

Excitatory synaptic transmission in the central nervous system (CNS) is mediated by either ionotropic (iGlus) or metabotropic glutamate receptors (mGlus) at the majority of synapses. The iGlus, e.g. NMDA, AMPA and kainate (Hollmann and Heinemann, 1994) are thought to mediate fast synaptic transmission whereas the mGlus are thought to be involved in slower G-protein mediated effects. mGlus can be divided into three subgroups according to sequence homology, pharmacology and signal transduction mechanisms determined in both neurones and diverse expression systems. Eight mGlus receptors have currently been identified and cloned. Group I mGlus consist of mGlu1 and mGlu5 and are linked to phosphoinositide hydrolysis. Group II mGlus, mGlu2 and mGlu3, and group III mGlus, mGlu4, mGlu6, mGlu7 and mGlu8, have been shown to inhibit adenylate cyclase (Pin and Duvoisin, 1995, Conn and Pin, 1997).

mGlus have been the subject of intense investigation over recent years and have been shown to play a role in phenomena such as neuroplasticity, neurotoxicity and neuromodulation (Pin and Duvoisin, 1995, Conn and Pin, 1997). Studies have shown that mGlus can modulate excitatory and inhibitory synaptic transmission in many, if not all, pathways in the CNS (Pin and Duvoisin, 1995, Conn and Pin, 1997, Nakanishi et al., 1998). These studies suggest that mGlus operate throughout the CNS as regulators of synaptic transmission.

Several mechanisms have been proposed as underlying the modulation of transmission by mGlus. It has been shown that inhibition of Ca2+ channels, activation of K+ and reduction in the frequency of mini EPSCs/IPSCs may account for the modulation of synaptic transmission observed (Pin and Duvoisin, 1995, Conn and Pin, 1997).

Autapses have been shown to be present in the brain by both morphological and electrophysiological criteria (Tamas et al., 1997, Pouzat and Marty, 1998). Recently several groups have utilised cultured autaptic hippocampal neurones to study synaptic transmission and have shown these synapses to have properties that are similar to, if not identical to, those found in hippocampal slices (Bekkers and Stevens, 1991, Reid et al., 1998).

In order to further understand how mGlus produce modulation of synaptic transmission, we have used electrophysiological and immunocytochemical techniques to study the modulation by and distribution of mGlus in both excitatory and inhibitory synaptic transmission in autaptic hippocampal cultures.

Section snippets

Cell culture

Primary non-autaptic hippocampal neuronal cultures were prepared as described previously (Scholz et al., 1987). Autaptic hippocampal cultures were prepared as follows. Hippocampi were dissected from foetal rats of 17 days gestation and incubated in 0.1% trypsin for 20 min (Worthington Biochemical Corporation, Lakewood, NJ). The hippocampi were then triturated approximately ten times using a Pasteur pipette, and a further ten times with a flame narrowed Pasteur pipette, in the presence of

Depression of evoked excitatory transmission at hippocampal autapses

The effects of agonists at all three subgroups of mGlus were investigated on evoked transmission in cultured excitatory autapses. These autapses were glutamatergic in nature as demonstrated by the block of transmission by the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM; n=5; data not shown).

Activation of all three groups of mGlus resulted in the inhibition of glutamatergic transmission. The group I selective agonist, (S)-3,5-dihydroxyphenylglycine (Ito et

Discussion

The data presented in this manuscript provide further evidence of a role for multiple mGlus in the modulation of neurotransmission. We observed that transmission in 10–17 DIV hippocampal glutamatergic autapses was inhibited by agonists at all three groups of mGlus. This data is in agreement with the previously reported modulation of transmission in various pathways of hippocampus (Pin and Duvoisin, 1995, Conn and Pin, 1997). These studies have described that specific pathways within the

Acknowledgements

This work was supported by a Wellcome International Travel Fellowship to TJB and by the Public Service Grants DA02121, MH40165, NS33502 and NS33826 for CCL and RJM. We are grateful to Dr Graeme I. Bell for use of his microscope for the antibody studies.

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