Excitatory amino acids and synaptic transmission in embryonic rat brainstem motoneurons in organotypic culture.

Abstract

We used brainstem motoneurons recorded in organotypic slice co-cultures maintained for more than 18 days in vitro, together with multibarrel ionophoretic applications of glutamate receptor agonists and bath applications of specific blocking agents, to study the responses of rat brainstem motoneurons to glutamate receptor activation, and the contribution of these receptors to synaptic transmission. Differentiated brainstem motoneurons in vitro are depolarized by glutamate, N-methyl-d-aspartate (NMDA) and dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) iontophoresis, and express NMDA, AMPA and also specific kainate receptors, as evidenced by (+/-)2-amino-5-phosphonovaleric acid (APV)- and (-)1-(4-aminophenyl)-3-methyl-carbamoyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzo-diazepine [GYKI 53784 (LY303070)]-resistant depolarizations. Electrical stimulations applied to the dorsal part of the explant trigger excitatory synaptic potentials with latencies distributed in three regularly spaced groups. Excitatory postsynaptic potentials (EPSPs) in the earliest group have a similar latency and time course and correspond to monosynaptic activation. EPSPs in later groups have more scattered latencies and time courses and correspond to polysynaptic activation. Monosynaptic EPSPs are insensitive to the specific NMDA blocker APV, and are completely and reversibly suppressed by the non-competitive AMPA receptor antagonist GYKI 53784 (LY303070). Detailed analysis of the spontaneous excitatory synaptic activity shows that APV decreases the frequency of spontaneous EPSPs without modifying their shape or amplitude. We conclude that excitatory synapses on brainstem motoneurons in vitro are mainly activated through AMPA receptors (AMPA-Rs). NMDA receptors (NMDA-Rs) are present in the membrane, but are located either at extrasynaptic sites or silent synapses, and are not directly involved in synaptic transmission on motoneurons. On the contrary, NMDA receptors contribute to synaptic transmission within the premotor interneuronal network.