Synaptic activity, induced rhythmic discharge patterns, and receptor subtypes in enriched primary cultures of embryonic rat motoneurones

Abstract

Long-term cultures of ventral horn neurones from embryonic rat spinal cord were established, after enrichment using density gradient centrifugation, to give a high proportion of cells (>82%) with motoneurone characteristics. Neurones were grown on spinal cord glial monolayers for 4-83 days and investigated using whole-cell patch clamp. Synaptic activity interrupted by periods of quiescence increased in frequency with culture age and was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and strychnine. However, strychnine (10 µM) or bicuculline (10-30 µM) or removal of Mg2+ alone induced patterned rhythmic bursting. Glutamate (3-300 µM), alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA, 0.3-30 µM), and kainate (1-300 µM) evoked inward currents, as did N-methyl-D-aspartic acid (NMDA, 100 µM) in the absence of Mg2+ and presence of glycine (3-10 µM). Inward currents carried by Cl- were elicited by glycine (10-300 µM) and GABA (1-300 µM), while adenosine (1-10 µM) and cyclopentyladenosine (10 nM - 1 µM) evoked a K+-dependent hyperpolarization. 5-HT, GABAB, purine A, and metabotropic glutamate receptors modulated synaptic excitation of presumed motoneurones. The results suggest that long-term cultures, containing more than 82% developing motoneurones, are able to generate rhythmic bursting; they respond to many of the neurotransmitters that are likely to be released onto motoneurones developing in vivo.Key words: embryonic rat motoneurones, culture, amino acid receptors, adenosine, spinal cord.