COBALT ACCUMULATION IN NEURONS EXPRESSING IONOTROPIC EXCITATORY AMINO-ACID RECEPTORS IN YOUNG-RAT SPINAL-CORD - MORPHOLOGY AND DISTRIBUTION

Excitatory amino acids (EAA) acting on N-methyl-D-aspartate (NMDA), al pha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kai nate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiment s suggest that NMDA receptors are localized mainly in lamina II while kainate and AMPA receptors are found on both dorsal and ventral horn n eurons. However the cell types expressing EAA receptors and their lami nar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressin g AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusio n of hemisected spinal cords of 14 day-old rat pups in vitro with exci tatory amino acid receptor ligands in the presence of CoCl2. Cobalt ha s been shown to enter cells through ligand-gated ion channels in place of Ca2+. Cells which accumulated cobalt ions following activation by ionotropic excitatory amino acid receptors were visualised histochemic ally. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric ac id (D-AP-5) (20 mu M) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-se lective excitatory amino acid receptor antagonist kynurenic acid (4 mM ). Passive opening of the voltage-gated Ca2+-channels by KCl (50 mM) d id not result in cobalt uptake, indicating that cobalt enters the cell s through ligand-gated Ca2+-channels. AMPA (500 mu M), kainate (500 mu M), or NMDA (500 mu M) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate ind uced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA sup erfusion, resulted in cobalt labeling of glial cells. Our results show that the cobalt uptake technique is a useful way to study the morphol ogy and distribution of cells expressing receptors with ligand-gated C a2+ channels. (C) 1994 Wiley-Liss, Inc.