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), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiments suggest that NMDA receptors are localized mainly in lamina II while kainate and AMPA receptors are found on both dorsal and ventral horn neurons. However the cell types expressing EAA receptors and their laminar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressing AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusion of hemisected spinal cords of 14 day-old rat pups in vitro with excitatory amino acid receptor ligands in the presence of CoCl2. Cobalt has 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 visualized histochemically. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric acid (D-AP-5) (20 microM) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-selective excitatory amino acid receptor antagonist kynurenic acid (4 mM). Passive opening of the voltage-gated Ca(2+)-channels by KCl (50 mM) did not result in cobalt uptake, indicating that cobalt enters the cells through ligand-gated Ca(2+)-channels. AMPA (500 microM), kainate (500 microM), or NMDA (500 microM) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA superfusion, resulted in cobalt labeling of glial cells. Our results show that the cobalt uptake technique is a useful way to study the morphology and distribution of cells expressing receptors with ligand-gated Ca2+ channels.
Nagy
I
I
Department of Anatomy and Developmental Biology, University College London England.
Woolf
C J
CJ
Dray
A
A
Urbán
L
L
D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism*; D016257:Fura-2; D015640:Ion Channel Gating / Q000187:drug effects; D009457:Neuroglia / Q000378:metabolism / Q000648:ultrastructure; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D017459:Receptors, Amino Acid / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D018092:Kainic Acid Receptors / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure
D015220:Calcium Channels; D003035:Cobalt; D016257:Fura-2; D018091:Receptors, AMPA; D017459:Receptors, Amino Acid; D018092:Kainic Acid Receptors; D016194:Receptors, N-Methyl-D-Aspartate