Cobalt accumulation in neurons expressing ionotropic excitatory amino acid receptors in young rat spinal cord: Morphology and distribution

Abstract

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.

In SituCa2+Imaging Reveals Neurotransmitter Receptors for Glutamate in Taste Receptor Cells

“…For instance, in the present study we recorded from a restricted sample of taste cells (those for which the apical processes reached the taste pore) that might have a larger proportion of glutamate-responsive cells. Furthermore, the present studies were able to reveal NMDA responses, whereas NMDA receptors generally cannot be visualized by using the Co 2ϩ technique [Caicedo et al (2000), but see Nagy et al (1994)]. …”

Impairment of the Ca2+‐Permeable AMPA/Kainate Receptors by Lead Exposure in Organotypic Rat Hippocampal Slice Cultures

“…Cellular accumulation of cobalt was assessed essentially as described by Pruss et al (1991) and Nagy et al (1994). After 3 to 5 days in vitro, the slices were placed in a cobalt-containing (CoCl 2 5 mM) buffer (NaCl 57.5 mM, KCl 5 mM, MgCl 2 2 mM, CaCl 2 1 mM, HEPES 10 mM, glucose 12 mM, sucrose 139 mM, tetrodotoxin 300 nM) for 20 min.…”

“…The cobalt accumulation was correlated with the accumulation of 45 Ca 2π in neurones within slice preparations of the hippocampus (Pruss & Akeson 1990;Pruss et al 1990). Cobalt uptake, followed by histochemical visualization, has been used to monitor calcium influx through AMPA/kainate-operated channels on neurons and glial cells in culture after stimulation by the respective glutamate receptor agonists (Pruss et al 1991;Nagy et al 1994). The objective of this study was to investigate the effects of lead exposure on Ca 2π -permeable AMPA/kainate receptors of rat hippocampus in organotypic slice culture using glutamate agonists-induced cobalt uptake.…”

Cardiovascular/Respiratory Control

“…Whilst both glutamate binding sites (Monaghan & Cotman, 1985) and excitatory amino acid (EAA) receptors (Robinson & Ellenberger, 1997) have been localized within the nucleus ambiguus, as with the majority of other putative neurotransmitters, their location with regard to specific functionally distinct populations of neurones remains to be determined. Therefore, the aim of the present study was to utilize a cobalt uptake technique (Nagy et al 1994) to determine the localization of specific ionotropic EAA receptors within the various subregions of the nucleus ambiguus.…”

“…In some experiments, the antagonists CNQX (100,uM), kynurenic acid (0'3-4 mM) or APV (100,uM) were added to the incubation media 10 min prior to the addition of the EAA agonist. Following the incubation period, the accumulated Co was precipitated out within the cell by incubation with an ACSF containing l' 2 % NH 4 S. The slices were fixed overnight in 4 % paraformaldehyde before resectioning and amplification of the Co precipitate utilizing a silver intensification process (Nagy et al 1994).…”