title: Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using arsenazo III.
abstract: Spinal cord neurons is dissociated cell culture were loaded with the calcium indicator arsenazo III using the whole-cell patch-clamp recording technique. Under voltage-clamp, depolarizing voltage steps evoked transient increases in absorbance at 660 nm, with no change at 570 nm, the isosbestic wavelength for calcium-arsenazo III complexes. The optical response occurred with a threshold depolarization to -30 mV, peaked at +10 mV, and decreased with further depolarization, consistent with an elevation of cytoplasmic free calcium resulting from Ca2+ flux through voltage-dependent calcium channels. Inward current responses to the excitatory amino acids N-methyl-D-aspartic acid (NMDA) and L-glutamate were also accompanied by calcium transients; these were dose-dependent, varied with the driving force for inward current, and were blocked by extracellular Mg2+ in a voltage-dependent manner, suggesting Ca2+ flux through NMDA-receptor channels. Responses to kainate, quisqualate, and GABA were not accompanied by comparable calcium transients. [Ca2+]i transients evoked by depolarizing voltage steps were of maximal amplitude at the start of recording and declined with time, reflecting rundown of voltage-dependent calcium channels. In contrast, [Ca2+]i transients evoked by NMDA gradually increased in amplitude during periods of whole-cell recording lasting 1-2 hr. Procedures resulting in loading of the neuron with Ca2+ accelerated the increase in amplitude of [Ca2+]i transients evoked by NMDA, but slowed the decay of [Ca2+]i transients evoked by voltage steps. Our results provide evidence for 2 independent sources of transmembrane Ca2+ flux in vertebrate neurons, through voltage-gated calcium channels and through NMDA-receptor channels. The Ca2+ flux gated by NMDA-receptor-specific agonists may play a role in synaptic plasticity, in regulating excitability, and in the excitotoxic response to excitatory amino acids.
mesh_terms: D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001150:Arsenazo III; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001391:Azo Compounds; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D007473:Ion Channels / Q000502:physiology; D051379:Mice / Q000196:embryology; D016202:N-Methylaspartate; D009474:Neurons / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism* / Q000502:physiology
chemical_list: D000596:Amino Acids; D001391:Azo Compounds; D007473:Ion Channels; D016194:Receptors, N-Methyl-D-Aspartate; D017981:Receptors, Neurotransmitter; D001150:Arsenazo III; D001224:Aspartic Acid; D016202:N-Methylaspartate; D002118:Calcium