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Journal of Neuroscience, Vol 7, 3230-3244, Copyright © 1987 by Society for Neuroscience
Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using arsenazo III
ML Mayer, AB MacDermott, GL Westbrook, SJ Smith and JL Barker
Laboratory of Developmental Neurobiology, NICHD, Bethesda, Maryland 20892.
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.
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