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Journal of Neuroscience, Vol 7, 1384-1400, Copyright © 1987 by Society for Neuroscience
Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures
JA Connor, HY Tseng and PE Hockberger
Digital imaging of the Ca indicator fura-2 has been used to study the
responses of developing granule cells in culture to depolarization and
transmitter action. Unstimulated cells bathed in Krebs saline exhibited
cytoplasmic Ca ion concentrations, [Ca2+], that were generally in the 30-60
nM range. Exposure of cells to high-potassium (25 mM) saline depolarized
the membrane potential and produced an immediate rise in [Ca2+] that
recovered within 2-3 min in normal saline. The response grew progressively
larger over the first 20 d in culture. Transient increases in [Ca2+] to
levels greater than 1 microM were observed after 12-14 d in vitro, at which
time the cells displayed intense electrical activity when exposed to high
K. At this stage, the increases were attenuated by blocking action
potential activity with TTX. In TTX- treated or immature cells, in which
the transient phase of the Ca change was relatively small, a second
exposure to high K typically produced a much larger Ca response that the
initial exposure. The duration of this facilitation of the response
persisted for periods longer than 5 min. Application of the
neurotransmitter GABA induced a transient increase in membrane conductance,
with a reversal potential near resting potential (approx. -60 mV), and
caused an intracellular Ca2+ increase that outlasted the exposure to GABA
by several minutes. Glutamate, or kainate, induced an increase in membrane
conductance but with a reversal potential more positive than spike
threshold. These agents also elevated intracellular Ca2+, but unlike the
case with GABA, this Ca response reversed rapidly upon removal of the
transmitter. The facilitatory effect of repeated exposures to high-K
saline, as well as the persistent Ca elevation following a brief GABA
application, suggests that granule cells possess the capability of
displaying activity-dependent changes in Ca levels in culture.
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