Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures

Abstract

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.

Inorganic mercury modifies Ca2+ signals, triggers apoptosis and potentiates NMDA toxicity in cerebellar granule neurons

“…After washing, normal Ca 2+ resting levels were reinstated. A second KCl addition triggered a similar Ca 2+ response in 90% of the cells, whereas in the remaining 10% the rise in [Ca 2+ ] i was smaller than that elicited by the first depolarization, in accordance with previous observations (Connor et al, 1987). When Hg 2+ (0.1 mM) was added after the first stimulation, the second Ca 2+ peak increased in the majority of the cells, while the plateau was unaffected (Figure1B).…”

“…While in immature neuronal cells, increased [Ca 2+ ] i following exposure to high K + protects cells lacking growth stimulation (Koike et al, 1989), Ca 2+ overload in differentiated neurons rapidly alters neurotransmitter release and can cause cell death (Hartley et al, 1993). Ca 2+ responses to high K + are amplified with cell differentiation (Connor et al, 1987) and more ion channels are expressed in mature and ageing neurons (Thibault and Landfield, 1996) than in neuroblasts or undifferentiated cell lines (Kalman et al, 1990;Janigro et al, 1989). According to the`Ca 2+ set point' hypothesis (Koike et al, 1989) increasing [Ca 2+ ] i may be required to prevent the adverse effects of growth factor deprivation in immature neurons, whereas a higher [Ca 2+ ] i setpoint would result in killing of mature post-mitotic neurons.…”

Regulation of Intracellular pH in Single Rat Cortical Neurons in vitro: A Microspectrofluorometric Study

“…We always focused upon the cell body of the neuron. Al though the neurons generally grew on a layer of glia cells, the contamination of signal from glia was low: As ob served by others, the uptake of dye was much lower in glia than in neurons (Connor et al, 1987;Nedergaard et al, 199 1). Glial cells were thin and well spread.…”

Bioenergetic analysis of cerebellar granule neurons undergoing apoptosis by potassium/serum deprivation

“…The FURA-PE3 ratio was not calibrated in this study, but from previous experiments, [40][41][42] þ buffers. Statistics: repeated measures one-way ANOVA for DHE oxidation rate indicated no significant treatment effect (P ¼ 0.15, 0.14) for experiments (a and c) but significance (P ¼ 0.02) for experiment (b), values sharing common superscripts are not different.…”