Measurement of intracellular Ca2+ in cerebellar Purkinje neurons in culture: resting distribution and response to glutamate.

Ca ion levels in cerebellar Purkinje neurons, in culture, have been measured using the fluorescent indicator, fura-2, and digital imaging. Cells were loaded with the indicator both by injecting the free acid form and by allowing the membrane permeant form (/AM) become deesterified and trapped. The two methods gave significantly different results in that the /AM loaded cells showed localized regions of high Ca2+ in the soma whereas the injected cells did not. Resting levels in the remainder of the cytoplasm were similar however, as were the excursions in Ca2+ induced by electrical or chemical stimulation. Comparison of the data from the two methods suggests that qualitative measures of Ca in intracellular stores can be derived from the /AM loading method. Injected cells showed high Ca2+ levels in the soma that persisted for 3-8 minutes following removal of the injection electrode. The dendrites of these cells however maintained low Ca2+ levels and differences of several hundred nM in Ca2+ were maintained between the soma and initial dendrite segment, demonstrating directly the large Ca pumping capacity of the dendrites. Localized regions of high Ca2+ in dendrites could be generated by applying glutamate from a microelectrode in TTX-Krebs saline. When studied in culture media with 4.7 mM K, the Purkinje neurons showed a biomodal distribution of Ca2+ with 35 to 40% showing stable Ca2+ levels between 250 and 350 nM, and the remainder 80 to 130 nM Ca2+. Granule neurons on the same coverslips had Ca2+ level in the lower range in greater than 95% of the examples observed.(ABSTRACT TRUNCATED AT 250 WORDS)