Fluorimetric determination of electrically evoked increase in intracellular calcium in cultured cerebellar granule cells

A technique is described to measure the electrically evoked increase in intracellular calcium in cerebellar granule cells cultured on glass coverslips and preloaded with FURA-2. To minimize light scattering, the coverslip containing the granules was placed in the fluorimeter cuvette at a 30° angle to the exciting light beam. The cuvette was provided with 2 platinum electrodes so as to stimulate the neurons with a tangential field. The [Ca2+]itransients were maximized by omitting Mg2+. The fluorescence peaks were directly related to the pulse (1 ms, 100 mA) frequency and to the train length. The responses were completely tetrodotoxin- and [Ca2+]o-dependent and could be replicated 5-6 times at 5-min intervals. At the stimulation rate of 20 Hz for 5 s, a condition ensuring submaximal peaks, the [Ca2+]irose from the basal levels of 41 ± 2.7 nmol/l to 89.6 ± 5.8 nmol/1. The participation of various membrane channels in the electrically induced [Ca2+]iincrease was demonstrated. 4-Aminopyridine (1 mM) increased the height of the peaks to 240%. Both nifedipine (10 μM) and ω-conotoxin (1 μM) reduced the transients by about 25%. The residual response (in the absence of Mg2+) depended mostly on the release of endogenous glutamate as it proved sensitive to NMDA, AMPA and t-ACPD receptor antagonists. Since a technique to measure the electrically evoked release of d-[3H]aspartate is presently available, the parallel determination of release and of [Ca2+]iin twin populations of cultured granule cells is possible. © 1994.