An improved cell isolation technique for studying intracellular Ca(2+) homeostasis in neurones of the cochlear...

Neurones isolated from various parts of the brain are used extensively for electrophysiological and immuncytochemical studies, as well as to investigate their Ca(2+) homeostasis. In this work we report on an isolation technique that yielded neurones suitable for functional studies targeting the investigation of their Ca(2+) handling mechanisms. The cell isolation involved enzymatic dissociation with combined collagenase/pronase treatment and gentle mechanical trituration. At the end of the isolation the cells were incubated in a cell culture incubator (CO2 concentration = 5.1%) at 37 degrees C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated horse serum. The vitality of the isolated cells was indicated by their low intracellular Ca(2+) concentrations (17.2 +/- 0.5 nM; n = 38) and by their ability to produce large Ca(2+) transients on depolarization. These Ca(2+) transients were rapidly terminated and the resting intracellular Ca(2+) concentration was quickly restored proving that isolation did not compromise the Ca(2+) homeostatic mechanisms of the nerve cells. The technique allowed reliable, long (45-60 min) and reproducible measurements of Ca(2+) currents on these neurones as well as the recording of their intracellular Ca(2+) concentration. Our results indicate that incubation in DMEM with horse serum markedly increases the number of surviving neurones after the enzyme treatment, and their Ca(2+) homeostasis can be studied for significantly longer periods of time.