An improved cell isolation technique for studying intracellular Ca2+ homeostasis in neurones of the cochlear nucleus

Neurones isolated from various parts of the brain are used extensively for electrophysiological and immuncytochemical studies, as well as to investigate their Ca2+ homeostasis. In this work we report on an isolation technique that yielded neurones suitable for functional studies targeting the investigation of their Ca2+ 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°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 Ca2+ concentrations (17.2±0.5 nM; n=38) and by their ability to produce large Ca2+ transients on depolarization. These Ca2+ transients were rapidly terminated and the resting intracellular Ca2+ concentration was quickly restored proving that isolation did not compromise the Ca2+ homeostatic mechanisms of the nerve cells. The technique allowed reliable, long (45–60 min) and reproducible measurements of Ca2+ currents on these neurones as well as the recording of their intracellular Ca2+ concentration. Our results indicate that incubation in DMEM with horse serum markedly increases the number of surviving neurones after the enzyme treatment, and their Ca2+ homeostasis can be studied for significantly longer periods of time.