Dissociation of postnatal hippocampal neurons for short term culture

A method is described to isolate hippocampal neurons from postnatal day 7 to postnatal day 21 rats for use in short term cultures using a combination of enzymatic and mechanical dissociation. A significant fraction of the cells (37.5 +/- 2.3%, n = 10 cultures) were labelled with anti-GABA antibodies and the neurons survived a minimum of 8 days in culture. Patch-clamp recording in the current clamp mode revealed an average membrane potential and input resistance of - 47.6 +/- 2.5 mV and 737 +/- 147 M omega respectively, after 24 h in culture. The cells exhibited normal excitability and fired multiple action potentials in response to a depolarizing pulse. The technique described here provides a good alternative to either the use of embryonic brain tissue for cultures used in electrophysiological studies which may not exhibit the mature phenotype or use of acutely isolated cells which may be unstable or have their channels and receptors modified by enzymatic treatment.