Preparation and maintenance of single-cell micro-island cultures of basal forebrain neurons

Micro-island cultures provide a simplified system for studying the expression of cellular phenotype, excitability, synapse formation and pre- and postsynaptic regulatory mechanisms without the usual problems that arise from complex interactions between large numbers of other cells. The technique relies on the ability to constrain the attachment and growth of either single or small groups of neurons to discrete (20-500 microm) 'islands' of cell-permissive substrate applied over a nonadherent background layer. Constrained in this way, neurons form large numbers of conventional synaptic and/or autaptic contacts that can be easily visualized, making them ideally suited for studying synaptic physiology using electrophysiological and/or high-resolution optical imaging techniques. The protocol described here requires approximately 2 h for preparation of the culture dishes and a further 3-4 h for isolation and plating out the cells. Once established, the cultures can be maintained for prolonged periods (>6 weeks) permitting manipulations to be made to their local environment and the effects on individually identified cells to be repeatedly monitored.