Isolated identified Aplysia neurons in cell culture

Abstract

Methods have been developed for primary culture of large identified Aplysia neurons. Aplysia ganglia were treated with neutral protease to soften the connective tissue sheath. Individual neurons were isolated either by manipulation with tungsten needles or by tying off their axons with fine nylon filament and were immobilized in a chick plasma clot or a solution of methylcellulose. Somata up to approximately 300 μm in diameter extended long processes within several hours in culture. A single neuron produced as many as 10 processes which could grow at different rates. Intracellular recordings showed spontaneous and evoked actin potentials in neurons cultured for up to 6 weeks. Electrical synapses formed between pairs of neurons in culture. In several culture dishes containing neurons from buccal ganglia, electrical coupling was observed between 90% of the cell pairs tested. This primary culture system currently is being used to compare the electrical and biochemical properties of neuronal processes with those of cell bodies and to study the conditions necessary for process regeneration and synapse formation between isolated identified neurons.