LOBSTER STOMATOGASTRIC NEURONS IN PRIMARY CULTURE .1. BASIC CHARACTERISTICS

1. A method for the isolation of stomatogastric neurons with neuropila r processes and an axon less-than-or-equal-to 2 mm long is described, Isolated neurons adhered to an uncoated plastic surface and demonstrat ed neurite outgrowth for greater-than-or-equal-to 7-10 days in a simpl e medium (salt-adjusted Leibovitz-15). Neurite outgrowth started immed iately after plating and was maximal during the first 2-3 days. The el ectrical activity of neurons and their responses to bath application o f pilocarpine were studied between 2 and 10 days after plating. 2. Ide ntified neurons [pyloric dilator (PD), pyloric (PY), and lateral pylor ic (LP) neurons from the pyloric pattern generator as well as gastric mill (GM) and lateral posterior gastric (LPG) neurons from the gastric mill pattern generator], isolated with neuropilar processes and axons , behaved in general like corresponding neurons in the isolated stomat ogastric ganglion (STG). PD neurons were tonically active or silent in culture; pilocarpine caused them to begin rhythmic activity, which at particular levels of imposed polarization was similar to the pyloric rhythm in vitro. PY and LP neurons were silent. Pilocarpine produced s ome rhythmicity in the PY neuron, whereas in LP neurons it decreased t he firing threshold to depolarizing current and accentuated postinhibi tory rebound. LPG neurons were tonically active. Pilocarpine depolariz ed the LPG neurons and accelerated their tonic activity; neuron hyperp olarization by current injection led to bursting pacemaker activity th at was similar to the gastric rhythm in vitro. GM neurons were silent; pilocarpine did not cause them to generate rhythmic activity but did lower their thresholds to depolarizing current. Simultaneous recording s from the soma and axon under direct visual control demonstrated that the intrasomatic spikes ( 15-20 mV in amplitude) were attenuated acti on potentials generated in the axon. 3. Neurons isolated with short pr imary neurites, including those without any noticeable primary neurite (in contrast to neurons isolated with longer neuropilar processes and axons), never generated any kind of electrical activity immediately a fter extraction from the STG. After 2 days in culture, these ''short-n eurite'' neurons became capable of generating different types of elect rical activity (e.g., fast spikes with amplitudes of less-than-or-equa l-to 40-45 mV, plateau potentials, bursting potentials, etc.). The cap ability of isolated somata to generate electrical activity did not dep end on whether or not the cell had adhered to the substrate and demons trated neurite outgrowth. Some cells isolated with (or without) short primary neurites expressed pacemaker-like properties without neuromodu latory substances. In this respect, they differed from neurons isolate d with longer neuropilar processes and axons which, like neurons in si tu, exhibited rhythmic activity only in the presence of pilocarpine. M ost neurons isolated with short primary neurites were sensitive to pil ocarpine, which evoked neuron depolarization and affected the pattern of their activity. 4. To examine the possibility of cultured neurons f orming electrical and chemical connections, the processes of two or mo re neurons were placed in close contact with one another. In two cases out of 19 trials, electrical coupling between neurons (2 GM and two P D, known to have electrical connections in situ) was noted, and in one case a chemically mediated interaction between two unidentified neuro ns was found.