LOBSTER STOMATOGASTRIC NEURONS IN PRIMARY CULTURE .1. BASIC CHARACTERISTICS

1. A method for the isolation of stomatogastric neurons with neuropilar processes and an axon less-than-or-equal-to 2 mm long is described, Isolated neurons adhered to an uncoated plastic surface and demonstrated neurite outgrowth for greater-than-or-equal-to 7-10 days in a simple medium (salt-adjusted Leibovitz-15). Neurite outgrowth started immediately after plating and was maximal during the first 2-3 days. The electrical activity of neurons and their responses to bath application of pilocarpine were studied between 2 and 10 days after plating. 2. Identified neurons [pyloric dilator (PD), pyloric (PY), and lateral pyloric (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 stomatogastric 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 some rhythmicity in the PY neuron, whereas in LP neurons it decreased the firing threshold to depolarizing current and accentuated postinhibitory rebound. LPG neurons were tonically active. Pilocarpine depolarized the LPG neurons and accelerated their tonic activity; neuron hyperpolarization by current injection led to bursting pacemaker activity that 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 recordings from the soma and axon under direct visual control demonstrated that the intrasomatic spikes ( 15-20 mV in amplitude) were attenuated action potentials generated in the axon. 3. Neurons isolated with short primary 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 after extraction from the STG. After 2 days in culture, these ''short-neurite'' neurons became capable of generating different types of electrical activity (e.g., fast spikes with amplitudes of less-than-or-equal-to 40-45 mV, plateau potentials, bursting potentials, etc.). The capability of isolated somata to generate electrical activity did not depend on whether or not the cell had adhered to the substrate and demonstrated neurite outgrowth. Some cells isolated with (or without) short primary neurites expressed pacemaker-like properties without neuromodulatory substances. In this respect, they differed from neurons isolated with longer neuropilar processes and axons which, like neurons in situ, exhibited rhythmic activity only in the presence of pilocarpine. Most neurons isolated with short primary neurites were sensitive to pilocarpine, which evoked neuron depolarization and affected the pattern of their activity. 4. To examine the possibility of cultured neurons forming electrical and chemical connections, the processes of two or more neurons were placed in close contact with one another. In two cases out of 19 trials, electrical coupling between neurons (2 GM and two PD, known to have electrical connections in situ) was noted, and in one case a chemically mediated interaction between two unidentified neurons was found.