for advice on the bioassay for serotonin; and Janis Weeks for her helpful criticism of this manuscript. ' Present address and that to which all reprint requests should be ,' Terms such as "swim" and "swimming" will be used as shorthand sent: Department of Neurobiology, Harvard Medical School, 25 Shat-for longer phrases such as "motor program for swimming." SII will be tuck Street, Boston, MA 02115. used as an abbreviation for swim-initiating interneuron.

Although rapid-onset, short-term regulation of neuronal Ca currents by neurotransmitters and second messengers is well documented, little is known about conditions that can cause longer-lasting changes in Ca channel function. We report here that persistent depolarization is accompanied by slowly developing long-term reduction of neuronal Ca currents. Rat myenteric neurons grown in cell culture for 1-7 d were studied with the tight-seal whole-cell recording technique. Macroscopic Ca-channel currents had decaying and sustained components at all days studied. When the neurons were grown in medium containing 25 mM KCl, which depolarized them to -40 mV and caused significant elevation of intracellular Ca, the densities of both components of Ca-channel current decreased by 40-80%. Several results suggest that different mechanisms underlie the downregulation of the two components. (1) The density of the decaying component decreased approximately four times faster than did that of the sustained component. (2) When neurons were returned to control medium, which contained 5 mM KCl, the density of the sustained component returned to control levels within 24 hr, while that of the decaying component did not recover significantly. (3) Inhibitors of RNA and protein synthesis reduced or prevented downregulation of the sustained but not of the decaying component. (4) The dihydropyridine antagonist nitrendipine, which prevented the sustained elevation of intracellular Ca in neurons grown in 25 mM KCl, prevented downregulation of the sustained component but had no effect on downregulation of the decaying component. We suggest that these forms of regulation of Ca current density could help neurons adapt to altered levels of electrical activity and may contribute to changes in synaptic strength that occur during periods of increased or decreased electrical activity.

Intracellular recording and immunochemical techniques were used to study synaptic transmission between individual pairs of rat myenteric plexus neurons in cell culture. This report describes the synaptic connections made by “dual function” presynaptic neurons that evoked slow postsynaptic depolarizations (slow EPSPs) in the same neurons in which they also evoked fast nicotinic cholinergic EPSPs. The slow EPSPs occurred only when presynaptic neurons were stimulated at frequencies of 5 Hz or higher. During the slow EPSPs, slope input resistance increased. The slow EPSPs were not detectably voltage-dependent, and they reversed sign at the estimated K+ equilibrium potential, suggesting that they resulted from a synaptically mediated decrease in resting K+ conductance. Several lines of evidence suggested that dual- function neurons evoke slow EPSPs by releasing a vasoactive intestinal peptide (VIP)-like cotransmitter. (1) Immunocytochemical staining revealed VIP-like immunoreactivity in all physiologically identified dual-function neurons. (2) Responses to exogenously applied VIP mimicked the slow EPSPs. (3) Superfusion of cultures with anti-VIP antisera blocked the slow EPSPs reversibly, as did application of desensitizing doses of VIP. These findings suggest that during periods of increased activity, subsets of cholinergic myenteric neurons release a VIP-like cotransmitter that enhances postsynaptic excitability. The effects of the cotransmitter may help to compensate for decreases in nicotinic EPSPs that occur during increased presynaptic activity.