A vasoactive intestinal peptide-like cotransmitter at cholinergic synapses between rat myenteric neurons in cell culture

Abstract

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.

Substance P mediates synaptic transmission between rat myenteric neurones in cell culture.

“…Previous studies have shown that individual neurones in cell cultures prepared from rat myenteric plexus can evoke slow EPSPs in other neurones (Willard & Nishi, 1985 Willard, 1990). In this study, immunocytochemical staining has been used to demonstrate that subsets of myenteric neurones that evoke EPSPs contain immunoreactive SP.…”

“…Cultures of myenteric neurones were prepared as described by Nishi & Willard (1985) and Willard (1990) from tissue strips removed from the small intestines of 1-to 3-day-old rat pups (CD strain; Charles River) that had been killed by decapitation.…”

“…Synaptically connected pairs of neurones were identified either by making recordings from randomly selected pairs of neurones and testing each for the ability to elicit synaptic potentials in the other or by the dye injection procedure described by Willard (1990). Briefly, patch pipettes were used to inject Lucifer Yellow into neurones.…”

“…This was accomplished by removing all fluid from the culture, except for about 30 Itl that remained in a paraffin ring surrounding cells in the centre of the culture dish. This ring, which could hold up to 300 /u1, was then superfused via a microperfusion system described by Willard (1990).…”

Regulatory mechanisms that govern nicotinic synapse formation in neurons

“…One of these factors, vasoactive intestinal peptide (VIP), is found at a variety of nerve terminals throughout the nervous system, frequently colocalizes with ACh, and may function as a cholinergic cotransmitter (Gozes and Brenneman, 1989;Whittaker, 1989;Willard, 1990). Treatment of CG neurons with VIP increases ACh responses, but not nAChR surface levels, by increasing intracellular cAMP (Gurantz et al, 1994).…”