Simultaneous whole-cell patch-clamp and calcium imaging on myenteric neurons

Abstract

Live calcium imaging is often used as a proxy for electrophysiological measurements and has been a valuable tool that allows simul-taneous analysis of neuronal activity in multiple cells at the population level. In the enteric nervous system, there are two main elec-trophysiological classes of neurons, after-hyperpolarizing (AH)-and synaptic (S)-neurons, which have been shown to have different calcium handling mechanisms. However, they are rarely considered separately in calcium imaging experiments. A handful of studies have shown that in guinea pig, a calcium transient will accompany a single action potential in AH-neurons, but multiple action poten-tials are required to generate a calcium transient in S-neurons. How this translates to different modes of cellular depolarization and whether this is consistent across species is unknown. In this study, we used simultaneous whole-cell patch-clamp electrophysiology together with calcium imaging to investigate how enteric neurons respond to different modes of depolarization. Using both traditional (4 Hz) and also high-speed (1,000 Hz) imaging techniques, we found that single action potentials elicit calcium transients in both AH-neurons and S-neurons. Subthreshold membrane depolarizations were also able to elicit calcium transients, although calcium responses were generally amplified if an action potential was present. Furthermore, we identified that responses to nicotinic acetyl-choline receptor stimulation can be used to distinguish between AH-and S-neurons in calcium imaging. NEW & NOTEWORTHY Live calcium imaging is an important tool for investigating enteric nervous system (ENS) function. Previous studies have shown that multiple action potentials are needed to generate a calcium response in S-neurons, which has important implications for the interpretation of calcium imaging data. Here, we show that in mouse myenteric neurons, calcium transients are elicited by single action potentials in both AH-and S-neurons. In addition, nicotinic acetylcholine receptor stimula-tion can be used to distinguish between these two classes.