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

Abstract

Background: Live Ca2+ imaging is a proxy for electrophysiological measurements and a valuable tool to analyze activity in multiple cells simultaneously. In the enteric nervous system (ENS), two main elec-trophysiological classes of neurons exist (AH and S). Although they have different Ca2+ handling mechanisms, they are rarely considered separately in Ca2+ imaging studies.Aim: To investigate whether the characteristics of a [Ca2+]i transient reflect the electrophysiological differences between murine AH and S neurons.Methods: Primary ENS cultures were made from adult Wnt1- Cre;R26R- GCaMP6f mice. After 4- 5 days in vitro, simultaneous Ca2+ imaging and patch- clamp recordings were performed. Cells were de-polarized by either 10 or 500 ms current pulses or high K+ (75 mM). DMPP (10 μM), a nicotinic receptor agonist, was used to mimic fast cholinergic transmission.Results: We recorded from 40 neurons, classed as AH (16) and S (24) based on the presence or absence of an “inflection” in the action po-tential's (AP) repolarizing phase. Unlike previous studies performed in guinea pig, S neurons exhibited a prominent [Ca2+]i transient ac-companying a single AP. In response to a 10 ms depolarization pulse, both the AP and [Ca2+]i transient amplitudes were significantly larger in AH neurons. However, with a 500 ms depolarization pulse or high K+, no significant difference persisted. Interestingly, when tetrodotoxin (1 μM) was applied to block APs, a reduced but distinct [Ca2+]i transient remained following the 500 ms depolarization pulse. In 10/12 AH neurons, DMPP did not elicit a membrane potential change or a [Ca2+]i transient. In 14/16 S neurons, DMPP triggered both a [Ca2+]i transient and either an AP or sub- threshold membrane potential change.Conclusions: [Ca2+]i transients were found to accompany single APs in both AH and S neurons. Although sub- threshold membrane de-polarizations could also elicit [Ca2+]i transients, these were gener-ally amplified if an AP was present. The [Ca2+]i response to DMPP was the most reliable way to optically distinguish between AH and S neurons.