Synaptic transmission induces transient Ca2+ concentration changes in cultured myenteric neurones

Authors


Tack Center for Gastroenterological Research, Catholic University Leuven, B-3000 Leuven, Belgium. Tel.: + 32 16 34 42 18; fax: + 32 16 34 44 19; e-mail: jan.tack@med.kuleuven.ac.be

Abstract

The enteric nervous system controls most of the gastrointestinal functions. We applied confocal microscopy and the Ca2+ indicator Fluo-3 as an optical approach to study synaptic activation in cultures of myenteric neurones. The optical recording of [Ca2+]i (the intracellular Ca2+ concentration) was used to monitor activation, since [Ca2+]i is crucial in the coupling between neuronal excitation and the activation of several intracellular events. Extracellular fibre tract stimulation (2 s, 30 Hz) caused a transient [Ca2+]i rise in a subset of neurones (50%). These transients lasted for 5.2 s (n=36), with an average amplitude of 3.4 ± 1.3 times the basal concentration. The removal of extracellular Ca2+ (n=15) or the application of 10–6M tetrodotoxin (n=16) blocked this response. The N-type Ca2+-channel blocker ω-conotoxin (5 × 10 –7M) abolished the [Ca2+]i increase, while blockade of L-type and P/Q type Ca2+ channels had no effect. Single stimuli evoked a [Ca2+]i rise in the processes. ω-conotoxin-sensitive postsynaptic events required repetitive stimulation. Cholinergic blockade did not inhibit the [Ca2+]i rise in all neurones, suggesting that, besides acetylcholine, other neurotransmitters are involved. Optical imaging of [Ca2+]i can be used to study synaptic spread of activation in enteric neuronal circuits expressed in culture.

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