Action potential-dependent calcium transients in myenteric S neurons of the guinea-pig ileum

Simultaneous intracellular microelectrode recording and Fura-2 imaging was used to investigate the relationship between intracellular calcium ion conc entration ([Ca2+](i)) and excitability of tonic S neurons in intact myenter ic plexus of the guinea-pig ileum, S neurons were impaled in myenteric gang lia, at locations near connections with internodal strands. The calcium ind icator Fura-2 was loaded via the recording microelectrode. The estimated [C a2+](i) of these neurons was approximately 95 nM (n = 25). Intracellular cu rrent injection (200 ms pulses, 0.2 nA, delivered at 0.05 Hz) resulted in a ction potential firing throughout the stimulus pulse, accompanied by transi ent increases in [Ca2+](i) (to approximately 240 nM, n = 12), Increasing th e number of evoked action potentials by increasing stimulus duration (100-5 00 ms) or intensity (0.05-0.3 nA) produced correspondingly larger [Ca2+](i) transients. Single action potentials rarely produced resolvable [Ca2+](i) events, while short bursts of action potentials (three to five events) inva riably produced resolvable [Ca2+](i) increases. Some neurons demonstrated s pontaneous action potential firing, which was accompanied by sustained [Ca2 +](i) increases. Action potential firing and [Ca2+](i) increases were also observed by activation of slow synaptic input to these neurons, in cases wh ere the slow depolarization initiated action potential firing. Action poten tials (evoked or spontaneous) and associated [Ca2+](i) transients were abol ished by tetrodotoxin (1 mu M). omega-conotoxin GVIA (100 nM) reduced [Ca2](i) transients by approximately 67%, suggesting that calcium influx throug h N-type calcium channels contributes to evoked [Ca2+](i) increases. The S neurons in this study showed prominent afterhyperpolarizations following bu rsts of action potential firing. The time-course of afterhyperpolarizations was correlated with the time-course of evoked [Ca2+](i) transients. Afterh yperpolarizations were blocked by tetrodotoxin and reduced by omega-conotox in GVIA, suggesting that calcium influx through N-type channels contributes to these events. The electrical properties of Fura-2-loaded neurons were n ot significantly different from properties of neurons recorded without Fura -2 injection, suggesting that Fura-2 injection alone does not significantly influence the electrical properties of these cells. These data indicate that myenteric S neurons in situ show prominent, activi ty-dependent increases in [Ca2+](i). These events can be generated spontane ously, or be evoked by intracellular current injection or synaptic activati on. [Ca2+](i) transients in these neurons appear to involve action potentia l-dependent opening of N-type calcium channels, and the elevation in [Ca2+] (i) increase may underlie afterhyperpolarizations and regulate excitability of these enteric neurons. (C) 1999 IBRO. Published by Elsevier Science Ltd .