Evaluation of the role of cholinergic and nitrergic myenteric neurons in colonic motility using genetically‐encoded optical tools

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Activity of neuronal subtypes within the myenteric plexus underlies coordinated patterns of gastro‐intestinal motility. Cholinergic neurons include intrinsic primary afferent neurons (IPAN), ascending interneurons, and excitatory motor neurons (eMNs) projecting to smooth muscle, whereas nitrergic neurons include descending interneurons and inhibitory motor neurons (iMNs) projecting to smooth muscle. The activity of cholinergic eMNs and nitrergic iMNs is believed to produce the two major patterns of motility underlying peristalsis in the colon, namely the periodic colonic migrating motor complex (CMMC) and the intervening periods of tonic inhibition, respectively. The objective of this study was to determine whether a.) such correlated activities of cholinergic eMNs and nitrergic iMNs could be discerned in the myenteric plexus of transgenic mice expressing the genetically‐encoded calcium indicator GCaMP6f in each of these two cell types (i.e., choline acetyltransferase (ChAT)‐GCaMP6f mice or neuronal nitric oxide synthase (nNOS)‐GCaMP6f mice), and b.) optogenetic activation of in these subtypes with genetically‐encoded optical actuator channelrhodopsin‐2 (ChR2) would produce CMMCs (in ChAT‐ChR2 mice) or tonic inhibition (in nNOS‐ChR2 mice). In order to correlate activity patterns, which require imaging of sliced colon, with defined states of motility, which require intact colon with force transducers, we took advantage of CAGGS‐GCaMP3 mice, which show widespread GCaMP3 expression. In loosely pinned preparations imaged at low power, we could easily discern CMMCs, which displayed pronounced movement together with robust smooth muscle Ca2+ responses. Then, we imaged the activity of nitrergic and cholinergic neurons against the backdrop of these movements. In nNOS‐GCaMP6f mice, as expected, we observed large numbers of cells that exhibited tonic Ca2+ responses in between CMMCs. We also observed a subpopulation of nitrergic neurons that exhibited a large phasic response just prior to the CMMC. Consistent with this finding, we found that optogenetic stimulation of the colon of nNOS‐ChR2 mice in between spontaneous CMMCs (i.e., during tonic inhibition) resulted in the generation of CMMCs. These ectopic CMMC were reduced by treatment with the ganglionic nicotinic AChR blocker hexamethonium, suggesting that they were primarily mediated by activation of nitrergic INs. In ChAT‐GCaMP6f mice, we observed fewer cells that exhibited tonic activity in between CMMCs. During CMMCs, we observed many cholinergic neurons that exhibited large phasic responses just after the CMMC was initiated. Similar to the effects in nNOS‐ChR2 mice, optogenetic stimulation of the colon of ChAT‐ChR2 mice in between spontaneous CMMCs produced ectopic CMMCs. Whether these effects are mediated by IPANs, ascending INs or eMNs is under investigation. These studies reveal unexpected complexity in the regulation of colonic motility by cholinergic and nitrergic myenteric neurons.Support or Funding InformationSPARC : OT2OD024899 (Director, Y Tache, UCLA)