Synaptic Components, Function and Modulation Characterized by GCaMP6f Ca²⁺ Imaging in Mouse Cholinergic Myenteric Ganglion Neurons

The peristaltic contraction and relaxation of intestinal circular and longitudinal smooth muscles is controlled by synaptic circuit elements that impinge upon phenotypically diverse neurons in the myenteric plexus. While electrophysiological studies provide useful information concerning the properties of such synaptic circuits, they typically involve tissue disruption and do not correlate circuit activity with biochemically defined neuronal phenotypes. To overcome these limitations, mice were engineered to express the sensitive, fast Ca²⁺ indicator GCaMP6f selectively in neurons that express the acetylcholine (ACh) biosynthetic enzyme choline acetyltransfarse (ChAT) thereby allowing rapid activity-driven changes in Ca²⁺ fluorescence to be observed without disrupting intrinsic connections, solely in cholinergic myenteric ganglion (MG) neurons. Experiments with selective receptor agonists and antagonists reveal that most mouse colonic cholinergic (i.e., GCaMP6f⁺/ChAT⁺) MG neurons express nicotinic ACh receptors (nAChRs), particularly the ganglionic subtype containing α3 and β4 subunits, and most express ionotropic serotonin receptors (5-HT₃Rs). Cholinergic MG neurons also display small, spontaneous Ca²⁺ transients occurring at ≈ 0.2 Hz. Experiments with inhibitors of Na⁺ channel dependent impulses, presynaptic Ca²⁺ channels and postsynaptic receptor function reveal that the Ca²⁺ transients arise from impulse-driven presynaptic activity and subsequent activation of postsynaptic nAChRs or 5-HT₃Rs. Electrical stimulation of axonal connectives to MG evoked Ca²⁺ responses in the neurons that similarly depended on nAChRs or/and 5-HT₃Rs. Responses to single connective shocks had peak amplitudes and rise and decay times that were indistinguishable from the spontaneous Ca²⁺ transients and the largest fraction had brief synaptic delays consistent with activation by monosynaptic inputs. These results indicate that the spontaneous Ca²⁺ transients and stimulus evoked Ca²⁺ responses in MG neurons originate in circuits involving fast chemical synaptic transmission mediated by nAChRs or/and 5-HT₃Rs. Experiments with an α7-nAChR agonist and antagonist, and with pituitary adenylate cyclase activating polypeptide (PACAP) reveal that the same synaptic circuits display extensive capacity for presynaptic modulation. Our use of non-invasive GCaMP6f/ChAT Ca²⁺ imaging in colon segments with intrinsic connections preserved, reveals an abundance of direct and modulatory synaptic influences on cholinergic MG neurons.