Optogenetic analysis of neuromuscular transmission in the colon of ChATChR2- YFP BAC transgenic mice

Abstract

Propulsion of luminal content along the gut requires coordinated contractions and relaxations of gastrointestinal smooth muscles controlled by the enteric nervous system. Activation of excitatory motor neurons (EMNs) causes muscle contractions, whereas inhibitory motor neuron (IMN) activation causes muscle relaxation. EMNs release acetylcholine (ACh), which acts at muscarinic receptors on smooth muscle cells and adjacent interstitial cells of Cajal, causing excitatory junction potentials (EJPs). IMNs release ATP (or another purine) and nitric oxide to cause inhibitory junction potentials (IJPs) and muscle relaxation. We used commercially available choline acetyltransferase (ChAT)-channelrhodopsin-2 (ChR2)-yellow fluorescent protein (YFP) bacterial artificial chromosome (BAC) transgenic mice, which express ChR2 in cholinergic neurons, to study cholinergic neuromuscular transmission in the colon. Intracellular microelectrodes were used to record IJPs and EJPs from circular muscle cells. We used blue light stimulation (BLS, 470 nm, 20 mW/mm2) and electrical field stimulation (EFS) to activate myenteric neurons. EFS evoked IJPs only, whereas BLS evoked EJPs and IJPs. Mecamylamine (10 μM, nicotinic cholinergic receptor antagonist) reduced BLS-evoked IJPs by 50% but had no effect on electrically evoked IJPs. MRS 2179 (10 μM, a P2Y1 receptor antagonist) blocked BLS-evoked IJPs. MRS 2179 and Nω-nitro-L-arginine (100 μM, nitric oxide synthase inhibitor) isolated the EJP, which was blocked by scopolamine (1 μM, muscarinic ACh receptor antagonist). Immunohistochemistry revealed ChAT expression in ∼88% of enhanced YFP (eYFP)-expressing neurons, whereas 12% of eYFP neurons expressed nitric oxide synthase. These data show that cholinergic interneurons synapse with EMNs and IMNs to cause contraction and relaxation of colonic smooth muscle.