Optogenetic analysis of neuromuscular transmission in the colon of ChAT-ChR2-YFP BAC transgenic mice

• Published in: American journal of physiology: Gastrointestinal and liver physiology Vol. 317; no. 5; pp. G569 - G579
• Main Authors: Perez-Medina, Alberto L, Galligan, James J
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.11.2019
• Series: Neurogastroenterology and Motility
• Subjects: Acetylcholine; Acetylcholine receptors; Acetylcholine receptors (muscarinic); Acetyltransferase; Animals; Arginine; Artificial chromosomes; Bacterial artificial chromosomes; Channelrhodopsins - genetics; Channelrhodopsins - metabolism; Choline; Choline O-acetyltransferase; Choline O-Acetyltransferase - genetics; Choline O-Acetyltransferase - metabolism; Cholinergic Neurons - metabolism; Cholinergic Neurons - physiology; Colon; Colon - innervation; Colon - physiology; Enteric nervous system; Female; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Immunohistochemistry; Interneurons; Interstitial cells; Interstitial cells of Cajal; Male; Mecamylamine; Mice; Mice, Inbred C57BL; Microelectrodes; Motor neurons; Motor Neurons - metabolism; Motor Neurons - physiology; Muscle Contraction; Myocytes, Smooth Muscle - metabolism; Myocytes, Smooth Muscle - physiology; Neuromuscular Junction - metabolism; Neuromuscular Junction - physiology; Neuromuscular junctions; Neurons; Nitric oxide; Nitric-oxide synthase; Optogenetics; Scopolamine; Smooth muscle; Synaptic Potentials; Transgenic animals; Transgenic mice; Yellow fluorescent protein; cholinergic interneurons; neuromuscular transmission; enteric nervous system; transgenic mice; optogenetics
• ISSN: 0193-1857; 1522-1547
• DOI: 10.1152/ajpgi.00089.2019

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/mm ) 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 P2Y receptor antagonist) blocked BLS-evoked IJPs. MRS 2179 and -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. Electrical stimulation of interganglionic connectives has been used widely to study synaptic transmission in the enteric nervous system. However, electrical stimulation will activate many types of neurons and nerve fibers, which complicates data interpretation. Optogenetic activation of enteric neurons using genetically modified mice expressing channelrhodopsin-2 in cholinergic neurons offers a new approach that provides more specificity for nerve stimulation when studying myenteric plexus nerve circuitry.