Optogenetic analysis of excitatory and inhibitory neurotransmission in the enteric nervous system

The enteric nervous system (ENS) is embedded within the gastrointestinal (GI) tract and controls GI function. Impaired ENS function leads to altered patterns of motility and secretion, causing GI disease. For instance, functional gastrointestinal disorders (FGID) are caused by poorly understood alterations in the structure and function of nerves, smooth muscle cells (SMC), and other cell types in the GI tract. It is estimated that these disorders comprise about 41% of the total GI complications in the United States. Also, altered patterns of motility that occur in the GI muscles is a hallmark characteristic of FGIDs. Although the ENS is fairly understood, further elucidation of the enteric circuitry that governs GI motility would help to understand the pathophysiology of FGID. For that reason, identifying the contributions of classes of enteric neurons that control GI motility and secretion could aid in the identification of novel therapeutic targets for the treatment of FGIDs. A widely used method to study neural control of GI motility is sharp-electrode electrophysiological recordings from the smooth muscle or enteric neurons. Conventional, intracellular electrophysiological recordings have relied on electrical stimulation of enteric neurons which will activate all neurons in an ex vivo preparation of the ENS, and does not allow cell-specific activation of individual subpopulations of myenteric neurons. To overcome this problem, we used immunohistochemical methods to identify subpopulations of myenteric neurons and the optogenetically activated protein channelrhodopsin-2 (ChR2) that can be selectively expressed in subsets of enteric neurons. In Chapter 3, immunohistochemical studies of the mouse enteric nervous system are performed using the purinergic neuronal marker, vesicular nucleotide transporter (VNUT) along with markers for specific subsets of myenteric neurons and nerve fibers (e.g., neuronal nitric oxide synthase, choline acetyltransferase, calretinin, calbindin, and tyrosine hydroxylase),. Chapter 4 compares electrical and optogenetic electrophysiology recordings from myenteric neurons of mice that express ChR2 in nNOS neurons. The studies described in Chapter 5 use ChAT-ChR2-YFP-BAC transgenic mice which have eYFP tagged ChR2 expressed in cholinergic neurons. Optogenetics was used to isolate the cholinergic component of the ENS. The findings discussed in this dissertation provides evidence of a more sophisticated enteric circuitry of GI motility. (1) Purinergic neurons are likely a separate subpopulation of enteric neurons. VNUT is only expressed in the form of punctate varicosities at the nerve fibers and is not endogenously expressed in the soma of enteric neurons. VNUT also does not appear to colocalize with other neuronal immunoreactive markers within the myenteric plexus (mp), the tertiary plexus (tp), or circular smooth muscle layer of all tested tissue preps. (2) BLS of ChR2 expressed in nNOS neurons induced a purinergic/nitrergic biphasic IJP, suggesting that nNOS IMNs co-releases a purine as a neurotransmitter. Ectopic expression of ChR2 in non-nNOS neurons, however, could explain the biphasic IJP responses during electrophysiology recordings. Hence, the existence of separate subset populations of IMN populations (e.g., nNOS only and purinergic only IMNs) can't be ruled out. (3) BLS of ChR2 expressed in ChAT positive neurons induced EJPs and IJP responses. Inhibition of the nicotinic ACh receptor (nAChR) with mecamylamine significantly reduced the light-evoked IJP. Bath application of the purinergic P2Y1 antagonist, MRS 2179, was sufficient to abolish the IJP response, while the muscarinic ACh receptor antagonist, Scopolamine, abolished the EJP response. The data suggest that BLS of ChR2 activates cholinergic EMNs and cholinergic interneurons, and that activation of the cholinergic interneurons activates purinergic only IMNs that supply the smooth muscle, resulting in a predominant purinergic only IJP. Taken together, this work provides evidence for a diverse and more complex enteric neural circuit of GI motility. Future experiments should, however, focus on studying these enteric circuits at the level of the neuron, as these studies can provide a more in-depth analysis of the enteric circuitry.