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. NEW & NOTEWORTHY 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.

ATP is both an important mediator of physiological gut functions such as motility and epithelial function, and a key danger signal that mediates cell death and tissue damage. The actions of extracellular ATP are regulated through the catalytic functions extracellular nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3, and -8, which ultimately generate nucleosides. Ectonucleotidases have distinct cellular associations, but the specific locations and functional roles of individual NTPDases in the intestine are still poorly understood. Here, we tested the hypothesis that differential and cell-selective regulation of purine hydrolysis by NTPDase1 and -2 plays important roles in gut physiology and disease. We studied Entpd1 and Entpd2 null mice in health and following colitis driven by 2% dextran sulfate sodium (DSS) administration using functional readouts of gut motility, epithelial barrier function, and neuromuscular communication. NTPDase1 is expressed by immune cells, and the ablation of Entpd1 altered glial numbers in the myenteric plexus. NTPDase2 is expressed by enteric glia, and the ablation of Entpd2 altered myenteric neuron numbers. Mice lacking either NTPDase1 or -2 exhibited decreased inhibitory neuromuscular transmission and altered components of inhibitory junction potentials. Ablation of Entpd2 increased gut permeability following inflammation. In conclusion, the location- and context-dependent extracellular nucleotide phosphohydrolysis by NTPDase1 and -2 substantially impacts gut function in health and disease. NEW & NOTEWORTHY Purines are important mediators of gastrointestinal physiology and pathophysiology. Nucleoside triphosphate diphosphohydrolases (NTPDases) regulate extracellular purines, but the roles of specific NTPDases in gut functions are poorly understood. Here, we used Entpd1- and Entpd2-deficient mice to show that the differential and cell-selective regulation of purine hydrolysis by NTPDase1 and -2 plays important roles in barrier function, gut motility, and neuromuscular communication in health and disease.

Chronic opioid exposure induces tolerance to the pain-relieving effects of opioids but sensitization to some other effects. While the occurrence of these adaptations is well-understood, the underlying cellular mechanisms are less clear. This study aimed to determine how chronic treatment with morphine, a prototypical opioid agonist, induced adaptations to subsequent morphine signaling in different subcellular contexts. Opioids acutely inhibit glutamatergic transmission from medial thalamic (MThal) inputs to the dorsomedial striatum (DMS) and anterior cingulate cortex (ACC) via activity at μ-opioid receptors (MORs). MORs are present in somatic and presynaptic compartments of MThal neurons terminating in both the DMS and ACC. We investigated the effects of chronic morphine treatment on subsequent morphine signaling at MThal-DMS synapses, MThal-ACC synapses, and MThal cell bodies in male and female mice. Surprisingly, chronic morphine treatment increased subsequent morphine inhibition of MThal-DMS synaptic transmission (morphine facilitation), but decreased subsequent morphine inhibition of transmission at MThal-ACC synapses (morphine tolerance) in a sex-specific manner; these adaptations were present in male but not female mice. Additionally, these adaptations were not observed in knockin mice expressing phosphorylation-deficient MORs, suggesting a role of MOR phosphorylation in mediating both facilitation and tolerance to morphine within this circuit. The results of this study suggest that the effects of chronic morphine exposure are not ubiquitous; rather adaptations in MOR function may be determined by multiple factors such as subcellular receptor distribution, influence of local circuitry and sex.

There is evidence that R-type Ca2+ channels contribute to synaptic transmission in the myenteric plexus. It is unknown if R-type Ca2+ channels contribute to neuromuscular transmission. We measured the effects of the nitric oxide synthase (NOS) inhibitor, nitro L-arginine (NLA), Ca2+ channel blockers and apamin (SK channel blocker) on neurogenic relaxations and contractions of the guinea pig ileum longitudinal muscle-myenteric plexus (LMMP) in vitro. We used intracellular recordings to measure inhibitory junction potentials (IJPs). Immunohistochemical and western blot techniques localized R-type Ca2+ channel protein in the LMMP and circular muscle. CdCl2 (pan Ca2+ channel blocker) blocked and NLA and NiCl2 (R-type Ca2+ channel blocker) reduced neurogenic relaxations in a non-additive manner. NiCl2 did not alter neurogenic cholinergic contractions but it potentiated neurogenic non-cholinergic contractions. Relaxations were inhibited by apamin, NiCl2 and NLA and were blocked by combined application of these drugs. Relaxations were reduced by NiCl2 or ω-conotoxin (ω-CTX, N-type Ca2+ channel blocker) and were blocked by combined application of these drugs. Longitudinal muscle IJPs were inhibited by NiCl2, but not MRS 2179 (P2Y1 receptor antagonist). Circular muscle IJPs were blocked by apamin, MRS 2179, ω-CTX and CdCl2 but not NiCl2. We conclude that neuronal R-type Ca2+ channels contribute to inhibitory neurotransmission to longitudinal muscle but less so or not all in the circular muscle of the guinea pig ileum.

Opioids modulate both sensory and affective components of pain, most significantly through agonist activity at the mu‐opioid receptor (MOR). While cellular mechanisms underlying opioid modulation of perception of stimulus intensity have been well characterized, mechanisms underlying opioid effects on pain‐related negative affect have not been well elucidated. The mediodorsal thalamus (MD) sends glutamatergic projections to the striatum as part of a thalamo‐cortical‐striatal loop that is involved in affective pain perception. Here, we determined the ability of various inhibitory coupled G‐protein coupled receptors (GPCRs), including MOR to inhibit glutamate release via presynaptic mechanisms. In mouse brain slices, MD terminals in the striatum were selectively activated using optogenetic techniques. Excitatory postsynaptic (EPSCs) were measured in striatal medium spiny neurons using whole cell patch‐clamp electrophysiology in the presence and absence of various inhibitory GPCR agonists. MOR full agonists met‐enkephalin (ME) and fentanyl, the delta opioid receptor agonist DPDPE, the adenosine A1 receptor agonist CPA, the GABAB receptor agonist baclofen, and the CB1 receptor agonist WIN 55,212 were tested. EPSC inhibition was seen with application of ME, fentanyl, CPA, and baclofen, indicating the presence of MORs, A1, and GABAB receptors. Inhibition was not seen with application of DPDPE or WIN 55,212, indicating delta opioid receptors and CB1 receptors are not present on these terminals. The effect of chronic opioid treatment on the function of these GPCRs will be discussed. Overall these data provide insight into how presynaptic GPCRs function to regulate neurotransmission in a circuit important for affective pain perception. Support or Funding Information NIH T32‐DA007281R01DA042779