Abnormal enteric nervous system and motor activity in the ganglionic proximal bowel of Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a congenital defect in which the enteric nervous system (ENS) does not develop in the distal bowel, requiring surgical removal of the portions of bowel without ENS ganglia ('aganglionic') and reattachment of the 'normal' proximal bowel with ENS ganglia. Unfortunately, many HSCR patients have persistent dysmotility (e.g., constipation, incontinence) and enterocolitis after surgery, suggesting that the remaining bowel is not normal despite having ENS ganglia. Anatomical and neurochemical alterations have been observed in the ENS-innervated proximal bowel from HSCR patients and mice, but no studies have recorded ENS activity to define the circuit mechanisms underlying post-surgical HSCR dysfunction. Here, we generated a HSCR mouse model with a genetically-encoded calcium indicator to map the ENS connectome in the proximal colon. We identified abnormal spontaneous and synaptic ENS activity in proximal colons from GCaMP-Ednrb -/- mice with HSCR that corresponded to motor dysfunction. Many HSCR-associated defects were also observed in GCaMP-Ednrb +/- mice, despite complete ENS innervation. Results suggest that functional abnormalities in the ENS-innervated bowel contribute to post-surgical bowel complications in HSCR patients, and HSCR-related mutations that do not cause aganglionosis may cause chronic colon dysfunction in patients without a HSCR diagnosis.

New Insights on Extrinsic Innervation of the Enteric Nervous System and Non-neuronal Cell Types That Influence Colon Function.

The enteric nervous system not only innervates the colon to execute various functions in a semi-autonomous manner but also receives neural input from three extrinsic sources, (1) vagal, (2) thoracolumbar (splanchnic), and (3) lumbosacral (pelvic) pathways, that permit bidirectional communication between the colon and central nervous system. Extrinsic pathways signal sensory input via afferent fibers, as well as motor autonomic output via parasympathetic or sympathetic efferent fibers, but the shared and unique roles for each pathway in executing sensory-motor control of colon function have not been well understood. Here, we describe the recently developed approaches that have provided new insights into the diverse mechanisms utilized by extrinsic pathways to influence colon functions related to visceral sensation, motility, and inflammation. Based on the cumulative results from anatomical, molecular, and functional studies, we propose pathway-specific functions for vagal, thoracolumbar, and lumbosacral innervation of the colon.

Unique Neural Circuit Connectivity of Mouse Proximal, Middle, and Distal Colon Defines Regional Colonic Motor Patterns.

BACKGROUND & AIMS: Colonic motor patterns have been described by a number of different groups, but the neural connectivity and ganglion architecture supporting patterned motor activity have not been elucidated. Our goals were to describe quantitatively, by region, the structural architecture of the mouse enteric nervous system and use functional calcium imaging, pharmacology, and electrical stimulation to show regional underpinnings of different motor patterns. METHODS: Excised colon segments from mice expressing the calcium indicator GCaMP6f or GCaMP6s were used to examine spontaneous and evoked (pharmacologic or electrical) changes in GCaMP-mediated fluorescence and coupled with assessment of colonic motor activity, immunohistochemistry, and confocal imaging. Three-dimensional image reconstruction and statistical methods were used to describe quantitatively mouse colon myenteric ganglion structure, neural and vascular network patterning, and neural connectivity. RESULTS: In intact colon, regionally specific myenteric ganglion size, architecture, and neural circuit connectivity patterns along with neurotransmitter-receptor expression underlie colonic motor patterns that define functional differences along the colon. Region-specific effects on spontaneous, evoked, and chemically induced neural activity contribute to regional motor patterns, as does intraganglionic functional connectivity. We provide direct evidence of neural circuit structural and functional regional differences that have only been inferred in previous investigations. We include regional comparisons between quantitative measures in mouse and human colon that represent an important advance in showing the usefulness and relevance of the mouse system for translation to the human colon. CONCLUSIONS: There are several neural mechanisms dependent on myenteric ganglion architecture and functional connectivity that underlie neurogenic control of patterned motor function in the mouse colon.

Optogenetic activation of the distal colon epithelium engages enteric nervous system circuits to initiate motility patterns.

Digestive functions of the colon depend on sensory-motor reflexes in the enteric nervous system (ENS), initiated by intrinsic primary afferent neurons (IPANs). IPAN terminals project to the mucosal layer of the colon, allowing communication with epithelial cells comprising the colon lining. The chemical nature and functional significance of this epithelial-neural communication in regard to secretion and colon motility are of high interest. Colon epithelial cells can produce and release neuroactive substances such as ATP and 5-hydroxytryptamine (5-HT), which can activate receptors on adjacent nerve fibers, including IPAN subtypes. In this study, we examined if stimulation of epithelial cells alone is sufficient to activate neural circuits that control colon motility. Optogenetics and calcium imaging were used in ex vivo preparations of the mouse colon to selectively stimulate the colon epithelium, measure changes in motility, and record activity of neurons within the myenteric plexus. Light-mediated activation of epithelial cells lining the distal, but not proximal, colon caused local contractions and increased the rate of colonic migrating motor complexes. Epithelial-evoked local contractions in the distal colon were reduced by both ATP and 5-HT receptor antagonists. Our findings indicate that colon epithelial cells likely use purinergic and serotonergic signaling to initiate activity in myenteric neurons, produce local contractions, and facilitate large-scale coordination of ENS activity responsible for whole colon motility patterns.NEW & NOTEWORTHY Using an all-optical approach to measure real-time cell-to-cell communication responsible for colon functions, we show that selective optogenetic stimulation of distal colon epithelium produced activity in myenteric neurons, as measured with red genetically encoded calcium indicators. The epithelial-induced neural response led to local contractions, mediated by both purinergic and serotonergic signaling, and facilitated colonic motor complexes that propagate from proximal to distal colon.

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 ß4 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.

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3.

BACKGROUND AND AIMS: Bowel function requires coordinated activity of diverse enteric neuron subtypes. Our aim was to define gene expression in these neuron subtypes to facilitate development of novel therapeutic approaches to treat devastating enteric neuropathies, and to learn more about enteric nervous system function. METHODS: To identify subtype-specific genes, we performed single-nucleus RNA-seq on adult mouse and human colon myenteric plexus, and single-cell RNA-seq on E17.5 mouse ENS cells from whole bowel. We used immunohistochemistry, select mutant mice, and calcium imaging to validate and extend results. RESULTS: RNA-seq on 635 adult mouse colon myenteric neurons and 707 E17.5 neurons from whole bowel defined seven adult neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially expressed genes. Manually dissected human colon myenteric plexus yielded RNA-seq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells of Cajal, and 2153 macrophages. Immunohistochemistry demonstrated differential expression for BNC2, PBX3, SATB1, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ∼50% of myenteric neurons with distinct effects on smooth muscle contractions. CONCLUSION: Single cell analyses defined genes differentially expressed in myenteric neuron subtypes and new roles for TBX3, GDNF and NRTN. These data facilitate molecular diagnostic studies and novel therapeutics for bowel motility disorders.

Optogenetic inhibition of the colon epithelium reduces hypersensitivity in a mouse model of inflammatory bowel disease.

ABSTRACT: Visceral pain is a prevalent symptom of inflammatory bowel disease that can be difficult to treat. Pain and hypersensitivity are mediated by extrinsic primary afferent neurons (ExPANs) that innervate the colon. Recent studies indicate that the colon epithelium contributes to initiating ExPAN firing and nociceptive responses. Based on these findings, we hypothesized that the epithelium contributes to inflammation-induced hypersensitivity. A key prediction of this hypothesis is that inhibition of the epithelium would attenuate nociceptive signaling and inflammatory hypersensitivity. To test this hypothesis, the inhibitory yellow light-activated protein archaerhodopsin was targeted to the intestinal epithelium (villin-Arch) or the ExPANs (TRPV1-Arch) that innervate the colon. Visceral sensitivity was assessed by measuring the visceromotor response (VMR) to colorectal distension (CRD), with and without yellow light illumination of the colon lumen. Inhibition of the colon epithelium in healthy villin-Arch mice significantly diminished the CRD-induced VMR. Direct inhibition of ExPANs during CRD using TRPV1-Arch mice showed that ExPAN and epithelial inhibition were similarly effective in reducing the VMR to CRD. We then investigated the effect of epithelial and ExPAN inhibition in the dextran sulfate sodium model of inflammatory bowel disease. Inhibition of the colon epithelium significantly decreased dextran sulfate sodium-induced hypersensitivity and was comparable with the inhibition of ExPANs. Together, these results reveal the potential of targeting the colon epithelium for the treatment of pain.

Sympathetic Input to Multiple Cell Types in Mouse and Human Colon Produces Region-Specific Responses.

BACKGROUND & AIMS: The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). METHODS: We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. RESULTS: Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. CONCLUSIONS: Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions.

Extrinsic Primary Afferent Neurons Link Visceral Pain to Colon Motility Through a Spinal Reflex in Mice.

BACKGROUND & AIMS: Proper colon function requires signals from extrinsic primary afferent neurons (ExPANs) located in spinal ganglia. Most ExPANs express the vanilloid receptor TRPV1, and a dense plexus of TRPV1-positive fibers is found around myenteric neurons. Capsaicin, a TRPV1 agonist, can initiate activity in myenteric neurons and produce muscle contraction. ExPANs might therefore form motility-regulating synapses onto myenteric neurons. ExPANs mediate visceral pain, and myenteric neurons mediate colon motility, so we investigated communication between ExPANs and myenteric neurons and the circuits by which ExPANs modulate colon function. METHODS: In live mice and colon tissues that express a transgene encoding the calcium indicator GCaMP, we visualized levels of activity in myenteric neurons during smooth muscle contractions induced by application of capsaicin, direct colon stimulation, stimulation of ExPANs, or stimulation of preganglionic parasympathetic neuron (PPN) axons. To localize central targets of ExPANs, we optogenetically activated TRPV1-expressing ExPANs in live mice and then quantified Fos immunoreactivity to identify activated spinal neurons. RESULTS: Focal electrical stimulation of mouse colon produced phased-locked calcium signals in myenteric neurons and produced colon contractions. Stimulation of the L6 ventral root, which contains PPN axons, also produced myenteric activation and contractions that were comparable to those of direct colon stimulation. Surprisingly, capsaicin application to the isolated L6 dorsal root ganglia, which produced robust calcium signals in neurons throughout the ganglion, did not activate myenteric neurons. Electrical activation of the ganglia, which activated even more neurons than capsaicin, did not produce myenteric activation or contractions unless the spinal cord was intact, indicating that a complete afferent-to-efferent (PPN) circuit was necessary for ExPANs to regulate myenteric neurons. In TRPV1-channel rhodopsin-2 mice, light activation of ExPANs induced a pain-like visceromotor response and expression of Fos in spinal PPN neurons. CONCLUSIONS: In mice, ExPANs regulate myenteric neuron activity and smooth muscle contraction via a parasympathetic spinal circuit, linking sensation and pain to motility.

Profound alteration in cutaneous primary afferent activity produced by inflammatory mediators.

Inflammatory pain is thought to arise from increased transmission from nociceptors and recruitment of 'silent' afferents. To evaluate inflammation-induced changes, mice expressing GCaMP3 in cutaneous sensory neurons were generated and neuronal responses to mechanical stimulation in vivo before and after subcutaneous infusion of an 'inflammatory soup' (IS) were imaged in an unanesthetized preparation. Infusion of IS rapidly altered mechanical responsiveness in the majority of neurons. Surprisingly, more cells lost, rather than gained, sensitivity and 'silent' afferents that were mechanically insensitive and gained mechanosensitivity after IS exposure were rare. However, the number of formerly 'silent' afferents that became mechanosensitive was increased five fold when the skin was heated briefly prior to infusion of IS. These findings suggest that pain arising from inflamed skin reflects a dramatic shift in the balance of sensory input, where gains and losses in neuronal populations results in novel output that is ultimately interpreted by the CNS as pain.