Enteric Control of the Sympathetic Nervous System.

The autonomic nervous system that regulates the gut is divided into sympathetic (SNS), parasympathetic (PNS), and enteric nervous systems (ENS). They inhibit, permit, and coordinate gastrointestinal motility, respectively. A fourth pathway, "extrinsic sensory neurons," connect gut to the central nervous system, mediating sensation. The ENS resides within the gut wall and its activities are critical for life; ENS failure to populate the gut in development is lethal without intervention."Viscerofugal neurons" are a distinctive class of enteric neurons, being the only type that escapes the gut wall. They form a unique circuit: their axons project out of the gut wall and activate sympathetic neurons, which then project back to the gut, and inhibit gut movements.For 80 years viscerofugal/sympathetic circuits were thought to have a restricted role, mediating simple sensory-motor reflexes. New data shows viscerofugal and sympathetic neurons behaving unexpectedly, compelling a re-evaluation of these circuits: both viscerofugal and sympathetic neurons transmit higher order, synchronized firing patterns that originate within the ENS. This identifies them as driving long-range motility control between different gut regions.There is need for gut motor control over distances beyond the range of ENS circuits, yet no mechanism has been identified to date. The entero-sympathetic circuits are ideally suited to meet this need. Here we provide an overview of the structure and functions of these peripheral sympathetic circuits, including new data showing the firing patterns generated by enteric networks can transmit through sympathetic neurons.

Analysis of Intestinal Movements with Spatiotemporal Maps: Beyond Anatomy and Physiology.

Over 150 years ago, methods for quantitative analysis of gastrointestinal motor patterns first appeared. Graphic representations of physiological variables were recorded with the kymograph after the mid-1800s. Changes in force or length of intestinal muscles could be quantified, however most recordings were limited to a single point along the digestive tract.In parallel, photography and cinematography with X-Rays visualised changes in intestinal shape, but were hard to quantify. More recently, the ability to record physiological events at many sites along the gut in combination with computer processing allowed construction of spatiotemporal maps. These included diameter maps (DMaps), constructed from video recordings of intestinal movements and pressure maps (PMaps), constructed using data from high-resolution manometry catheters. Combining different kinds of spatiotemporal maps revealed additional details about gut wall status, including compliance, which relates forces to changes in length. Plotting compliance values along the intestine enabled combined DPMaps to be constructed, which can distinguish active contractions and relaxations from passive changes. From combinations of spatiotemporal maps, it is possible to deduce the role of enteric circuits and pacemaker cells in the generation of complex motor patterns. Development and application of spatiotemporal methods to normal and abnormal motor patterns in animals and humans is ongoing, with further technical improvements arising from their combination with impedance manometry, magnetic resonance imaging, electrophysiology, and ultrasonography.

Anatomical distribution of CGRP-containing lumbosacral spinal afferent neurons in the mouse uterine horn.

Sensory stimuli from the uterus are detected by spinal afferent neurons whose cell bodies arise from thoracolumbar and lumbosacral dorsal root ganglia (DRG). Using an in vivo survival surgical technique developed in our laboratory to remove select DRG from live mice, we recently quantified the topographical distribution of thoracolumbar spinal afferents innervating the mouse uterine horn, revealed by loss of immunoreactivity to calcitonin gene-related peptide (CGRP). Here, we used the same technique to investigate the distribution of lumbosacral uterine spinal afferents, in which L5-S1 DRG were unilaterally removed from adult female C57BL/6J mice (N = 6). Following 10-12 days recovery, CGRP immunoreactivity was quantified along the length of uterine horns using fluorescence immunohistochemistry. Relative to myometrial thickness, overall CGRP density in uterine tissues ipsilateral to L5-S1 DRG removal was reduced compared to the DRG-intact, contralateral side (P = 0.0265). Regionally, however, myometrial CGRP density was unchanged in the cranial, mid, and caudal portions. Similarly, CGRP-expressing nerve fiber counts, network lengths, junctions, and the proportion of area occupied by CGRP immunoreactivity were unaffected by DRG removal (P ≥ 0.2438). Retrograde neuronal tracing from the caudal uterine horn revealed fewer spinal afferents here arise from lumbosacral than thoracolumbar DRG (P = 0.0442) (N = 4). These data indicate that, unlike thoracolumbar DRG, lumbosacral spinal afferent nerves supply relatively modest sensory innervation across the mouse uterine horn, with no regional specificity. We conclude most sensory information between the mouse uterine horn and central nervous system is likely relayed via thoracolumbar spinal afferents.

The human enteric nervous system. Historical and modern advances. Collaboration between science and surgery.

BACKGROUND: There are considerable advantages and opportunities for surgeons and trainee surgeons in conducting a period of research allied with basic scientists. Such clinicians are well placed to define relevant clinical questions, provide human material (tissue, biopsy and blood) and translate the techniques derived in experimental animals to human subjects. METHODS: This small review explores research conducted on the nervous system of the intestines, with an emphasis on the translation of findings from animal to human. RESULTS: This work shows that new techniques of immunohistochemistry and retrograde tracing, developed in animal tissue, have greatly expanded our knowledge of the structure of the human enteric nervous system. CONCLUSIONS: Such findings have sparked therapeutic trials for the treatment of gastrointestinal disorders in patients.

Modification of Neurogenic Colonic Motor Behaviours by Chemogenetic Ablation of Calretinin Neurons.

How the enteric nervous system determines the pacing and propagation direction of neurogenic contractions along the colon remains largely unknown. We used a chemogenetic strategy to ablate enteric neurons expressing calretinin (CAL). Mice expressing human diphtheria toxin receptor (DTR) in CAL neurons were generated by crossing CAL-ires-Cre mice with Cre-dependent ROSA26-DTR mice. Immunohistochemical analysis revealed treatment with diphtheria toxin incurred a 42% reduction in counts of Hu-expressing colonic myenteric neurons (P = 0.036), and 57% loss of CAL neurons (comprising ∼25% of all Hu neurons; P = 0.004) compared to control. As proportions of Hu-expressing neurons, CAL neurons that contained nitric oxide synthase (NOS) were relatively spared (control: 15 ± 2%, CAL-DTR: 13 ± 1%; P = 0.145), while calretinin neurons lacking NOS were significantly reduced (control: 26 ± 2%, CAL-DTR: 18 ± 5%; P = 0.010). Colonic length and pellet sizes were significantly reduced without overt inflammation or changes in ganglionic density. Interestingly, colonic motor complexes (CMCs) persisted with increased frequency (mid-colon interval 111 ± 19 vs. 189 ± 24 s, CAL-DTR vs. control, respectively, P < 0.001), decreased contraction size (mid-colon AUC 26 ± 24 vs. 59 ± 13 gram/seconds, CAL-DTR vs. control, respectively, P < 0.001), and lacked preferential anterograde migration (P < 0.001). The functional effects of modest calretinin neuron ablation, particularly increased neurogenic motor activity frequencies, differ from models that incur general enteric neuron loss, and suggest calretinin neurons may contribute to pacing, force, and polarity of CMCs in the large bowel.

How should we define a nociceptor in the gut-brain axis?

In the past few years, there has been extraordinary interest in how the gut communicates with the brain. This is because substantial and gathering data has emerged to suggest that sensory nerve pathways between the gut and brain may contribute much more widely in heath and disease, than was originally presumed. In the skin, the different types of sensory nerve endings have been thoroughly characterized, including the morphology of different nerve endings and the sensory modalities they encode. This knowledge is lacking for most types of visceral afferents, particularly spinal afferents that innervate abdominal organs, like the gut. In fact, only recently have the nerve endings of spinal afferents in any visceral organ been identified. What is clear is that spinal afferents play the major role in pain perception from the gut to the brain. Perhaps surprisingly, the majority of spinal afferent nerve endings in the gut express the ion channel TRPV1, which is often considered to be a marker of "nociceptive" neurons. And, a majority of gut-projecting spinal afferent neurons expressing TRPV1 are activated at low thresholds, in the "normal" physiological range, well below the normal threshold for detection of painful sensations. This introduces a major conundrum regarding visceral nociception. How should we define a "nociceptor" in the gut? We discuss the notion that nociception from the gut wall maybe a process encrypted into multiple different morphological types of spinal afferent nerve ending, rather than a single class of sensory ending, like free-endings, suggested to underlie nociception in skin.

Effects of optogenetic activation of the enteric nervous system on gastrointestinal motility in mouse small intestine.

BACKGROUND AND AIMS: Recently, it was demonstrated that optogenetics could be used to stimulate enteric calretinin neurons, leading to increased colonic transit in vitro and in vivo. The aim of the current study was to determine if similar approaches could be used to stimulate the isolated mouse small intestine, with the aim of potentially also improving transit in the small bowel. METHODS: Cre-Lox recombination was used to generate transgenic mice expressing the light-sensitive ion channel channelrhodopsin-2 (ChR2) in calretinin neurons. RESULTS: Spontaneous migrating motor complexes were recorded from isolated terminal small intestine in both CalCre+ mice expressing ChR2 in calretinin-expressing neurons and experimental controls, CalCre-. Trains of blue light pulses (20 ms, 5 Hz, 20s) evoked a brief local contraction of circular muscle, but never a premature MMC, irrespective of light intensity (1-40 mV/mm2) or the region of ileum stimulated. However, MMCs were readily evoked by calretinin neuron activation in colon, consistent with our previous study. Light-evoked contractions of the terminal small bowel were hexamethonium-resistant (300 µM), but blocked by tetrodotoxin (0.6 µM). Light-evoked smooth muscle contraction did not change the pacemaker frequency underlying MMCs. CONCLUSION: Focal illumination of the small intestine does not appear as effective at inducing propulsive motor activity as has been demonstrated in the colon of the same colony mice. This study suggests caution should be exercised when assuming optogenetic technology is equally effective at increasing GI transit in the small intestine as in the large intestine of mice.

Distinct patterns of myogenic motor activity identified in isolated human distal colon with high-resolution manometry.

BACKGROUND: Colonic high-resolution manometry (HRM) has been used to reveal discrete, propagating colonic motor patterns. To help determine mechanisms underlying these patterns, we used HRM to record contractile activity in human distal colon ex vivo. METHODS: Surgically excised segments of descending (n = 30) or sigmoid colon (n = 4) were immersed in oxygenated Krebs solution at 36°C (n = 34; 16 female; 67.6 ± 12.4 years; length: 24.7 ± 3.5 cm). Contractility was recorded by HRM catheters. After 30 minutes of baseline recording, 0.3 mM lidocaine and/or 1 mM hexamethonium were applied. Ascending neural pathways were activated by electrical field stimulation (EFS; 10 Hz, 0.5 ms, 50 V, 5-s duration) applied to the anal end before and after drug application. RESULTS: Spontaneous propagating contractions were recorded in all specimens (0.1-1.5 cycles/minute). Most contractions occurred synchronously across all recording sites. In five specimens, rhythmic antegrade contractions propagated across the full length of the preparation. EFS evoked local contractions at the site of stimulation (latency: 5.5 ± 2.4 seconds) with greater amplitude than spontaneous contractions (EFS; 29.3 ± 26.9 vs 12.1 ± 14.8 mm Hg; P = .02). Synchronous or retrograde propagating motor patterns followed EFS; 71% spanned the entire preparation length. Hexamethonium and lidocaine modestly and only temporarily inhibited spontaneous contractions, whereas TTX increased the frequency of contractile activity while inhibiting EFS-evoked contractions. CONCLUSIONS AND INFERENCES: Our study suggests that the propagated contractions recorded in the organ bath have a myogenic origin which can be regulated by neural input. Once activated at a local site, the contractions do not require the propulsion of fecal content to sustain long-distance propagation.

Optogenetic control of the enteric nervous system and gastrointestinal transit.

INTRODUCTION: There are limited effective therapies available for improving gastrointestinal (GI) transit in mammals with intractable or chronic constipation. Current therapeutics to improve GI-transit usually require oral ingestion of therapeutic drugs, such as the serotonin receptor agonist prucalopride. However, most receptors are distributed all over the body and unsurprisingly drugs like prucalopride stimulate multiple organs, often leading to unwanted side effects. There is a desperate need in the community to improve GI-transit selectively without effects on other organs. Areas covered: We performed a systematic review of the literature on Pubmed and report significant technical advances in optogenetic control of the GI-tract. We discuss recent demonstrations that optogenetics can be used to potently control the activity of subsets of enteric neurons. Special focus is made of the first recent demonstration that wireless optogenetics can be used to stimulate the colon in conscious, freely-moving, untethered mice causing a significant increase in fecal pellet output. This is a significant technical breakthrough with a major therapeutic potential application to improve GI-transit. Expert opinion: The ability to selectively stimulate the ENS to modulate GI-transit in live mammals using light, avoids the need for oral consumption of any drugs and side effects; by stimulating only the GI-tract.

Visceral pain - Novel approaches for optogenetic control of spinal afferents.

Painful stimuli arising within visceral organs are detected by peripheral nerve endings of spinal afferents, whose cell bodies are located in dorsal root ganglia (DRG). Recent technical advances have made it possible to reliably expose and inject single DRG with neuronal tracers or viruses in vivo. This has facilitated, for the first time, unequivocal identification of different types of spinal afferent endings in visceral organs. These technical advances paved the way for a very exciting series of in vivo experiments where individual DRG are injected to facilitate opsin expression (e.g. Archaerhodopsin). Organ-specific expression of opsins in sensory neurons may be achieved by retrograde viral transduction. This means activity of target-specific populations of sensory neurons, within single DRG, can be modulated by optogenetic photo-stimulation. Using this approach we implanted micro light-emitting diodes (micro-LEDs) adjacent to DRG of interest, thereby allowing focal DRG-specific control of visceral and/or somatic afferents in conscious mice. This is vastly different from broad photo-illumination of peripheral nerve endings, which are dispersed over much larger surface areas across an entire visceral organ; and embedded deep within multiple anatomical layers. Focal DRG photo-stimulation also avoids the potential that wide-field illumination of the periphery could inadvertently activate other closely apposed organs, or co-activate different classes of axons in the same organ (e.g. enteric and spinal afferent endings in the gut). It is now possible to selectively control nociceptive and/or non-nociceptive pathways to specific visceral organs in vivo, using wireless optogenetics and micro-LEDs implanted adjacent to DRG, for targeted photo-stimulation.

Identifying unique subtypes of spinal afferent nerve endings within the urinary bladder of mice.

Spinal afferent neurons are responsible for the transduction and transmission of noxious (painful) stimuli and innocuous stimuli that do not reach conscious sensations from visceral organs to the central nervous system. Although the location of the nerve cell bodies of spinal afferents is well known to reside in dorsal root ganglia (DRG), the morphology and location of peripheral nerve endings of spinal afferents that transduce sensory stimuli into action potentials is poorly understood. The individual nerve endings of spinal afferents that innervate the urinary bladder have never been unequivocally identified in any species. We used an anterograde tracing technique developed in our laboratory to selectively label only spinal afferents. Mice were anesthetized and unilateral injections of dextran-amine made into lumbosacral DRGs (L5-S2). Seven to nine days postsurgery, mice were euthanized, the urinary bladder removed, then fresh-fixed and stained for immunoreactivity to calcitonin-gene-related-peptide (CGRP). Four distinct morphological types of spinal afferent ending in the bladder were identified. Three types existed in the detrusor muscle and one major type in the sub-urothelium and urothelium. Most nerve endings were located in detrusor muscle where the three types could be identified as having: "branching", "simple", or "complex" morphology. The majority of spinal afferent nerve endings were CGRP-immunoreactive. Single spinal afferent axons bifurcated many times upon entering the bladder and developed varicosities along their axon terminal endings. We present the first morphological identification of spinal afferent nerve endings in the mammalian urinary bladder.

Techniques to identify and temporally correlate calcium transients between multiple regions of interest in vertebrate neural circuits.

Calcium imaging is commonly used to record dynamic changes in excitability from axons or cell bodies in the nervous system of vertebrates. These recordings often reveal discrete calcium transients that have variable amplitudes, durations, and rates of rise and decay, all of which can arise from an unstable or "noisy" baseline. This often leads to considerable ambiguity about how to discriminate and quantify calcium transients. We describe an analytical methodology that objectively identifies multiple calcium transients from multiple recording sites and quantifies the degree of temporal synchrony between each event. The methodology consists of multiple steps. The first step involves baselining, to either preserve the underlying shape of calcium transients or remove unwanted frequency components and transform the peaks of calcium transients into more easily detectable patterns. The second step is the application of at least one of two different spike detection algorithms, one based on a gradient estimate and the other on template matching. The third step is the quantification of synchrony between pairs of recordings using at least one of two time lag correlation measures. The fourth step is the identification of statistically significant coincident firing patterns. This allows discrimination of neuronal firing patterns between different sites that appear to occur simultaneously and that statistically could not be attributed to chance. The analytical methods we have demonstrated can be applied not only to calcium imaging but also to many other physiological recordings, where discrimination and temporal correlation of biological signals from multiple sites is required, particularly when arising from unstable baselines, with variable signal-to-noise ratios.NEW & NOTEWORTHY Dynamic imaging of intracellular calcium is commonly used to record changes in excitability in central and peripheral neurons. We describe a novel analytical methodology that objectively discriminates calcium transients from low signal-to-noise recordings from multiple sites and quantifies the degree of temporal synchrony between events. These new methods can be applied not only to calcium imaging but also to many other physiological recordings where discrimination and temporal correlation of biological signals from multiple sites is required.

Spinal afferent nerve endings in visceral organs: recent advances.

Spinal afferent neurons play a major role in detection and transduction of painful stimuli from internal (visceral) organs. Recent technical advances have made it possible to visualize the endings of spinal afferent axons in visceral organs. Although it is well known that the sensory nerve cell bodies of spinal afferents reside within dorsal root ganglia (DRG), identifying their endings in internal organs has been especially challenging because of a lack of techniques to distinguish them from endings of other extrinsic and intrinsic neurons (sympathetic, parasympathetic, and enteric). We recently developed a surgical approach in live mice that allows selective labeling of spinal afferent axons and their endings, revealing a diverse array of different types of varicose and nonvaricose terminals in visceral organs, particularly the large intestine. In total, 13 different morphological types of endings were distinguished in the mouse distal large intestine, originating from lumbosacral DRG. Interestingly, the stomach, esophagus, bladder, and uterus had less diversity in their types of spinal afferent endings. Taken together, spinal afferent endings (at least in the large intestine) appear to display greater morphological diversity than vagal afferent endings that have previously been extensively studied. We discuss some of the new insights that these findings provide.

Imaging activation of peptidergic spinal afferent varicosities within visceral organs using novel CGRPα-mCherry reporter mice.

In vertebrates, visceral pain from internal organs is detected by spinal afferents, whose cell bodies lie in dorsal root ganglia (DRG). Until now, all recordings from spinal afferents have been restricted to recording transmission of action potentials along axons, or from cell bodies lying outside their target organ, which is not where sensory transduction occurs. Our aim was to record directly from a major class of spinal afferent within visceral organs, where transduction of sensory stimuli into action potentials occurs. Using novel calcitonin gene-related peptide (CGRP)α reporter mice, DRG neurons expressed mCherry, including nerve axons within viscera. In colon, a minority of total CGRP immunoreactivity was attributed CGRPα. In isolated unstretched colon, calcium imaging from CGRPα-expressing varicose axons did not detect resolvable calcium transients. However, noxious levels of maintained circumferential stretch to the colon induced repetitive calcium transients simultaneously in multiple neighboring varicosities along single mCherry-expressing axons. Discrete varicosities could generate unitary calcium transients independently of neighboring varicosities. However, axons expressing mCherry only generated coordinated calcium transients when accompanied by simultaneous activation of multiple varicosities along that axon. Simultaneous imaging from different classes of myenteric neurons at the same time as mCherry-expressing axons revealed coordinated calcium transients in multiple myenteric neurons, independent of activity in mCherry-expressing axons. CGRPα-expressing axon terminals preferentially responded to heat, capsaicin, and low pH. We show that direct recordings can be made from the major class of peptidergic spinal afferent that contributes to visceral nociception. This approach can provide powerful insights into transduction of stimuli in viscera.

Different types of spinal afferent nerve endings in stomach and esophagus identified by anterograde tracing from dorsal root ganglia.

In visceral organs of mammals, most noxious (painful) stimuli as well as innocuous stimuli are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRGs). One of the major unresolved questions is the location, morphology, and neurochemistry of the nerve endings of spinal afferents that actually detect these stimuli in the viscera. In the upper gastrointestinal (GI) tract, there have been many anterograde tracing studies of vagal afferent endings, but none on spinal afferent endings. Recently, we developed a technique that now provides selective labeling of only spinal afferents. We used this approach to identify spinal afferent nerve endings in the upper GI tract of mice. Animals were anesthetized, and injections of dextran-amine were made into thoracic DRGs (T8-T12). Seven days post surgery, mice were euthanized, and the stomach and esophagus were removed, fixed, and stained for calcitonin gene-related peptide (CGRP). Spinal afferent axons were identified that ramified extensively through many rows of myenteric ganglia and formed nerve endings in discrete anatomical layers. Most commonly, intraganglionic varicose endings (IGVEs) were identified in myenteric ganglia of the stomach and varicose simple-type endings in the circular muscle and mucosa. Less commonly, nerve endings were identified in internodal strands, blood vessels, submucosal ganglia, and longitudinal muscle. In the esophagus, only IGVEs were identified in myenteric ganglia. No intraganglionic lamellar endings (IGLEs) were identified in the stomach or esophagus. We present the first identification of spinal afferent endings in the upper GI tract. Eight distinct types of spinal afferent endings were identified in the stomach, and most of them were CGRP immunoreactive. J. Comp. Neurol. 524:3064-3083, 2016. © 2016 Wiley Periodicals, Inc.