Mechanisms underlying the gut-brain communication: How enterochromaffin (EC) cells activate vagal afferent nerve endings in the small intestine.

How the gastrointestinal tract communicates with the brain, via sensory nerves, is of significant interest for our understanding of human health and disease. Enterochromaffin (EC) cells in the gut mucosa release a variety of neurochemicals, including the largest quantity of 5-hydroxytryptamine (5-HT) in the body. How 5-HT and other substances released from EC cells activate sensory nerve endings in the gut wall remains a major unresolved mystery. We used in vivo anterograde tracing from nodose ganglia to determine the spatial relationship between 5-HT synthesizing and peptide-YY (PYY)-synthesizing EC cells and their proximity to vagal afferent nerve endings that project to the mucosa of mouse small intestine. The shortest mean distances between single 5-HT- and PYY-synthesizing EC cells and the nearest vagal afferent nerve endings in the mucosa were 33.1 ± 14.4 µm (n = 56; N = 6) and 70.3 ± 32.3 µm (n = 16; N = 6). No morphological evidence was found to suggest that 5-HT- or PYY-containing EC cells form close morphological associations with vagal afferents endings, or varicose axons of passage. The large distances between EC cells and vagal afferent endings are many hundreds of times greater than those known to underlie synaptic transmission in the nervous system (typically 10-15 nm). Taken together, the findings lead to the inescapable conclusion that communication between 5-HT-containing EC cells and vagal afferent nerve endings in the mucosa of the mouse small intestinal occurs in a paracrine fashion, via diffusion. New and Noteworthy None of the findings here are consistent with a view that close physical contacts occur between 5-HT-containing EC cells and vagal afferent nerve endings in mouse small intestine. Rather, the findings suggest that gut-brain communication between EC cells and vagal afferent endings occurs via passive diffusion. The morphological data presented do not support the view that EC cells are physically close enough to vagal afferent endings to communicate via fast synaptic transmission.

Identification of vagal afferent nerve endings in the mouse colon and their spatial relationship with enterochromaffin cells.

Understanding how the gut communicates with the brain, via sensory nerves, is of significant interest to medical science. Enteroendocrine cells (EEC) that line the mucosa of the gastrointestinal tract release neurochemicals, including the largest quantity of 5-hydroxytryptamine (5-HT). How the release of substances, like 5-HT, from enterochromaffin (EC) cells activates vagal afferent nerve endings is unresolved. We performed anterograde labelling from nodose ganglia in vivo and identified vagal afferent axons and nerve endings in the mucosa of whole-mount full-length preparations of mouse colon. We then determined the spatial relationship between mucosal-projecting vagal afferent nerve endings and EC cells in situ using 3D imaging. The mean distances between vagal afferent nerve endings in the mucosa, or nearest varicosities along vagal afferent axon branches, and the nearest EC cell were 29.6 ± 19.2 µm (n = 107, N = 6) and 25.7 ± 15.2 µm (n = 119, N = 6), respectively. No vagal afferent endings made close contacts with EC cells. The distances between EC cells and vagal afferent endings are many hundreds of times greater than known distances between pre- and post-synaptic membranes (typically 10-20 nm) that underlie synaptic transmission in vertebrates. The absence of any close physical contacts between 5-HT-containing EC cells and vagal afferent nerve endings in the mucosa leads to the inescapable conclusion that the mechanism by which 5-HT release from ECs in the colonic mucosa occurs in a paracrine fashion, to activate vagal afferents.

TRPV4 is expressed by enteric glia and muscularis macrophages of the colon but does not play a prominent role in colonic motility.

Background: Mechanosensation is an important trigger of physiological processes in the gastrointestinal tract. Aberrant responses to mechanical input are associated with digestive disorders, including visceral hypersensitivity. Transient Receptor Potential Vanilloid 4 (TRPV4) is a mechanosensory ion channel with proposed roles in visceral afferent signaling, intestinal inflammation, and gut motility. While TRPV4 is a potential therapeutic target for digestive disease, current mechanistic understanding of how TRPV4 may influence gut function is limited by inconsistent reports of TRPV4 expression and distribution. Methods: In this study we profiled functional expression of TRPV4 using Ca2+ imaging of wholemount preparations of the mouse, monkey, and human intestine in combination with immunofluorescent labeling for established cellular markers. The involvement of TRPV4 in colonic motility was assessed in vitro using videomapping and contraction assays. Results: The TRPV4 agonist GSK1016790A evoked Ca2+ signaling in muscularis macrophages, enteric glia, and endothelial cells. TRPV4 specificity was confirmed using TRPV4 KO mouse tissue or antagonist pre-treatment. Calcium responses were not detected in other cell types required for neuromuscular signaling including enteric neurons, interstitial cells of Cajal, PDGFRα+ cells, and intestinal smooth muscle. TRPV4 activation led to rapid Ca2+ responses by a subpopulation of glial cells, followed by sustained Ca2+ signaling throughout the enteric glial network. Propagation of these waves was suppressed by inhibition of gap junctions or Ca2+ release from intracellular stores. Coordinated glial signaling in response to GSK1016790A was also disrupted in acute TNBS colitis. The involvement of TRPV4 in the initiation and propagation of colonic motility patterns was examined in vitro. Conclusions: We reveal a previously unappreciated role for TRPV4 in the initiation of distension-evoked colonic motility. These observations provide new insights into the functional role of TRPV4 activation in the gut, with important implications for how TRPV4 may influence critical processes including inflammatory signaling and motility.

Early Australian neuroscientists and the tyranny of distance.

Australian neuroscientists at the turn of the twentieth century and in the succeeding decades faced formidable obstacles to communication and supply due to their geographical isolation from centers of learning in Europe and North America. Consequently, they had to spend significant periods of their lives overseas for training and experience. The careers of six pioneers-Laura Forster, James Wilson, Grafton Elliot Smith, Alfred Campbell, Raymond Dart, and John Eccles-are presented in the form of vignettes that address their lives and most enduring scientific contributions. All six were medically trained and, although they never collaborated directly with one another, they were linked by their neuroanatomical interests and by shared mentors, who included Nobelists Ramon y Cajal and Charles Sherrington. By the 1960s, as the so-called "tyranny of distance" was overcome by advances in communication and transport technology, local collaborative groups of neuroscientists emerged in several Australian university departments that built on the individual achievements of these pioneers. This in turn led to the establishment of the Australasian Neuroscience Society in 1981.

From the organ bath to the whole person: a review of human colonic motility.

Motor function of the colon is essential for health. Our current understanding of the mechanisms that underlie colonic motility are based upon a range of experimental techniques, including molecular biology, single cell studies, recordings from muscle strips, analysis of part or whole organ ex vivo through to in vivo human recordings. For the surgeon involved in the clinical management of colonic conditions this amounts to a formidable volume of material. Here, we synthesize the key findings from these various experimental approaches so that surgeons can be better armed to deal with the complexities of the colon.

Circadian rhythms in colonic function.

A rhythmic expression of clock genes occurs within the cells of multiple organs and tissues throughout the body, termed "peripheral clocks." Peripheral clocks are subject to entrainment by a multitude of factors, many of which are directly or indirectly controlled by the light-entrainable clock located in the suprachiasmatic nucleus of the hypothalamus. Peripheral clocks occur in the gastrointestinal tract, notably the epithelia whose functions include regulation of absorption, permeability, and secretion of hormones; and in the myenteric plexus, which is the intrinsic neural network principally responsible for the coordination of muscular activity in the gut. This review focuses on the physiological circadian variation of major colonic functions and their entraining mechanisms, including colonic motility, absorption, hormone secretion, permeability, and pain signalling. Pathophysiological states such as irritable bowel syndrome and ulcerative colitis and their interactions with circadian rhythmicity are also described. Finally, the classic circadian hormone melatonin is discussed, which is expressed in the gut in greater quantities than the pineal gland, and whose exogenous use has been of therapeutic interest in treating colonic pathophysiological states, including those exacerbated by chronic circadian disruption.

Types of Neurons in the Human Colonic Myenteric Plexus Identified by Multilayer Immunohistochemical Coding.

BACKGROUND AND AIMS: Gut functions including motility, secretion, and blood flow are largely controlled by the enteric nervous system. Characterizing the different classes of enteric neurons in the human gut is an important step to understand how its circuitry is organized and how it is affected by disease. METHODS: Using multiplexed immunohistochemistry, 12 discriminating antisera were applied to distinguish different classes of myenteric neurons in the human colon (2596 neurons, 12 patients) according to their chemical coding. All antisera were applied to every neuron, in multiple layers, separated by elutions. RESULTS: A total of 164 combinations of immunohistochemical markers were present among the 2596 neurons, which could be divided into 20 classes, with statistical validation. Putative functions were ascribed for 4 classes of putative excitatory motor neurons (EMN1-4), 4 inhibitory motor neurons (IMN1-4), 3 ascending interneurons (AIN1-3), 6 descending interneurons (DIN1-6), 2 classes of multiaxonal sensory neurons (SN1-2), and a small, miscellaneous group (1.8% of total). Soma-dendritic morphology was analyzed, revealing 5 common shapes distributed differentially between the 20 classes. Distinctive baskets of axonal varicosities surrounded 45% of myenteric nerve cell bodies and were associated with close appositions, suggesting possible connectivity. Baskets of cholinergic terminals and several other types of baskets selectively targeted ascending interneurons and excitatory motor neurons but were significantly sparser around inhibitory motor neurons. CONCLUSIONS: Using a simple immunohistochemical method, human myenteric neurons were shown to comprise multiple classes based on chemical coding and morphology and dense clusters of axonal varicosities were selectively associated with some classes.

Slowed gastrointestinal transit is associated with an altered caecal microbiota in an aged rat model.

Gastrointestinal (GI) motility is largely dependent upon activity within the enteric nervous system (ENS) and is an important part of the digestive process. Dysfunction of the ENS can impair GI motility as is seen in the case of constipation where gut transit time is prolonged. Animal models mimicking symptoms of constipation have been developed by way of pharmacological manipulations. Studies have reported an association between altered GI motility and gut microbial population. Little is known about the changes in gut microbiota profile resulting specifically from pharmacologically induced slowed GI motility in rats. Moreover, the relationship between gut microbiota and altered intestinal motility is based on studies using faecal samples, which are easier to obtain but do not accurately reflect the intestinal microbiome. The aim of this study was to examine how delayed GI transit due to opioid receptor agonism in the ENS modifies caecal microbiota composition. Differences in caecal microbial composition of loperamide-treated or control male Sprague Dawley rats were determined by 16S rRNA gene amplicon sequencing. The results revealed that significant differences were observed at both genus and family level between treatment groups. Bacteroides were relatively abundant in the loperamide-induced slowed GI transit group, compared to controls. Richness and diversity of the bacterial communities was significantly lower in the loperamide-treated group compared to the control group. Understanding the link between specific microbial species and varying transit times is crucial to design interventions targeting the microbiome and to treat intestinal motility disorders.

Endocannabinoids, anandamide and 2-AG, regulate mechanosensitivity of mucosal afferents in the Guinea pig bladder.

Bladder afferents play a crucial role in urine storage and voiding, and conscious sensations from the bladder. Endocannabinoids, anandamide (AEA) and 2-arachidonolylglycerol (2-AG), are endogenous ligands of G-protein coupled cannabinoid receptors 1 and 2 (CB1 and CB2) found in the CNS and peripheral organs. They also have off-target effects on some ligand- and voltage-gated channels. The aim of this study is to determine the role of AEA and 2-AG in regulation of mechanosensitivity of probable nociceptive neurons innervating the bladder - capsaicin-sensitive mucosal afferents. The activity of these afferents was determined by ex vivo single unit extracellular recordings in the guinea pig bladder. A stable analogue of anandamide, methanandamide (mAEA) evoked initial excitatory response of mucosal afferents followed by potentiation of their responses to mechanical stimulation. In the presence of TRPV1 antagonist (AMG9810), mAEA's effect on mechanosensitivity switched from excitatory to inhibitory. The inhibitory effect of mAEA is due to activation of both CB1 and CB2 cannabinoid receptors since it was abolished by combined application of selective CB1 (NESS0327) and CB2 (SR144528) antagonists. 2-AG application evoked a brief excitation of mucosal afferents, without potentiation of their mechanosensitivity, followed by the inhibition of their responses to mechanical stimulation. CB2 receptor antagonist, SR144528 abolished the inhibitory effect of 2-AG. Our data indicated that anandamide and 2-AG have opposite effects on mechanosensitivity of mucosal capsaicin-sensitive afferents in the guinea pig bladder; mAEA potentiated while 2-AG inhibited responses of mucosal afferents to mechanical stimulation. These findings are important for understanding of the role of endocannabinoids in regulating bladder sensation and function.

Stimulation of extrinsic sympathetic nerves differentially affects neurogenic motor activity in guinea pig distal colon.

The speed of pellet propulsion through the isolated guinea pig distal colon in vitro significantly exceeds in vivo measurements, suggesting a role for inhibitory mechanisms from sources outside the gut. The aim of this study was to investigate the effects of sympathetic nerve stimulation on three different neurogenic motor behaviors of the distal colon: transient neural events (TNEs), colonic motor complexes (CMCs), and pellet propulsion. To do this, segments of guinea pig distal colon with intact connections to the inferior mesenteric ganglion (IMG) were set up in organ baths allowing for simultaneous extracellular suction electrode recordings from smooth muscle, video recordings for diameter mapping, and intraluminal manometry. Electrical stimulation (1-20 Hz) of colonic nerves surrounding the inferior mesenteric artery caused a statistically significant, frequency-dependent inhibition of TNEs, as well as single pellet propulsion, from frequencies of 5 Hz and greater. Significant inhibition of CMCs required stimulation frequencies of 10 Hz and greater. Phentolamine (3.6 µM) abolished effects of colonic nerve stimulation, consistent with a sympathetic noradrenergic mechanism. Sympathetic inhibition was constrained to regions with intact extrinsic nerve pathways, allowing normal motor behaviors to continue without modulation in adjacent extrinsically denervated regions of the same colonic segments. The results demonstrate differential sensitivities to sympathetic input among distinct neurogenic motor behaviors of the colon. Together with findings indicating CMCs activate colo-colonic sympathetic reflexes through the IMG, these results raise the possibility that CMCs may paradoxically facilitate suppression of pellet movement in vivo, through peripheral sympathetic reflex circuits.

Piezo2 channels expressed by colon-innervating TRPV1-lineage neurons mediate visceral mechanical hypersensitivity.

Inflammatory and functional gastrointestinal disorders such as irritable bowel syndrome (IBS) and obstructive bowel disorder (OBD) underlie the most prevalent forms of visceral pain. Although visceral pain can be generally provoked by mechanical distension/stretch, the mechanisms that underlie visceral mechanosensitivity in colon-innervating visceral afferents remain elusive. Here, we show that virally mediated ablation of colon-innervating TRPV1-expressing nociceptors markedly reduces colorectal distention (CRD)-evoked visceromotor response (VMR) in mice. Selective ablation of the stretch-activated Piezo2 channels from TRPV1 lineage neurons substantially reduces mechanically evoked visceral afferent action potential firing and CRD-induced VMR under physiological conditions, as well as in mouse models of zymosan-induced IBS and partial colon obstruction (PCO). Collectively, our results demonstrate that mechanosensitive Piezo2 channels expressed by TRPV1-lineage nociceptors powerfully contribute to visceral mechanosensitivity and nociception under physiological conditions and visceral hypersensitivity under pathological conditions in mice, uncovering potential therapeutic targets for the treatment of visceral pain.

Characterization of viscerofugal neurons in human colon by retrograde tracing and multi-layer immunohistochemistry.

Background and Aims: Viscerofugal neurons (VFNs) have cell bodies in the myenteric plexus and axons that project to sympathetic prevertebral ganglia. In animals they activate sympathetic motility reflexes and may modulate glucose metabolism and feeding. We used rapid retrograde tracing from colonic nerves to identify VFNs in human colon for the first time, using ex vivo preparations with multi-layer immunohistochemistry. Methods: Colonic nerves were identified in isolated preparations of human colon and set up for axonal tracing with biotinamide. After fixation, labeled VFN cell bodies were subjected to multiplexed immunohistochemistry for 12 established nerve cell body markers. Results: Biotinamide tracing filled 903 viscerofugal nerve cell bodies (n = 23), most of which (85%) had axons projecting orally before entering colonic nerves. Morphologically, 97% of VFNs were uni-axonal. Of 215 VFNs studied in detail, 89% expressed ChAT, 13% NOS, 13% calbindin, 9% enkephalin, 7% substance P and 0 of 123 VFNs expressed CART. Few VFNs contained calretinin, VIP, 5HT, CGRP, or NPY. VFNs were often surrounded by dense baskets of axonal varicosities, probably reflecting patterns of connectivity; VAChT+ (cholinergic), SP+ and ENK+ varicosities were most abundant around them. Human VFNs were diverse; showing 27 combinations of immunohistochemical markers, 4 morphological types and a wide range of cell body sizes. However, 69% showed chemical coding, axonal projections, soma-dendritic morphology and connectivity similar to enteric excitatory motor neurons. Conclusion: Viscerofugal neurons are present in human colon and show very diverse combinations of features. High proportions express ChAT, consistent with cholinergic synaptic outputs onto postganglionic sympathetic neurons in prevertebral ganglia.

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.

Activation of ENS Circuits in Mouse Colon: Coordination in the Mouse Colonic Motor Complex as a Robust, Distributed Control System.

The characteristic motor patterns of the colon are coordinated by the enteric nervous system (ENS) and involve enterochromaffin (EC) cells, enteric glia, smooth muscle fibers, and interstitial cells. While the fundamental control mechanisms of colonic motor patterns are understood, greater complexity in the circuitry underlying motor patterns has been revealed by recent advances in the field. We review these recent advances and new findings from our laboratories that provide insights into how the ENS coordinates motor patterns in the isolated mouse colon. We contextualize these observations by describing the neuromuscular system underling the colonic motor complex (CMC) as a robust, distributed control system. Framing the colonic motor complex as a control system reveals a new perspective on the coordinated motor patterns in the colon. We test the control system by applying electrical stimulation in the isolated mouse colon to disrupt the coordination and propagation of the colonic motor complex.

Identifying Types of Neurons in the Human Colonic Enteric Nervous System.

Distinguishing and characterising the different classes of neurons that make up a neural circuit has been a long-term goal for many neuroscientists. The enteric nervous system is a large but moderately simple part of the nervous system. Enteric neurons in laboratory animals have been extensively characterised morphologically, electrophysiologically, by projections and immunohistochemically. However, studies of human enteric nervous system are less advanced despite the potential availability of tissue from elective surgery (with appropriate ethics permits). Recent studies using single cell sequencing have confirmed and extended the classification of enteric neurons in mice and human, but it is not clear whether an encompassing classification has been achieved. We present preliminary data on a means to distinguish classes of myenteric neurons in specimens of human colon combining immunohistochemical, morphological, projection and size data on single cells. A method to apply multiple layers of antisera to specimens was developed, allowing up to 12 markers to be characterised in individual neurons. Applied to multi-axonal Dogiel type II neurons, this approach demonstrated that they constitute fewer than 5% of myenteric neurons, are nearly all immunoreactive for choline acetyltransferase and tachykinins. Many express the calcium-binding proteins calbindin and calretinin and they are larger than average myenteric cells. This methodology provides a complementary approach to single-cell mRNA profiling to provide a comprehensive account of the types of myenteric neurons in the human colon.

Rhythmicity in the Enteric Nervous System of Mice.

The enteric nervous system (ENS) is required for many cyclical patterns of motor activity along different regions of the gastrointestinal (GI) tract. What has remained mysterious is precisely how many thousands of neurons within the ENS are temporally activated to generate cyclical neurogenic contractions of GI-smooth muscle layers. This has been an especially puzzling conundrum, since the ENS consists of an extensive network of small ganglia, with each ganglion consisting of a heterogeneous population of neurons, with diverse cell soma morphologies, neurochemical and biophysical characteristics, and neural connectivity. Neuronal imaging studies of the mouse large intestine have provided major new insights into how the different classes of myenteric neurons are activated during cyclical neurogenic motor patterns, such as the colonic motor complex (CMC). It has been revealed that during CMCs (in the isolated mouse whole colon), large populations of myenteric neurons, across large spatial fields, coordinate their firing, via bursts of fast synaptic inputs at ~2 Hz. This coordinated firing of many thousands of myenteric neurons synchronously over many rows of interconnected ganglia occurs irrespective of the functional class of neuron. Aborally directed propulsion of content along the mouse colon is due, in large part, to polarity of the enteric circuits including the projections of the intrinsic excitatory and inhibitory motor neurons but still involves the fundamental ~2 Hz rhythmic activity of specific classes of enteric neurons. What remains to be determined are the mechanisms that initiate and terminate the patterned firing of large ensembles of enteric neurons during cyclic activity. This remains an exciting challenge for future studies.

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.

Disengaging spinal afferent nerve communication with the brain in live mice.

Our understanding of how abdominal organs (like the gut) communicate with the brain, via sensory nerves, has been limited by a lack of techniques to selectively activate or inhibit populations of spinal primary afferent neurons within dorsal root ganglia (DRG), of live animals. We report a survival surgery technique in mice, where select DRG are surgically removed (unilaterally or bilaterally), without interfering with other sensory or motor nerves. Using this approach, pain responses evoked by rectal distension were abolished by bilateral lumbosacral L5-S1 DRG removal, but not thoracolumbar T13-L1 DRG removal. However, animals lacking T13-L1 or L5-S1 DRG both showed reduced pain sensitivity to distal colonic distension. Removal of DRG led to selective loss of peripheral CGRP-expressing spinal afferent axons innervating visceral organs, arising from discrete spinal segments. This method thus allows spinal segment-specific determination of sensory pathway functions in conscious, free-to-move animals, without genetic modification.

Quantification of CGRP-immunoreactive myenteric neurons in mouse colon.

Quantitative data of biological systems provide valuable baseline information for understanding pathology, experimental perturbations, and computational modeling. In mouse colon, calcitonin gene-related peptide (CGRP) is expressed by myenteric neurons with multiaxonal (Dogiel type II) morphology, characteristic of intrinsic primary afferent neurons (IPANs). Analogous neurons in other species and gut regions represent 5-35% of myenteric neurons. We aimed to quantify proportions of CGRP-immunopositive (CGRP+) myenteric neurons. Colchicine-treated wholemount preparations of proximal, mid, and distal colon were labeled for HuC/D, CGRP, nitric oxide synthase (NOS), and peripherin (Per). The pan-neuronal markers (Hu+/Per+) co-labeled 94% of neurons. Hu+/Per- neurons comprised ∼6%, but Hu-/Per+ cells were rare. Thus, quantification was based on Hu+ myenteric neurons (8576 total; 1225 ± 239 per animal, n = 7). CGRP+ cell bodies were significantly larger than the average of all Hu+ neurons (329 ± 13 vs. 261 ± 12 µm2 , p < .0001). CGRP+ neurons comprised 19% ± 3% of myenteric neurons without significant regional variation. NOS+ neurons comprised 42% ± 2% of myenteric neurons overall, representing a lower proportion in proximal colon, compared to mid and distal colon (38% ± 2%, 44% ± 2%, and 44% ± 3%, respectively). Peripherin immunolabeling revealed cell body and axonal morphology in some myenteric neurons. Whether all CGRP+ neurons were multiaxonal could not be addressed using peripherin immunolabeling. However, of 118 putatively multiaxonal neurons first identified based on peripherin immunoreactivity, all were CGRP+ (n = 4). In conclusion, CGRP+ myenteric neurons in mouse colon were comprehensively quantified, occurring within a range expected of a putative IPAN marker. All Per+ multiaxonal neurons, characteristic of Dogiel type II/IPAN morphology, were CGRP+.