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The sensory innervation of the distal colorectum includes mechanically insensitive afferents (MIAs; ~25%), which acquire mechanosensitivity in persistent visceral hypersensitivity and thus generate de novo input to the central nervous system. We utilized an optogenetic approach to bypass the process of transduction (generator potential) and focus on transformation (spike initiation) at colorectal MIA sensory terminals, which is otherwise not possible in typical functional studies. From channelrhodopsin2-expressing mice (driven by Advillin-Cre), the distal colorectum with attached pelvic nerve was harvested for ex vivo single-fiber recordings. Afferent receptive fields (RFs) were identified by electrical stimulation and tested for response to mechanical stimuli (probing, stroking, and stretch), and afferents were classified as either MIAs or mechanosensitive afferents (MSAs). All MIA and MSA RFs were subsequently stimulated optically and MIAs were also tested for activation/ sensitization with inflammatory soup (IS), acidic hypertonic solution (AHS), and/or bile salts (BS). Responses to pulsed optical stimuli (1-10 Hz) were comparable between MSAs and MIAs whereas 43% of MIAs compared with 86% of MSAs responded tonically to stepped optical stimuli. Tonic-spiking MIAs responded preferentially to AHS (an osmotic stimulus) whereas non-tonicspiking MIAs responded to IS (an inflammatory stimulus). A significant proportion of MIAs were also sensitized by BS. These results reveal transformation as a critical factor underlying the differences between MIAs (osmosensors vs. inflammatory sensors), revealing a previously unappreciated heterogeneity of MIA endings. The current study draws attention to the sensory encoding of MIA nerve endings that likely contribute to afferent sensitization and thus have important roles in visceral pain. [ABSTRACT FROM AUTHOR]

Carbohydrate malabsorption such as in lactose intolerance or enteric infection causes symptoms that include abdominal pain. Because this digestive disorder increases intracolonic osmolarity and acidity by accumulation of undigested carbohydrates and fermented products, we tested whether these two factors (hypertonicity and acidity) would modulate colorectal afferents in association with colorectal nociception and hypersensitivity. In mouse colorectum-pelvic nerve preparations in vitro, afferent activities were monitored after application of acidic hypertonic saline (AHS; pH 6.0, 800 mosM). In other experiments, AHS was instilled intracolonically to mice and behavioral responses to colorectal distension (CRD) measured. Application of AHS in vitro excited 80% of serosal and 42% of mechanically-insensitive colorectal afferents (MIAs), sensitizing a proportion of MIAs to become mechanically sensitive and reversibly inhibiting stretch-sensitive afferents. Acute intracolonic AHS significantly increased expression of the neuronal activation marker pERK in colon sensory neurons and augmented noxious CRD-induced behavioral responses. After three consecutive daily intracolonic AHS treatments, mice were hypersensitive to CRD 4-15 days after the first treatment. In complementary single fiber recordings in vitro, the proportion of serosal class afferents increased at day 4; the proportion of MIAs decreased, and muscular class stretch-sensitive afferents were sensitized at days 11-15 in mice receiving AHS. These results indicate that luminal hypertonicity and acidity, two outcomes of carbohydrate malabsorption, can induce colorectal hypersensitivity to distension by altering the excitability and relative proportions of colorectal afferents, suggesting the potential involvement of these factors in the development of abdominal pain. [ABSTRACT FROM AUTHOR]

Inflammation of the distal bowel is often associated with abdominal pain and hypersensitivity, but whether and which colorectal afferents contribute to the hypersensitivity is unknown. Using a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, we investigated colorectal hypersensitivity following intracolonic TNBS and associated changes in colorectum and afferent functions. C57BL/6 mice were treated intracolonically with TNBS or saline. Visceromotor responses to colorectal distension (15-60 mmHg) were recorded over 8 wk in TNBS- and saline-treated (control) mice. In other mice treated with TNBS or saline, colorectal inflammation was assessed by myeloperoxidase assay and immunohistological staining. In vitro single-fiber recordings were conducted on both TNBS and saline-treated mice to assess colorectal afferent function. Mice exhibited significant colorectal hypersensitivity through day 14 after TNBS treatment that resolved by day 28 with no resensitization through day 56. TNBS induced a neutrophil- and macrophage-based colorectal inflammation as well as loss of nerve fibers, all of which resolved by days 14-28. Single-fiber recordings revealed a net increase in afferent drive from stretch-sensitive colorectal afferents at day 14 post-TNBS and reduced proportions of mechanically insensitive afferents (MIAs) at days 14-28. Intracolonic TNBS-induced colorectal inflammation was associated with the development and recovery of hypersensitivity in mice, which correlated with a transient increase and recovery of sensitization of stretch-sensitive colorectal afferents and MIAs. These results indicate that the development and maintenance of colorectal hypersensitivity following inflammation are mediated by peripheral drive from stretch-sensitive colorectal afferents and a potential contribution from MIAs. [ABSTRACT FROM AUTHOR]

Irritable bowel syndrome (IBS) is characterized as functional because a pathobiological cause is not readily apparent. Considerable evidence, however, documents that sensitizing proinflammatory and lipotoxic lipids, mast cells and their products, tryptases, enteroendocrine cells, and mononuclear phagocytes and their receptors are increased in tissues of IBS patients with colorectal hypersensitivity. It is also clear from recordings in animals of the colorectal afferent innervation that afferents exhibit long-term changes in models of persistent colorectal hypersensitivity. Such changes in afferent excitability and responses to mechanical stimuli are consistent with relief of discomfort and pain in IBS patients, including relief of referred abdominal hypersensitivity, upon intra-rectal instillation of local anesthetic. In the aggregate, these experimental outcomes establish the importance of afferent drive in IBS, consistent with a larger literature with respect to other chronic conditions in which pain is a principal complaint (e.g., neuropathic pain, painful bladder syndrome, fibromyalgia). Accordingly, colorectal afferents and the environment in which these receptive endings reside constitute the focus of this review. That environment includes understudied and incompletely understood contributions from immune-competent cells resident in and recruited into the colorectum. We close this review by highlighting deficiencies in existing knowledge and identifying several areas for further investigation, resolution of which we anticipate would significantly advance our understanding of neural and neuro-immune contributions to IBS pain and hypersensitivity. [ABSTRACT FROM AUTHOR]

Afferent input contributes significantly to the pain and colorectal hypersensitivity that characterize irritable bowel syndrome. In the present study, we investigated the contributions of mechanically sensitive and mechanically insensitive afferents (MIAs; or silent afferents) to colorectal hypersensitivity. The visceromotor response to colorectal distension (CRD; 15-60 mmHg) was recorded in mice before and for weeks after intracolonic treatment with zymosan or saline. After CRD tests, the distal colorectum with the pelvic nerve attached was removed for single-fiber electrophysiological recordings. Colorectal afferent endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. Intracolonic zymosan produced persistent colorectal hypersensitivity (>24 days) associated with brief colorectal inflammation. Pelvic nerve muscular-mucosal but not muscular mechanosensitive afferents recorded from mice with colorectal hypersensitivity exhibited persistent sensitization. In addition, the proportion of MIAs (relative to control) was significantly reduced from 27% to 13%, whereas the proportion of serosal afferents was significantly increased from 34% to 53%, suggesting that MIAs acquired mechanosensitivity. PGP9.5 immunostaining revealed no significant loss of colorectal nerve fiber density, suggesting that the reduction in MIAs is not due to peripheral fiber loss after intracolonic zymosan. These results indicate that colorectal MIAs and sensitized muscularmucosal afferents that respond to stretch contribute significantly to the afferent input that sustains hypersensitivity to CRD, suggesting that targeted management of colorectal afferent input could significantly reduce patients' complaints of pain and hypersensitivity. [ABSTRACT FROM AUTHOR]

Hypersensitivity in inflammatory/irritable bowel syndrome is contributed to in part by changes in the receptive properties of colorectal afferent endings, likely including mechanically insensitive afferents (MIAs; silent afferents) that have the ability to acquire mechanosensitivity. The proportion and attributes of colorectal MIAs, however, have not previously been characterized. The distal ~3 cm of colorectum with either pelvic (PN) or lumbar splanchnic (LSN) nerve attached was removed, opened longitudinally, pinned flat in a recording chamber, and perfused with oxygenated Krebs solution. Colorectal receptive endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. MIA endings were tested for response to and acquisition of mechanosensitivity by localized exposure to an inflammatory soup (IS). Colorectal afferents were also tested with twin-pulse and repetitive electrical stimulation paradigms. PN MIAs represented 23% of 211 afferents studied, 71% (30/42) of which acquired mechanosensitivity after application of IS to their receptive ending. LSN MIAs represented 33% of 156 afferents studied, only 23% (11/48) of which acquired mechanosensitivity after IS exposure. Mechanosensitive PN endings uniformly exhibited significant twin-pulse slowing whereas LSN endings showed no significant twin-pulse difference. PN MIAs displayed significantly greater activity-dependent slowing than LSN MIAs. In conclusion, significant proportions of MIAs are present in the colorectal innervation; significantly more in the PN than LSN acquire mechanosensitivity in an inflammatory environment. This knowledge contributes to our understanding of the possible roles of MIAs in colon-related disorders like inflammatory/irritable bowel syndrome. [ABSTRACT FROM AUTHOR]

Information about colorectal distension (i.e., colorectal dilation by increased intraluminal pressure) is primarily encoded by stretch-sensitive colorectal afferents in the pelvic nerve (PN). Despite anatomic differences between rectum and distal colon, little is known about the functional roles of colonic vs. rectal afferents in the PN pathway or the quantitative nature of mechanosensory encoding. We utilized an in vitro mouse colorectum-PN preparation to investigate pressure-encoding characteristics of colorectal afferents. The colorectum with PN attached was dissected, opened longitudinally, and pinned flat in a Sylgard-lined chamber. Action potentials of afferent fibers evoked by circumferential stretch (servo-controlled force actuator) were recorded from the PN. Stretch-sensitive fibers were categorized into the following four groups: colonic muscular, colonic muscular/mucosal, rectal muscular, and rectal muscular/mucosal. Seventy-nine stretch-sensitive PN afferents evenly distributed into the above four groups were studied. Rectal muscular afferents had significantly greater stretch-responses than the other three groups. Virtually all rectal afferents (98%) had low thresholds for response and encoded stimulus intensity into the noxious range without obvious saturation. Most colonic afferents (72%) also had low thresholds (<14 mmHg), but a significant proportion (28%) had high thresholds (>18 mml-lg) for response. These high-threshold colonic afferents were sensitized to stretch by inflammatory soup; response threshold was significantly reduced (from 23 to 12 mmHg), and response magnitude significantly increased. These results suggest that the encoding of mechanosensory information differs between colonic and rectal stretch-sensitive PN afferents. Rectal afferents have a wide response range to stretch, whereas high-threshold colonic afferents likely contribute to visceral nociception. [ABSTRACT FROM AUTHOR]

The enteric nervous system (ENS) functions largely independently of the central nervous system (CNS). Glutamate, the dominant neurotransmitter in the CNS and sensory afferents, is not a primary neurotransmitter in the ENS. Only a fraction (-2%) of myenteric neurons in the mouse distal colon and rectum (colorectum) are positive for vesicular glutamate transporter type 2 (VGLUT2), the structure and function of which remain undetermined. Here, we systematically characterized VGLUT2-positive enteric neurons (VGLUT2-ENs) through sparse labeling with adeno-associated virus, single-cell mRNA sequencing (scRNAseq), and GCaMP6f calcium imaging. Our results reveal that the majority of VGLUT2-ENs (29 of 31, 93.5%) exhibited Dogiel type I morphology with a single aborally projecting axon; most axons (26 of 29, 89.7%) are between 4 and 10 mm long, each traversing 19 to 34 myenteric ganglia. These anatomical features exclude the VGLUT2-ENs from being intrinsic primary afferent or motor neurons. The scRNA-seq conducted on 52 VGLUT2-ENs suggests different expression profiles from conventional descending interneurons. Ex vivo GCaMP6f recordings from flattened colorectum indicate that almost all VGLUT2-EN (181 of 215, 84.2%) are indirectly activated by colorectal stretch via nicotinic cholinergic neural transmission. In conclusion, VGLUT2-ENs are a functionally unique group of enteric neurons with single aborally projecting long axons that traverse multiple myenteric ganglia and are activated indirectly by colorectal mechanical stretch. This knowledge will provide a solid foundation for subsequent studies on the potential interactions of VGLUT2-EN with extrinsic colorectal afferents via glutamatergic neurotransmission. [ABSTRACT FROM AUTHOR]

Sex differences in visceral nociception have been reported in clinical and preclinical studies, but the potential differences in sensory neural encoding of the colorectum between males and females are not well understood. In this study, we systematically assessed sex differences in colorectal neural encoding by conducting high-throughput optical recordings in intact dorsal root ganglia (DRGs) from control and visceral hypersensitive mice. We found an apparent sex difference in zymosan-induced behavioral visceral hypersensitivity: enhanced visceromotor responses to colorectal distension were observed only in male mice, not in female mice. In addition, a higher number of mechanosensitive colorectal afferents were identified per mouse in the zymosantreated male group than in the saline-treated male group, whereas the mechanosensitive afferents identified per mouse were comparable between the zymosan- and saline-treated female groups. The increased number of identified afferents in zymosantreated male mice was predominantly from thoracolumbar (TL) innervation, which agrees with the significant increase in the TL afferent proportion in the zymosan group as compared with the control group in male mice. In contrast, female mice showed no difference in the proportion of colorectal neurons between saline- and zymosan-treated groups. Our results revealed a significant sex difference in colorectal afferent innervation and sensitization in the context of behavioral visceral hypersensitivity, which could drive differential clinical symptoms in male and female patients. NEW & NOTEWORTHY We used high-throughput GCaMP6f recordings to study 2,275 mechanosensitive colorectal afferents in mice. Our results revealed significant sex differences in the zymosan-induced behavioral visceral hypersensitivity, which were present in male but not female mice. Male mice also showed sensitization of colorectal afferents in the thoracolumbar pathway, whereas female mice did not. These findings highlight sex differences in sensory neural anatomy and function of the colorectum, with implications for sex-specific therapies for treating visceral pain. [ABSTRACT FROM AUTHOR]

Colorectal hypersensitivity and sensitization of both mechanosensitive and mechanically insensitive afferents develop after intracolonic instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in the mouse, a model of postinfectious irritable bowel syndrome. In mice in which ∼80% of extrinsic colorectal afferents were labeled genetically using the promotor for vesicular glutamate transporter type 2 (VGLUT2), we systematically quantified the morphology of VGLUT2-positive axons in mouse colorectum 7-28 days following intracolonic TNBS treatment. After removal, the colorectum was distended (20 mmHg), fixed with paraformaldehyde, and optically cleared to image VGLUT2-positive axons throughout the colorectal wall thickness. We conducted vector path tracing of individual axons to allow systematic quantification of nerve fiber density and shape. Abundant VGLUT2-positive nerve fibers were present in most layers of the colorectum, except the serosal and longitudinal muscular layers. A small percentage of VGLUT2-positive myenteric plexus neurons was also detected. Intracolonic TNBS treatment significantly reduced the number of VGLUT2-positive nerve fibers in submucosal, myenteric plexus, and mucosal layers at day 7 post-TNBS, which mostly recovered by day 28. We also found that almost all fibers in the submucosa were meandering and curvy, with ∼10% showing pronounced curviness (quantified by the linearity index). TNBS treatment resulted in a significant reduction of the proportions of pronounced curvy fibers in the rectal region at 28 days post-TNBS. Altogether, the present morphological study reveals profound changes in the distribution of VGLUT2-positive fibers in mouse colorectum undergoing TNBS-induced colitis and draws attention to curvy fibers in the submucosa with potential roles in visceral nociception. NEW & NOTEWORTHY We conducted genetic labeling and optical clearing to visualize extrinsic sensory nerve fibers in whole-mount colorectum, which revealed widespread presence of axons in the submucosal layer. Remarkably, axons in the submucosa were meandering and curvy, in contrast to axons in other layers generally aligned with the basal tissues. Intracolonic TNBS treatment led to pronounced changes of nerve fiber density and curviness, suggesting nerve fiber morphologies as potentially contributing factors to sensory sensitization. [ABSTRACT FROM AUTHOR]

Mechanical distension beyond a particular threshold evokes visceral pain from distal colon and rectum (colorectum), and thus biomechanics plays a central role in visceral nociception. In this study we focused on the layered structure of the colorectum through the wall thickness and determined the biomechanical properties of layer-separated colorectal tissue. We harvested the distal 30 mm of mouse colorectum and dissected this tissue into inner and outer composite layers. The inner composite consists of the mucosa and submucosa, whereas the outer composite includes the muscular layers and serosa. We divided each composite axially into three 10-mm-long segments and conducted biaxial mechanical extension tests and opening-angle measurements for each tissue segment. In addition, we quantified the thickness of the rich collagen network in the submucosa by nonlinear imaging via second-harmonic generation (SHG). Our results reveal that the inner composite is slightly stiffer in the axial direction, whereas the outer composite is stiffer circumferentially. The stiffness of the inner composite in the axial direction is about twice that in the circumferential direction, consistent with the orientations of collagen fibers in the submucosa approximately ±30° to the axial direction. Submucosal thickness measured by SHG showed no difference from proximal to distal colorectum under the load-free condition, which likely contributes to the comparable tension stiffness of the inner composite along the colorectum. This, in turn, strongly indicates the submucosa as the load-bearing structure of the colorectum. This further implies nociceptive roles for the colorectal afferent endings in the submucosa, which likely encode tissue-injurious mechanical distension. [ABSTRACT FROM AUTHOR]

Cholestasis of pregnancy endangers fetal and neonatal survival, yet systematic knowledge of the cause and effect of disrupted bile acid (BA) homeostasis in pregnancy is limited. Here we show that gestation stage-associated BA dysregulation in swine correlated with fetal death resulting from compromised capacity for BA secretion and increased alternative systemic efflux. The balance of BA input and output in the developing uterus suggested little uptake and metabolism of maternal BA by the placenta-fetus unit, implying a protection role of placenta in preventing maternal BA transported into the fetus. We showed that the maternal origin of BA accounted for the increase in placental total BA, leading to dysregulated expression of genes involved in BA transport and potentially impaired transplacental export of fetus-derived BA. Correspondingly, the secondary BA, mainly derived from the mother, gradually decreased in the fetus. Finally, we identified that sulfation rather than glucuronidation played pivotal roles in maintaining BA homeostasis of the developing fetus. These novel and systemic findings contribute to a whole picture of BA metabolism in pregnancy and provide new insights into mechanisms responsible for maternal and fetal BA homeostasis. [ABSTRACT FROM AUTHOR]

Visceral pain is one of the principal complaints of patients with irritable bowel syndrome, and this pain is reliably evoked by mechanical distension and stretch of distal colon and rectum (colorectum). This study focuses on the biomechanics of the colorectum that could play critical roles in mechanical neural encoding. We harvested the distal 30 mm of the colorectum from mice, divided evenly into three 10-mm-long segments (colonic, intermediate and rectal), and conducted biaxial mechanical stretch tests and opening-angle measurements for each tissue segment. In addition, we determined the collagen fiber orientations and contents across the thickness of the colorectal wall by nonlinear imaging via second harmonic generation (SHG). Our results reveal a progressive increase in tissue compliance and prestress from colonic to rectal segments, which supports prior electrophysiological findings of distinct mechanical neural encodings by afferents in the lumbar splanchnic nerves (LSN) and pelvic nerves (PN) that dominate colonic and rectal innervations, respectively. The colorectum is significantly more viscoelastic in the circumferential direction than in the axial direction. In addition, our SHG results reveal a rich collagen network in the submucosa and orients approximately 30° to the axial direction, consistent with the biaxial test results presenting almost twice the stiffness in axial direction versus the circumferential direction. Results from current biomechanical study strongly indicate the prominent roles of local tissue biomechanics in determining the differential mechanical neural encoding functions in different regions of the colorectum. [ABSTRACT FROM AUTHOR]