Extrinsic Fibers Research Articles

Nodose afferent fibers require PIEZO2 for esophageal mechanosensation

The esophagus receives dual innervation of extrinsic afferent fibers from both the dorsal root ganglia as well as the vagal ganglia. These are polymodal sensory afferents that are able to sense a variety of stimuli that include chemical and mechanical stimuli. Previous research has demonstrated that the esophagus is innervated by low threshold mechanoreceptors arising from the nodose ganglia, which provide critical peripheral feedback that is required for proper swallowing and peristaltic reflexes. We know that in the vagal system, PIEZO2 plays a critical role in lung inflation and baroreceptor function in the aorta and carotid sinus. Hence, we hypothesize that PIEZO2 is the mechanotransducer in these nodose esophageal low threshold mechanoreceptors. We used a two-pronged approach to elucidate PIEZO2’s role in esophageal mechanosensation: first, GCaMP6s imaging and then single fiber electrophysiology to understand the kinetics of nodose esophageal afferent fiber activation. Both of these approaches used our ex-vivo intact vagal esophageal preparation. Our proof-of-principle studies were conducted in PIRT-GCaMP6s mice, obtained through the crossbreeding of Pirt-Cre (Cre expressed in all sensory neurons) mice with Cre-dependent, ROSA26 based, GCaMP6s reporter (B6.129S6-Gt(ROSA)26Sor-tm96(CAG-CGaMP6s)Hze). Our preliminary data in Pirt-GCaMP6s mice showed a profound increase in the Ca2+ influx in a subset of nodose neurons in response to mechanical distention of the esophagus at 5, 10, and 30mmHg pressures. For our knockout group, we obtained nodose-specific knockout of PIEZO2 by crossing P2X2-Cre mice (P2X2 is expressed in all nodose neurons) with PIEZO2fl/fl mice (P2X2/PIEZO2 KO mice). Upon performing our GCaMP6s imaging experiments in this group, we saw that there was a significant reduction in the number of nodose neurons that were responsive to esophageal distention (58% down to 17%). Comparing the fluorescence intensities of all esophageal neurons between the control and KO groups, we observed a significant reduction at all three intraesophageal pressures (p<0.05, n=458 and 395 neurons in control and KO groups, respectively). In our single fiber electrophysiology experiments, our control and KO groups were PIEZO2fl/fl mice and P2X2/PIEZO2 KO respectively. We were able to successfully record APs from A-fiber mechanosensitive esophageal neurons. The number of APs recorded from KO group was reduced at all the three intraesophageal pressures when compared to the control PIEZO2fl/fl mice. Our findings support the hypothesis that PIEZO2 is the mechanotransduction protein involved in vagal nodose afferent fibers innervating the esophagus. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Clinical Relevance of Pathological Diagnosis of Hirschsprung's Disease with Acetylcholine-Esterase Histochemistry or Calretinin Immunohistochemistry.

Hirschsprung disease (HD) manifests as a developmental anomaly affecting the enteric nervous system, where there is an absence of ganglion cells in the lower part of the intestine. This deficiency leads to functional blockages within the intestines. HD is usually confirmed or ruled out through rectal biopsy. The identification of any ganglion cells through hematoxylin and eosin (H&E) staining rules out HD. If ganglion cells are absent, further staining with acetylcholine-esterase (AChE) histochemistry or calretinin immunohistochemistry (IHC) forms part of the standard procedure for determining a diagnosis of HD. In 2017, our Institute of Pathology at University Hospital of Heidelberg changed our HD diagnostic procedure from AChE histochemistry to calretinin IHC. In this paper, we report the impact of the diagnostic procedure change on surgical HD therapy procedures and on the clinical outcome of HD patients. We conducted a retrospective review of the diagnostic procedures, clinical data, and postoperative progress of 29 patients who underwent surgical treatment for HD in the Department of Pediatric Surgery, University of Heidelberg, between 2012 and 2021. The patient sample was divided into two groups, each covering a treatment period of 5 years. In 2012-2016, HD diagnosis was performed exclusively using AChE histochemistry (AChE group, n = 17). In 2017-2021, HD diagnosis was performed exclusively using calretinin IHC (CR group, n = 12). There were no significant differences between the groups in sex distribution, weeks of gestation, birth weight, length of the aganglionic segment, or associated congenital anomalies. Almost half of the children in the AChE group, twice as many as in the CR group, required an enterostomy before transanal endorectal pull-through procedure (TERPT). In the AChE group, 4 patients (23.5%) required repeat bowel sampling to confirm the diagnosis. Compared to the AChE group, more children in the CR group suffered from constipation post TERPT. Elevated AChE expression is linked to hypertrophied extrinsic cholinergic nerve fibers in the aganglionic segment in the majority of patients with HD. The manifestation of increased AChE expression develops over time. Therefore, in neonatal patients with HD, especially those in the first 3 weeks of life, an increase in AChE reaction is not detected. Calretinin IHC reliably identifies the presence or absence of ganglion cells and offers multiple benefits over AChE histochemistry. These include the ability to perform the test on paraffin-embedded tissue sections, a straightforward staining pattern, a clear binary interpretation (negative or positive), cost-effectiveness, and utility regardless of patient age. The ability of calretinin IHC to diagnose HD early and time-independently prevented repeated intestinal biopsies in our patient population and allowed us to perform a one-stage TERPT in the first months of life, reducing the number of enterostomies and restoring colonic continuity early. Patients undergoing transanal pull-through under the age of 3 months require a close follow-up to detect cases with bowel movement problems.

Acellular Extrinsic Fiber Cementum Is Invariably Present in the Superficial Layer of Apical Cementum in Mouse Molar.

The cementum is a highly mineralized tissue that covers the tooth root. The regional differences among the types of cementum, especially in the extrinsic fibers that contribute to tooth support, remain controversial. Therefore, this study used second harmonic generation imaging in conjunction with automated collagen extraction and image analysis algorithms to facilitate the quantitative examination of the fiber characteristics and the changes occurring in these fibers over time. Acellular extrinsic fiber cementum (AEFC) was invariably observed in the superficial layer of the apical cementum in mouse molars, indicating that this region of the cementum plays a crucial role in supporting the tooth. The apical AEFC exhibited continuity and fiber characteristics comparable with the cervical AEFC, suggesting a common cellular origin for their formation. The cellular intrinsic fiber cementum present in the inner layer of the apical cementum showed consistent growth in the apical direction without layering. This study highlights the dynamic nature of the cementum in mouse molars and underscores the requirement for re-examining its structure and roles. The findings of the present study elucidate the morphophysiological features of cementum and have broader implications for the maintenance of periodontal tissue health.

Schwann Cells in the Aganglionic Colon of Hirschsprung Disease Can Generate Neurons for Regenerative Therapy.

Cell therapy offers the potential to replace the missing enteric nervous system (ENS) in patients with Hirschsprung disease (HSCR) and to restore gut function. The Schwann cell (SC) lineage has been shown to generate enteric neurons pre- and post-natally. Here, we aimed to isolate SCs from the aganglionic segment of HSCR and to determine their potential to restore motility in the aganglionic colon. Proteolipid protein 1 (PLP1) expressing SCs were isolated from the extrinsic nerve fibers present in the aganglionic segment of postnatal mice and patients with HSCR. Following 7-10 days of in vitro expansion, HSCR-derived SCs were transplanted into the aganglionic mouse colon ex vivo and in vivo. Successful engraftment and neuronal differentiation were confirmed immunohistochemically and calcium activity of transplanted cells was demonstrated by live cell imaging. Organ bath studies revealed the restoration of motor function in the recipient aganglionic smooth muscle. These results show that SCs isolated from the aganglionic segment of HSCR mouse can generate functional neurons within the aganglionic gut environment and restore the neuromuscular activity of recipient mouse colon. We conclude that HSCR-derived SCs represent a potential autologous source of neural progenitor cells for regenerative therapy in HSCR.

Pachyosteosclerosis, rhamphotheca and enhanced sensory capabilities of the premaxillae of Hyperodapedon (Archosauromorpha, Rhynchosauria): implications for foraging at the sediment–water interface

AbstractThe external morphology and microanatomy of 32 partial and complete Hyperodapedon premaxillae was examined to assess their functional attributes. This revealed morphological correlates for innervation of Hyperodapedon premaxillae in the form of posteriorly opening enlarged neurovascular foramina associated with several grooves, and a prominent neurovascular sulcus. Scanning electron microscopy shows numerous small, circular foramina in clusters along the lateroventral surface towards the anterior tip and along the ventral edge, often in a preferred orientation. These are found associated with high rugosity along the elongated anterolateral depression, and were related to nutrient supply and/or part of the neurovascular system. Selected premaxillae show extremely high bone compactness indices (especially at the anterior end) suggesting specialized osteosclerotic conditions, and dense and compact bone microstructure with almost no clear transition between the outer compact cortex and inner core. With ontogeny, the premaxillae became lateromedially thickened by deposition of lamellar zonal bone, and highly vascularized and dense from intense Haversian remodelling, suggesting pachyosteosclerosis of the premaxillae. Other characteristic features include profuse open vascular channels or a frayed margin at the anteroventral tip, and dense bundles of long and wavy extrinsic fibres. These features, along with high bone compactness, decrease posteriorly towards the naris. It is proposed that the Hyperodapedon premaxillae were covered by keratinized epithelium or rhamphotheca at the anterior end, and had heightened sensory capabilities that aided foraging for mussels and other invertebrates in soft sediments under shallow water. Such enhanced sensory capability is reported for the first time in an early‐diverging archosauromorph.

Down-Regulation of Double C2 Domain Alpha Promotes the Formation of Hyperplastic Nerve Fibers in Aganglionic Segments of Hirschsprung's Disease.

Hirschsprung’s disease (HSCR) is a common developmental anomaly of the gastrointestinal tract in children. The most significant characteristics of aganglionic segments in HSCR are hyperplastic extrinsic nerve fibers and the absence of endogenous ganglion plexus. Double C2 domain alpha (DOC2A) is mainly located in the nucleus and is involved in Ca2+-dependent neurotransmitter release. The loss function of DOC2A influences postsynaptic protein synthesis, dendrite morphology, postsynaptic receptor density and synaptic plasticity. It is still unknown why hyperplastic extrinsic nerve fibers grow into aganglionic segments in HSCR. We detected the expression of DOC2A in HSCR aganglionic segment colons and established three DOC2A-knockdown models in the Neuro-2a cell line, neural spheres and zebrafish separately. First, we detected the protein and mRNA expression of DOC2A and found that DOC2A was negatively correlated with AChE+ grades. Second, in the Neuro-2a cell lines, we found that the amount of neurite outgrowth and mean area per cell were significantly increased, which suggested that the inhibition of DOC2A promotes nerve fiber formation and the neuron’s polarity. In the neural spheres, we found that the DOC2A knockdown was manifested by a more obvious connection of nerve fibers in neural spheres. Then, we knocked down Doc2a in zebrafish and found that the down-regulation of Doc2a accelerates the formation of hyperplastic nerve fibers in aganglionic segments in zebrafish. Finally, we detected the expression of MUNC13-2 (UNC13B), which was obviously up-regulated in Grade3/4 (lower DOC2A expression) compared with Grade1/2 (higher DOC2A expression) in the circular muscle layer and longitudinal muscle layer. The expression of UNC13B was up-regulated with the knocking down of DOC2A, and there were protein interactions between DOC2A and UNC13B. The down-regulation of DOC2A may be an important factor leading to hyperplastic nerve fibers in aganglionic segments of HSCR. UNC13B seems to be a downstream molecule to DOC2A, which may participate in the spasm of aganglionic segments of HSCR patient colons.

Immunohistochemical characterization of nerve elements in porcine intrinsic laryngeal ganglia.

The present study investigated the chemical coding of neurons and nerve fibres in local laryngeal ganglia in pigs (n=5) using double-labelling immunohistochemistry. Virtually all the neurons were cholinergic in nature (ChAT- or VAChT-positive). Only very solitary, small nerve cells (presumably representing interneurons) stained intensely for adrenergic marker, DβH. Many neurons also contained immunoreactivity for NOS (91%), VIP (62.7%), NPY (24.7%), galanin (10%), SP (1.3%) and CGRP (5.3%). No neurons expressing somatostatin or Leu-enkephalin were observed. Nearly all the neuronal somata were densely supplied with varicose cholinergic nerve terminals, which presumably represented preganglionic axons, and some of them were also closely apposed with CGRP- and/or SP-positive varicose nerve endings, which were putative collaterals of extrinsic primary sensory fibres. In conclusion, this study has revealed that intrinsic neurons in the porcine larynx, like in many other mammalian species studied, should be classified as parasympathetic cholinergic neurons expressing biologically active substances, predominantly NOS and VIP. Furthermore, they are likely to receive inputs from not only preganglionic neurons but also primary sensory nerve cells. Finally, it appears that the information on the occurrence of the local laryngeal ganglia should be regularly included in textbooks dealing with the cranial portion of the parasympathetic nervous system in mammals.

Constipation Caused by Anti-calcitonin Gene-Related Peptide Migraine Therapeutics Explained by Antagonism of Calcitonin Gene-Related Peptide's Motor-Stimulating and Prosecretory Function in the Intestine.

The development of small-molecule calcitonin gene-related peptide (CGRP) receptor antagonists (gepants) and of monoclonal antibodies targeting the CGRP system has been a major advance in the management of migraine. In the randomized controlled trials before regulatory approval, the safety of these anti-CGRP migraine therapeutics was considered favorable and to stay within the expected profile. Post-approval real-world surveys reveal, however, constipation to be a major adverse event which may affect more than 50% of patients treated with erenumab (an antibody targeting the CGRP receptor), fremanezumab or galcanezumab (antibodies targeting CGRP). In this review article we address the question whether constipation caused by inhibition of CGRP signaling can be mechanistically deduced from the known pharmacological actions and pathophysiological implications of CGRP in the digestive tract. CGRP in the gut is expressed by two distinct neuronal populations: extrinsic primary afferent nerve fibers and distinct neurons of the intrinsic enteric nervous system. In particular, CGRP is a major messenger of enteric sensory neurons which in response to mucosal stimulation activate both ascending excitatory and descending inhibitory neuronal pathways that enable propulsive (peristaltic) motor activity to take place. In addition, CGRP is able to stimulate ion and water secretion into the intestinal lumen. The motor-stimulating and prosecretory actions of CGRP combine in accelerating intestinal transit, an activity profile that has been confirmed by the ability of CGRP to induce diarrhea in mice, dogs and humans. We therefore conclude that the constipation elicited by antibodies targeting CGRP or its receptor results from interference with the physiological function of CGRP in the small and large intestine in which it contributes to the maintenance of peristaltic motor activity, ion and water secretion and intestinal transit.

Poreklo ćelija enteričkog nervnog sistema i putevi migracije tokom embrionalnog razvoja

The enteric nervous system (ENS) is represented by a complex network of neurons, glial and other cells within the wall of the digestive tract. ENS is responsible for numerous, vital functions in our body. Thus, ENS regulates motility of the digestive tract, secretion into the intestinal lumen, exchange of fluid and electrolytes through the mucosa, as well as mucosal perfusion. In order to perform these important functions, proper embryonic development of ENS is necessary. ENS cells are derived from precursor cells of the neural crest (NCCs - neural crest cells). Two cell populations that contribute to the largest number of future ENS cells are the vagal and sacral NCCs. Vagal NCCs enter the primitive gut tube in the region of the future esophagus (foregut), and begin their migration, through the midgut towards the hindgut and the future anal region. Sacral NCCs enter the hindgut region following the extrinsic nerve fibers and continue their migration rostrally, towards vagal NCCs. Along with the migration process, these cells undergo other important processes, such as proliferation, neuro-glial differentiation, gangliogenesis, axonal pathway formation and synaptogenesis. All these processes are strictly regulated by numerous signaling pathways, which are still being actively researched. Modern lineage tracing and other technologies, that enabled following of individual precursor cells through their development pathways, will significantly contribute to the better understanding of development of ENS. This may have repercussions in improving the diagnosis and treatment of some developmental (Hirschsprung disease) and other ENS disorders.

Step-by-Step Cadaver Dissection and Surgical Technique for Compartmental Tongue and Floor of Mouth Resection.

BackgroundThe aim of oral cancer surgery is tumor removal within clear margins of healthy tissue: the latter definition in the literature, however, may vary between 1 and 2 cm, and should be intended in the three dimensions, which further complicates its precise measurement. Moreover, the biological behavior of tongue and floor of mouth cancer can be unpredictable and often eludes the previously mentioned safe surgical margins concept due to the complexity of tongue anatomy, the intricated arrangements of its intrinsic and extrinsic muscle fibers, and the presence of rich neurovascular and lymphatic networks within it. These structures may act as specific pathways of loco-regional tumor spread, allowing the neoplasm to escape beyond its visible macroscopic boundaries. Based on this concept, in the past two decades, compartmental surgery (CS) for treatment of oral tongue and floor of mouth cancer was proposed as an alternative to more traditional transoral resections.MethodsThe authors performed three anatomical dissections on fresh-frozen cadaver heads that were injected with red and blue-stained silicon. All procedures were documented by photographs taken with a professional reflex digital camera.ResultsOne of these step-by-step cadaver dissections is herein reported, detailing the pivotal points of CS with the aim to share this procedure at benefit of the youngest surgeons.ConclusionsWe herein present the CS step-by-step technique to highlight its potential in improving loco-regional control by checking all possible routes of tumor spread. Correct identification of the anatomical space between tumor and nodes (T-N tract), spatial relationships of extrinsic tongue muscles, as well as neurovascular bundles of the floor of mouth, are depicted to improve knowledge of this complex anatomical area.

Optical clearing reveals TNBS-induced morphological changes of VGLUT2-positive nerve fibers in mouse colorectum.

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.

A new capsulotomy-based dorsal approach to the wrist: A cadaver study

Using a cadaver study, we described a new dorsal approach to the wrist joint using a "U-shaped with proximal base" capsulotomy. Six fresh adult cadaveric wrists were dissected after intra-arterial silicone injection. We did a dorsal approach to expose the dorsal joint capsule. It was then possible to identify the dorsal radiocarpal and intercarpal ligaments, the dorsal radiocarpal and intercarpal arterial arches, the dorsal branch of the anterior interosseous artery and the terminal branch of the posterior interosseous nerve. Wrist arthrotomy was done using our capsulotomy. In each dissected capsular flap, we always found the individual ligament, vascular, and nerve structures, implying they were intact over their trajectories. The mean surface area of the articular exposure was 945 mm2 (range 725–1102 mm2) allowing easy access to the carpal bones and the radiocarpal and midcarpal joint spaces. This surgical approach to the wrist is technically feasible and avoids damaging the dorsal extrinsic ligaments fibers. Keeping the vascularization intact could improve capsular healing, while preserving innervation could maintain wrist proprioception.

Chemical-neuroanatomical organization of peripheral sensory-efferent systems in the pond snail (Lymnaea stagnalis)

Perception and processing of chemical cues are crucial for aquatic gastropods, for proper elaboration of adaptive behavior. The pond snail, Lymnaea stagnalis, is a model species of invertebrate neurobiology, in which peripheral sensory neurons with different morphology and transmitter content have partly been described, but we have little knowledge regarding their functional morphological organization, including their possible peripheral intercellular connections and networks. Therefore the aim of our study was to characterize the sensory system of the tentacles and the lip, as primary sensory regions, and the anterior foot of Lymnaea with special attention to the transmitter content of the sensory neurons, and their relationship to extrinsic elements of the central nervous system. Numerous bipolar sensory cells were demonstrated in the epithelial layer of the peripheral organs, displaying immunoreactivity to antibodies raised against tyrosine hydroxylase, histamine, glutamate and two molluscan type oligopeptides, FMRFamide and Mytilus inhibitory peptide. A subepithelial plexus was formed by extrinsic serotonin and FMRFamide immunoreactive fibers, whereas in deeper regions axon processess of different origin with various immunoreactivities formed networks, too. HPLC–MS assay confirmed the presence of the low molecular weight signal molecules in the three examined areas. Following double-labeling immunohistochemistry, close arrangements were observed, formed by sensory neurons and extrinsic serotonergic (and FMRFamidergic) fibers at axo-dendritic, axo-somatic and axo-axonic levels. Our results suggest the involvement of a much wider repertoire of signal molecules in peripheral sensory processes of Lymnaea, which can locally be modified by central input, hence influencing directly the responses to environmental cues.

Elevated activity levels do not influence extrinsic fiber attachment morphology on the surface of muscle-attachment sites.

Extrinsic fibers (EFs) are a type of penetrating collagenous fiber, closely related to the periodontal ligament, which help anchor soft tissue into bone. These fibers are associated with muscle attachment sites (entheses). Their size and grouping patterns are thought to be indicative of the loading history of the muscle. EFs are of particular significance in anthropology as potential tools for the reconstruction of behavior from skeletal remains and, specifically, entheses. In this study, we used a mouse model to experimentally test how activity level alters the morphology of EF insertion sites on the bone surface of a fibrocartilaginous enthesis, the biceps brachii insertion. Further, we adapted surface metrological techniques from studies of dental wear to perform automated, quantitative and non-destructive analysis of bone surface histology. Our results show that experimentally increased activity had no significant effect on the quantity or density of EF insertions at the enthesis, nor on the size of those insertions. Although EF presence does indicate muscle attachment, activity did not have an observable effect on EF morphology.

Innervation and nerve-immune cell contacts in mouse Peyer's patches.

Neural regulation of the function of the gastrointestinal tract (GIT) relies on a delicate balance of the two divisions of its nervous system, namely, the intrinsic and extrinsic divisions. The intrinsic innervation is provided by the enteric nervous system (ENS), whereas the extrinsic innervation includes sympathetic/parasympathetic nerve fibers and extrinsic sensory nerve fibers. In the present study, we used immunofluorescent staining of neurofilament-heavy (NF-H) to reveal the distribution of nerve fibers and their associations with immune cells inside mouse Peyer's patches (PP), an essential part of gut-associated lymphoid tissue (GALT). Our results demonstrate (1) the presence of an extensive meshwork of NF-H-immunoreactive presumptive nerve fibers in all PP compartments including the lymphoid nodules, interfollicular region, follicle-associated epithelium, and subepithelial dome; (2) close associations/contacts of nerve fibers with blood vessels including high endothelial venules, indicating neural control of blood flow and immune cell dynamics inside the PP; (3) close contacts between nerve fibers/endings and B/T cells and various subsets of dendritic cells ( e.g., B220⁻, B220⁺, CD4⁻, CD4⁺, CD8⁻, and CD8⁺). Our novel findings concerning PP innervation and nerve-immune-cell contacts in situ should facilitate our understanding of bi-directional communications between the PNS and GALT. Since the innervation of the gut, including PP, might be important in the pathogenesis and progression of some neurological, infectious, and autoimmune diseases, e.g., prion diseases and inflammatory bowel disease, better knowledge of PNS-immune system interactions in the GALT (including PP) should benefit the development of potential treatments for these diseases via neuroimmune manipulations.

Tyrosine hydroxylase-immunoreactive neurons in the mushroom body of the field cricket, Gryllus bimaculatus.

The mushroom body of the insect brain participates in processing and integrating multimodal sensory information and in various forms of learning. In the field cricket, Gryllus bimaculatus, dopamine plays a crucial role in aversive memory formation. However, the morphologies of dopamine neurons projecting to the mushroom body and their potential target neurons, the Kenyon cells, have not been characterized. Golgi impregnations revealed two classes of Kenyon cells (types I and II) and five different types of extrinsic fibers in the mushroom body. Type I cells, which are further divided into two subtypes (types I core and I surface), extend their dendrites into the anterior calyx, whereas type II cells extend many bushy dendritic branches into the posterior calyx. Axons of the two classes bifurcate between the pedunculus and lobes to form the vertical, medial and γ lobes. Immunocytochemistry to tyrosine hydroxylase (TH), a rate-limiting enzyme in dopamine biosynthesis, revealed the following four distinct classes of neurons: (1) TH-SLP projecting to the distal vertical lobe; (2) TH-IP1 extending to the medial and γ lobes; (3) TH-IP2 projecting to the basal vertical lobe; and (4) a multiglomerular projection neuron invading the anterior calyx and the lateral horn (TH-MPN). We previously proposed a model in the field cricket in which the efficiency of synapses from Kenyon cells transmitting a relevant sensory stimulus to output neurons commanding an appropriate behavioral reaction can be modified by dopaminergic neurons mediating aversive signals and here, we provide putative neural substrates for the cricket's aversive learning. These will be instrumental in understanding the principle of aversive memory formation in this model species.

Spinal Afferent Innervation of the Colon and Rectum.

Despite their seemingly elementary roles, the colon and rectum undertake a variety of key processes to ensure our overall wellbeing. Such processes are coordinated by the transmission of sensory signals from the periphery to the central nervous system, allowing communication from the gut to the brain via the “gut-brain axis”. These signals are transmitted from the peripheral terminals of extrinsic sensory nerve fibers, located within the wall of the colon or rectum, and via their axons within the spinal splanchnic and pelvic nerves to the spinal cord. Recent studies utilizing electrophysiological, anatomical and gene expression techniques indicate a surprisingly diverse set of distinct afferent subclasses, which innervate all layers of the colon and rectum. Combined these afferent sub-types allow the detection of luminal contents, low- and high-intensity stretch or contraction, in addition to the detection of inflammatory, immune, and microbial mediators. To add further complexity, the proportions of these afferents vary within splanchnic and pelvic pathways, whilst the density of the splanchnic and pelvic innervation also varies along the colon and rectum. In this review we traverse this complicated landscape to elucidate afferent function, structure, and nomenclature to provide insights into how the extrinsic sensory afferent innervation of the colon and rectum gives rise to physiological defecatory reflexes and sensations of discomfort, bloating, urgency, and pain.

Shape change throughout the body of the tongue during drinking in the striped skunk (Mephitis mephitis)

In mammals, the tongue plays a central role in many oral behaviors, but it is particularly essential for feeding and drinking. During feeding, the tongue is used for ingestion, manipulation and positioning of food within the oral cavity, and swallowing. When mammals drink, they do so by lapping, licking, or sucking, and the tongue is vital to uptake and transport of fluid into and through the oral cavity and oropharynx. All of these behaviors are performed with only a partial reliance on a bony support system; instead the tongue engages in complex movements as a muscular hydrostat – where selective contraction of intrinsic and extrinsic muscle fibers produce movements and deformations of the tongue. The tongue's anatomical complexity and its location inside the oral cavity make it particularly difficult to visualize and study the movements that contribute to these and other oral behaviors. The objective of this study is to test the ability of marker‐based XROMM (X‐ray reconstruction of moving morphology) to track shape changes throughout the body of the tongue. The striped skunk (Mephitis mephitis), a mammalian omnivore belonging to the order Carnivora, was used as a model for rhythmic lapping while drinking a barium liquid. Data was collected using XROMM with markers implanted in the bones of the skull as well as in the body of the tongue. Tongue markers were used to calculate length changes throughout the body of the tongue. Timing and movement parameters are also registered to the gape cycle derived from the rigid body motions derived from skeletal markers. Based on an analysis of 150 cycles, during rhythmic drinking, the middle and posterior tongue underwent the most variation in length change, while the anterior tongue underwent the least amount of variation in length. In contrast, the posterior tongue underwent the least amount of variation in tongue width, as compared to the anterior and middle tongue. Maximum overall tongue length occurred 2.74±29.3 msec before maximum gape, whereas minimal overall tongue length occurred 20.24±7.38 msec before minimum gape. Based on these preliminary results, this method shows promise for producing quantitative data for studying tongue deformations within the oral cavity. Future studies incorporating spatial parameters of the tongue to the rest of the feeding apparatus across mammals will further our understanding of how the tongue functions during feeding and drinking behaviors.Support or Funding InformationNational Science Foundation Grant DBI‐0922988, National Science Foundation Grant IOS‐1456810This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation.

Background & AimsTachykinins are involved in physiological and pathophysiological mechanisms in the gastrointestinal tract. The major sources of tachykinins in the gut are intrinsic enteric neurons in the enteric nervous system and extrinsic nerve fibers from the dorsal root and vagal ganglia. Although tachykinins are important mediators in the enteric nervous system, how they contribute to neuroinflammation through effects on neurons and glia is not fully understood. Here, we tested the hypothesis that tachykinins contribute to enteric neuroinflammation through mechanisms that involve intercellular neuron-glia signaling.MethodsWe used immunohistochemistry and quantitative real-time polymerase chain reaction, and studied cellular activity using transient-receptor potential vanilloid-1 (TRPV1)tm1(cre)Bbm/J::Polr2atm1(CAG-GCaMP5g,-tdTomato)Tvrd and Sox10CreERT2::Polr2atm1(CAG-GCaMP5g,-tdTomato)Tvrd mice or Fluo-4. We used the 2,4-di-nitrobenzene sulfonic acid (DNBS) model of colitis to study neuroinflammation, glial reactivity, and neurogenic contractility. We used Sox10::CreERT2+/-/Rpl22tm1.1Psam/J mice to selectively study glial transcriptional changes.ResultsTachykinins are expressed predominantly by intrinsic neuronal varicosities whereas neurokinin-2 receptors (NK2Rs) are expressed predominantly by enteric neurons and TRPV1-positive neuronal varicosities. Stimulation of NK2Rs drives responses in neuronal varicosities that are propagated to enteric glia and neurons. Antagonizing NK2R signaling enhanced recovery from colitis and prevented the development of reactive gliosis, neuroinflammation, and enhanced neuronal contractions. Inflammation drove changes in enteric glial gene expression and function, and antagonizing NK2R signaling mitigated these changes. Neurokinin A–induced neurodegeneration requires glial connexin-43 hemichannel activity.ConclusionsOur results show that tachykinins drive enteric neuroinflammation through a multicellular cascade involving enteric neurons, TRPV1-positive neuronal varicosities, and enteric glia. Therapies targeting components of this pathway could broadly benefit the treatment of dysmotility and pain after acute inflammation in the intestine.

Internal anal sphincter nerves - a macroanatomical and microscopic description of the extrinsic autonomic nerve supply of the internal anal sphincter.

The internal anal sphincter (IAS) contributes substantially to anorectal functions. While its autonomic nerve supply has been studied at the microscopic level, little information is available concerning the macroscopic topography of extrinsic nerve fibres. This study was designed to identify neural connections between the pelvic plexus and the IAS, provide a detailed topographical description, and give histological proof of autonomic nerve tissue. Macroscopic dissection of pelvic autonomic nerves was performed under magnification in seven (five male, two female) hemipelvises obtained from body donors (67-92years). Candidate structures were investigated by histological and immunohistochemical staining protocols to visualize nerve tissue. Nerve fibres could be traced from the anteroinferior edge of the pelvic plexus to the anorectal junction running along the neurovascular bundle anterolaterally to the rectum and posterolaterally to the prostate/vagina. Nerve fibres penetrated the longitudinal rectal muscle layer just above the fusion with the levator ani muscle (conjoint longitudinal muscle) and entered the intersphincteric space to reach the IAS. Histological and immunohistochemical findings confirmed the presence of nerve tissue. Autonomic nerve fibres supplying the IAS emerge from the pelvic plexus and are distinct to nerves entering the rectum via the lateral pedicles. Thus, they should be classified as IAS nerves. The identification and precise topographical location described provides a basis for nerve-sparing rectal resection procedures and helps to prevent postoperative functional anorectal disorders.