Afferent Innervation Research Articles

Stellate Ganglion Sympathetic postganglionic efferent innervation in the flat-mount of the whole heart of rats: Anterograde Tracing

The autonomic nervous system (sympathetic and parasympathetic) plays an essential role in controlling cardiac functions. Previously, we used anterograde tracing to examine the vagal afferent and efferent (preganglionic) innervations of the normal heart, their anatomical remodeling and their functional changes in disease models of rats and mice. In addition, we used vesicular acetylcholine transporter (VAChT, a parasympathetic marker;) and tyrosine hydroxylase (TH, a sympathetic marker,) to determine the parasympathetic and sympathetic postganglionic innervation of the whole heart (atria and ventricles). Immunohistochemical labeling with TH is useful for determining the broad distribution of sympathetic efferent axons in the heart. However, it is difficult to distinguish its extrinsic and intrinsic origins using TH labeling. Furthermore, the topographical organization and morphological terminal structures of cardiac sympathetic postganglionic efferent axons from the stellate ganglia (SG) remains unknown. To address this we anterogradely labeled individual sympathetic postganglionic axons projecting to the heart, using tracer Dextran-Biotin microinjections into the right SG of Sprague-Dawley rats (male, n=6, 3-6 months). Fourteen days after the injection, animals were euthanized, and their hearts were dissected into the left/right atria (LA and RA) and ventricles and prepared as flat-mounts. The tracer-labeled axons were digitized and analyzed using The Neurolucida 3D tracing system and imaged using a Zeiss M2 Imager. We found that stellate sympathetic neurons had projections to the LA and RA and left and right ventricles. Individual sympathetic axons formed complex arbors of varicose neurites in the areas of the sinoatrial node (SAN) and atrioventricular node (AVN) as well as the other areas of the atria and throughout the ventricles. Sympathetic axons also innervated blood vessels. Using the Neurolucida Digitization and Tracer system, we determined the regional density and characterized different types of axons. Some axons formed relatively simple terminal structures with only a few branches, whereas other axons produced very complex terminals with extensive branching. For the first time, we anterogradely labeled the sympathetic postganglionic axons and characterized their terminal architecture in the whole heart. These results will provide an anatomical foundation for future study This study was supported by NIH HEAL/SPARC U01 NS113867-01 and NIH R15HL137143-01A1. This is the full abstract presented at the American Physiology Summit 2023 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.

Vagal Afferent Innervation of the Atria and Ventricles of the Rat Heart: Anterograde Tracing

Vagal afferent innervation of the atria from the nodose ganglion has been shown in previous studies but the innervation of the ventricles remains unknown. In this study, tracer Dextran-Biotin was injected into the left nodose ganglion of SD male rats (n=6; 3 months). The tracer was allowed to transport for 14 days, then the rats were perfused through the left ventricle with 40°C phosphate-buffered saline (PBS, 0.1 M, pH = 7.4). Tissues were then fixed by perfusion with 4% paraformaldehyde in 0.1M PBS (4°C, pH 7.4). Whole rat hearts were harvested, and the atria and ventricles were separated. Then, a cut was made along the interatrial septum to separate the left and right atrium, then either a DAB staining or fluorescent immunohistochemistry staining was performed. Intrinsic cardiac ganglionic (ICG) neurons were stained with Cuprolinic Blue or Fluoro-Gold. Labeled single vagal afferent axons in the flat mounts of the whole heart were traced using the Neurolucida system. Consistent with previous findings using fluorescent tracer 1,1’-dioleyl-3,3,3’,3’ tetramethylindocarbocyaninemethanesulfonate (DiI) injection, afferent axons have been observed in the epi-, myo-, and endocardium and vagal axons formed “flower-spray” and “end-net” terminals in the atria. Also, the afferents displayed contact with cardiac muscles. For the first time, the “flower-spray” and “end-net” vagal afferent terminals have been found in both the left and right ventricles. Neither the “flower-spray” and “end-net” vagal afferent terminals on the heart, nor the vagal afferent terminal structures on the stomach such as intraganglionic laminar endings ( IGLEs) and intramuscular arrays ( IMAs) were found to co-localize with calcitonin gene-related peptide (CGRP) or Substance P (SP) in the three rats with injection followed fluorescent labeling and immunohistochemical analysis. In conclusion, our study provides the foundation for further functional studies of vagal afferent innervation of the whole heart (atria and ventricles) in normal and pathological states. This study was supported by NIH HEAL/SPARC U01 NS113867-01 (ZJC), NIH R15HL137143-01A1 (ZJC). This is the full abstract presented at the American Physiology Summit 2023 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.

About The Physiology of Hearing

The membrana basilaris plays an important role in the theory of traveling wave. It originates from the ectoblast of the connective tissue, and is bereft of both afferent or efferent innervation. Vibrations of the membrana basilaris formed into a transverse wave occur together with the organ of Corti and a band of connective tissue on the inferior membrane surface. Vibrations take place in a fluid which has suppressive properties. The length and the mass of the membrana basilaris in mammals and birds are very different and there is no relationship between the membrane basilaris length and the length of the sound wave, especially in the range of low frequencies. Small sound intensities, supraliminal, have amplitudes within the limits of a few picometers upon the entrance into the auditory meatus. This amplitude disappears in cochlear fluids and cannot reach the receptor through cochlear fluids. It cannot be intensified by OHC contractions, since there is no depolarization of the external acoustic cell. Short sounds, whose duration time is equal to tenth parts of milliseconds, are perceived by the receptor and recognized is the frequency of those waves, although they do not generate any resonance with the membrana basilaris. Attention was paid to molecular, intracellular transformations responsible for the transfer of information and intracellular amplification. An important role in those processes is played by ion channels of sodium, potassium, calcium and chlorine in the lateral and inferior wall of the acoustic cell. They are responsible for the polarization and depolarization of a cell. Underlined is the role of calcium in the signal transmission and its amplification in an acoustic cell

Neurochemical alterations of intrinsic cardiac ganglionated nerve plexus caused by arterial hypertension developed during ageing in spontaneously hypertensive and Wistar Kyoto rats.

The acknowledged hypothesis of the cause of arterial hypertension is the emerging disbalance in sympathetic and parasympathetic regulations of the cardiovascular system. This disbalance manifests in a disorder of sustainability of endogenous autonomic and sensory neural substances including calcitonin gene-related peptide (CGRP). This study aimed to examine neurochemical alterations of intrinsic cardiac ganglionated nerve plexus (GP) triggered by arterial hypertension during ageing in spontaneously hypertensive rats of juvenile (prehypertensive, 8-9 weeks), adult (early hypertensive, 12-18 weeks) and elderly (persistent hypertensive, 46-60 weeks) age in comparison with the age-matched Wistar-Kyoto rats as controls. Parasympathetic, sympathetic and sensory neural structures of GP were analysed and evaluated morphometrically in tissue sections and whole-mount cardiac preparations. Both the elevated blood pressure and the evident ultrasonic signs of heart failure were identified for spontaneously hypertensive rats and in part for the aged control rats. The amount of adrenergic and immunoreactive to CGRP neural structures was increased in the adult group of spontaneously hypertensive rats along with the significant alterations that occurred during ageing. In conclusion, the revealed chemical alterations of GP support the hypothesis about the possible disbalance of efferent and afferent heart innervation and may be considered as the basis for the emergence and progression of arterial hypertension and perhaps even as a consequence of hypertension in the aged spontaneously hypertensive rats. The determined anatomical changes in the ageing Wistar-Kyoto rats suggest this breed being as inappropriate for its use as control animals for hypertension studies in older animal age.

About the Physiology of Hearing

The membrane basilaris plays an important role in the theory of traveling wave.It originates from the ectoblast of the connective tissue, and is bereft of both afferent or efferent innervation. Vibrations of the membrana basilaris formed into a transverse wave occur together with the organ of Corti and a band of connective tissue on the inferior membrane surface. Vibrations take place in a fluid which has suppressive properties. The length and the mass of the membrana basilaris in mammals and birds are very different and there is no relationship between the membrane basilaris length and the length of the sound wave, especially in the range of low frequencies. Small sound intensities, supraliminal, have amplitudes within the limits of a few picometers upon the entrance into the auditory meatus. This amplitude disappears in cochlear fluids and cannot reach the receptor through cochlear fluids. It cannot be intensified by OHC contractions, since there is no depolarization of the external acoustic cell. Short sounds, whose duration time is equal to tenth parts of milliseconds, are perceived by the receptor and recognized is the frequency of those waves, although they do not generate any resonance with the membrana basilaris. Attention was paid to molecular, intracellular transformations responsible for the transfer of information and intracellular amplification. An important role in those processes is played by ion channels of sodium, potassium, calcium and chlorine in the lateral and inferior wall of the acoustic cell. They are responsible for the polarization and depolarization of a cell. Underlined is the role of calcium in the signal transmission and its amplification in an acoustic cell. Słowa kluczowe: Komórka słuchowa, wzmocnienie sygnału, receptor, kanały jonowe

Analysis of the spinal and vagal afferent innervation of the mouse colon using neuronal retrograde tracers.

The gut-brain axis has received increasing attention recently due to evidence that colonic microbes can affect brain function and behavior. However, little is known about the innervation of the colon by a major component of the gut-brain axis, vagal afferent neurons. Furthermore, it is currently unknown whether individual NG neurons or DRG neurons innervate both the proximal and distal colon. We aimed to quantify the number of vagal and spinal afferent neurons that innervate the colon; and determine whether these individual neurons simultaneously innervate the mouse proximal and distal colon. C57Bl/6 mice received injections of a combination of retrograde tracers that were either injected into the muscularis externa of the proximal or the distal colon: fast blue, DiI and DiO. Five to seven percent of lumbosacral and thoracolumbar spinal afferent neurons, and 25% of vagal afferent neurons were labelled by injections of DiI and DiO into the colon. We also found that approximately 8% of NG neurons innervate the distal colon. Ten percent of labeled thoracolumbar and 15% of labeled lumbosacral DRG neurons innervate both the distal and proximal colon. Eighteen percent of labeled NG neurons innervated both the distal and proximal colon. In conclusion, vagal afferent innervation of the distal colon is less extensive than the proximal colon, whereas a similar gradient was not observed for the spinal afferent innervation. Furthermore, overlap appears to exist between the receptive fields of vagal and spinal afferent neurons that innervate the proximal and distal colon.

MP03-05 DETRUSOR NERVE RADIOFREQUENCY ABLATION (DENERA): EXPERIMENTAL STUDY OF SAFETY AND EFFICACY IN OVINE MODEL

You have accessJournal of UrologyCME1 Apr 2023MP03-05 DETRUSOR NERVE RADIOFREQUENCY ABLATION (DENERA): EXPERIMENTAL STUDY OF SAFETY AND EFFICACY IN OVINE MODEL Gamal Ghoniem, Rongwei Mao, David Csuka, Bilal Farhan, and Sami Souccar Gamal GhoniemGamal Ghoniem More articles by this author , Rongwei MaoRongwei Mao More articles by this author , David CsukaDavid Csuka More articles by this author , Bilal FarhanBilal Farhan More articles by this author , and Sami SouccarSami Souccar More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The sub trigonal peri-vesical nerve plexus has a considerable proportion of the bladder afferent and autonomic innervation. It has been a targeted area to treat Overactive Bladder (OAB) and Bladder Painful Syndrome (BPS). The transurethral approach for radiofrequency ablation (RFA) of this region has been successful in denervating it to alleviate symptoms. However, there were some adverse effects because of the induced scarring. Here, we report a novel transvaginal approach of low-temperature RFA device (DENERA) and assess its feasibility and efficacy and safety of transvaginal ablation of peri-vesical nerve plexus in Vivo ovine model. METHODS: We used 14 adult female sheep in this prospective study. They were divided into control (n=4) without ablation. Post-ablation groups were: 1 week (n=3), 4 weeks (n=4), and 12 weeks (n=3). RFA probe inserted into the vagina aiming to deploy RFA needles through the anterior vaginal wall into the sub trigonal peri-vesical plexus. Pulsed RF energy is applied for three cycles of 4 minutes with 30 seconds rest between each cycle, maintaining the tissue temperature at 45°C. After termination, the bladder and proximal urethra were harvested and subjected to gross and histological assessment, including neural histopathology analysis with H&E, S100, TH, and VAcht immunostaining. To quantify the tissue's neural density and neural vacuolization, they were graded on a 0-3 scale proportional to the number of vacuoles or nerves counted on the microscope slide, with higher scores being a greater effect of ablation (more vacuoles and fewer nerves) vice versa. RESULTS: The ablation effects were most pronounced as neural vacuolization at one week and decreased neural density the most at four weeks (Figure 1). Both variables displayed a slight rebound towards baseline at 12 weeks. The pathology analysis also showed that the needles penetrated deep into the sub trigonal detrusor muscle. There was no urothelium damage or injury reported in the post-ablation group. The sheep recovered from the DENERA procedure with no signs of pain or complications. CONCLUSIONS: This study shows that DENERA device can cause sub trigonal perivesical nerve denervation, with some rebound at 12 weeks. The ease of the procedure and using low temperature offers DENERA as an encouraging new office treatment option for OAB and or BPS. Future clinical data is needed. Source of Funding: University of California Innovation grant © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e22 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gamal Ghoniem More articles by this author Rongwei Mao More articles by this author David Csuka More articles by this author Bilal Farhan More articles by this author Sami Souccar More articles by this author Expand All Advertisement PDF downloadLoading ...

Alterations in Pulmonary Physiology with Lung Transplantation.

Lung transplant is a treatment option for patients with end-stage lung diseases; however, survival outcomes continue to be inferior when compared to other solid organs. We review the several anatomic and physiologic changes that result from lung transplantation surgery, and their role in the pathophysiology of common complications encountered by lung recipients. The loss of bronchial circulation into the allograft after transplant surgery results in ischemia-related changes in the bronchial artery territory of the allograft. We discuss the role of bronchopulmonary anastomosis in blood circulation in the allograft posttransplant. We review commonly encountered complications related to loss of bronchial circulation such as allograft airway ischemia, necrosis, anastomotic dehiscence, mucociliary dysfunction, and bronchial stenosis. Loss of dual circulation to the lung also increases the risk of pulmonary infarction with acute pulmonary embolism. The loss of lymphatic drainage during transplant surgery also impairs the management of allograft interstitial fluid, resulting in pulmonary edema and early pleural effusion. We discuss the role of lymphatic drainage in primary graft dysfunction. Besides, we review the association of late posttransplant pleural effusion with complications such as acute rejection. We then review the impact of loss of afferent and efferent innervation from the allograft on control of breathing, as well as lung protective reflexes. We conclude with discussion about pulmonary function testing, allograft monitoring with spirometry, and classification of chronic lung allograft dysfunction phenotypes based on total lung capacity measurements. We also review factors limiting physical exercise capacity after lung transplantation, especially impairment of muscle metabolism. © 2023 American Physiological Society. Compr Physiol 13:4269-4293, 2023.

Olov Oscarsson (1931-1996) of Lund University, a Pioneer in Cerebellar Neurobiology.

The present Cerebellar Classic highlights the experimental work of the Swedish neurophysiologist Olov Oscarsson (1931-1996) on the afferent innervation of the cerebellum by axons emanating from neurons in the spinal cord and the inferior olive. Historically, the schemes of cerebellar division had been principally based on the external morphology of lobules and fissures. However, the macroscopic anatomical division of the cerebellum does not coincide with its pattern of functional organization. By defining a system of longitudinal somatotopy, Oscarsson contributed to the much needed plan of cerebellar division that correlates experimental information on axonal connections with physiology. His contribution has ultimately led to the currently accepted microzonal modular scheme of cerebellar corticonuclear microcomplexes.

High‐resolution mapping of sensory fibers at the healthy and post‐myocardial infarct whole transgenic hearts

The sensory nervous system is critical to maintain cardiac function. As opposed to efferent innervation, less is known about cardiac afferents. For this, we mapped the VGLUT2-expressing cardiac afferent fibers of spinal and vagal origin by using the VGLUT2::tdTomato double transgenic mouse as an approach to visualize the whole hearts both at the dorsal and ventral sides. For comparison, we colabeled mixed-sex transgenic hearts with either TUJ1 protein for global cardiac innervation or tyrosine hydroxylase for the sympathetic network at the healthy state or following ischemic injury. Interestingly, the nerve density for global and VGLUT2-expressing afferents was found significantly higher on the dorsal side compared to the ventral side. From the global nerve innervation detected by TUJ1 immunoreactivity, VGLUT2 afferent innervation was detected to be 15-25% of the total network. The detailed characterization of both the atria and the ventricles revealed a remarkable diversity of spinal afferent nerve ending morphologies of flower sprays, intramuscular endings, and end-net branches that innervate distinct anatomical parts of the heart. Using this integrative approach in a chronic myocardial infarct model, we showed a significant increase in hyperinnervation in the form of axonal sprouts for cardiac afferents at the infarct border zone, as well as denervation at distal sites of the ischemic area. The functional and physiological consequences of the abnormal sensory innervation remodeling post-ischemic injury should be further evaluated in future studies regarding their potential contribution to cardiac dysfunction.

Population coding strategies in human tactile afferents.

Sensory information is conveyed by populations of neurons, and coding strategies cannot always be deduced when considering individual neurons. Moreover, information coding depends on the number of neurons available and on the composition of the population when multiple classes with different response properties are available. Here, we study population coding in human tactile afferents by employing a recently developed simulator of mechanoreceptor firing activity. First, we highlight the interplay of afferents within each class. We demonstrate that the optimal afferent density to convey maximal information depends on both the tactile feature under consideration and the afferent class. Second, we find that information is spread across different classes for all tactile features and that each class encodes both redundant and complementary information with respect to the other afferent classes. Specifically, combining information from multiple afferent classes improves information transmission and is often more efficient than increasing the density of afferents from the same class. Finally, we examine the importance of temporal and spatial contributions, respectively, to the joint spatiotemporal code. On average, destroying temporal information is more destructive than removing spatial information, but the importance of either depends on the stimulus feature analyzed. Overall, our results suggest that both optimal afferent innervation densities and the composition of the population depend in complex ways on the tactile features in question, potentially accounting for the variety in which tactile peripheral populations are assembled in different regions across the body.

Specification of neuronal subtypes in the spiral ganglion begins prior to birth in the mouse

The afferent innervation of the cochlea is comprised of spiral ganglion neurons (SGNs), which are characterized into four subtypes (Type 1A, B, and C and Type 2). However, little is known about the factors and/or processes that determine each subtype. Here, we present a transcriptional analysis of approximately 5,500 single murine SGNs collected across four developmental time points. All four subtypes are transcriptionally identifiable prior to the onset of coordinated spontaneous activity, indicating that the initial specification process is under genetic control. Trajectory analysis indicates that SGNs initially split into two precursor types (Type 1A/2 and Type 1B/C), followed by subsequent splits to give rise to four transcriptionally distinct subtypes. Differential gene expression, pseudotime, and regulon analyses were used to identify candidate transcription factors which may regulate the subtypes specification process. These results provide insights into SGN development and comprise a transcriptional atlas of SGN maturation across the prenatal period.

Anatomical characterization of vagal nodose afferent innervation and ending morphologies at the murine heart using a transgenic approach.

Heart is an extensively innervated organ and its function is strictly coordinated by autonomic neural circuits. After pathological events such as myocardial infarction (MI), cardiac nerves undergo a structural and functional remodeling contributing to cardiac dysfunction. Although the efferent component of the cardiac nerves has been well described, sensory innervation of the heart has not been defined in detail. Considering its importance, comprehensive description of vagal afferent innervation on the whole heart would enable a better description of autonomic imbalances manifesting as sympathoexcitation and vagal withdrawal in post-ischemic states. To address this issue, we globally mapped the vagal nodose afferent fibers innervating the whole murine heart with unprecedented resolution. By using the Phox2b-Cre::tdTomato transgenic mouse line, we described the detailed distribution and distinct vagal sensory ending morphologies at both the dorsal and ventral sides of the mouse heart. By neural tracing analysis, we quantitated the distribution and prevalence of vagal afferent nerve fibers with varying diameters across dorsal and ventral surfaces of the heart. Moreover, we demonstrated that vagal afferents formed flower spray and end-net-like endings within the atria and ventricles. As distinct from the atria, vagal afferents formed intramuscular array-like endings within the ventricles. Furthermore, we showed that vagal afferents undergo structural remodeling by forming axonal sprouts around the infarct area in post-MI hearts. These findings improve our understanding of the potential effect of vagal afferent remodeling on autonomic imbalance and generation of cardiac arrhythmias and could prospectively contribute to the development of more effective neuromodulatory therapies.

Noise-induced hearing loss vulnerability in type III intermediate filament peripherin gene knockout mice.

In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the ‘cochlear amplifier' selectively express the type III intermediate filament peripherin gene (Prph). Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the PrphKO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, “contralateral suppression” of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the PrphKO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and PrphKO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24–32 kHz) in PrphKO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the PrphKO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.

Intermittent hypoxia in a mouse model of apnea of prematurity leads to a retardation of cerebellar development and long-term functional deficits

BackgroundApnea of prematurity (AOP) is caused by respiratory control immaturity and affects nearly 50% of premature newborns. This pathology induces perinatal intermittent hypoxia (IH), which leads to neurodevelopmental disorders. The impact on the brain has been well investigated. However, despite its functional importance and immaturity at birth, the involvement of the cerebellum remains poorly understood. Therefore, this study aims to identify the effects of IH on cerebellar development using a mouse model of AOP consisting of repeated 2-min cycles of hypoxia and reoxygenation over 6 h and for 10 days starting on postnatal day 2 (P2).ResultsAt P12, IH-mice cerebella present higher oxidative stress associated with delayed maturation of the cerebellar cortex and decreased dendritic arborization of Purkinje cells. Moreover, mice present with growth retardation and motor disorders. In response to hypoxia, the developing cerebellum triggers compensatory mechanisms resulting in the unaltered organization of the cortical layers from P21 onwards. Nevertheless, some abnormalities remain in adult Purkinje cells, such as the dendritic densification, the increase in afferent innervation, and axon hypomyelination. Moreover, this compensation seems insufficient to allow locomotor recovery because adult mice still show motor impairment and significant disorders in spatial learning.ConclusionsAll these findings indicate that the cerebellum is a target of intermittent hypoxia through alterations of developmental mechanisms leading to long-term functional deficits. Thus, the cerebellum could contribute, like others brain structures, to explaining the pathophysiology of AOP.

Cornea: anatomical and functional features, new methods of in vivo diagnostics of abnormalities

The cornea is a highly organized, transparent part of fibrous tunic of an eyeball. It acts as the primary infectious and structural barrier of the eye. The cornea is the major refractive element of an adult eye. It consists of epithelium, Bowman's membrane, stroma, Descemet's membrane and endothelium. Although the normal human cornea is avascular, it is supplied via perilimbal blood vessels, the aqueous humor (AqH) and tear film. Afferent innervation to the cornea is provided by long ciliary nerves, which form subepithelial and subbasal nerve plexus. Epithelium is a stratified, non-keratinizing squamous layer that consists of various cell types. Epithelial cells are connected to each other by zonula adherens, and to the basement membrane via hemidesmosomes. Bowman's membrane is composed of randomly-oriented type I and V collagen fibrils and anchoring type IV and VII collagen fibrils. The stroma consists of cells (principally keratocytes) and distinct lamella formed by collagen fibers, proteoglycans, elastin and glycoproteins. Descemet's membrane is a basal membrane, secreted by endothelial cells. It is a network organized by type VIII collagen molecules, which modulate the passage of growth factors, cytokines and nutrients from the AqH into the corneal stroma and backward. Corneal endothelium is a monolayer of hexagonal cells tightly adherent to one another. In vivo endothelial cells are arrested in G1-phase of cell cycle. The endothelium forms a physiological barrier between the nutrient-rich AqH and the corneal stroma. Tight and gap cell junctions and dynamic pump-leak system maintains corneal deturgescence and permit sufficient nutrient delivery into the stroma and epithelium. Disruption of the endothelial cells results in corneal edema. Modern non-contact real-time imaging of the cornea include specular microscopy, optical coherence tomography and in vivo confocal laser scanning microscopy. These methods can help to visualize corneal layers (during keratorefractive surgery, pre- and postoperative periods), detect localization and etiology of pathological changes.

Single-cell multiplex qPCR evidence for sex-dimorphic glutamate decarboxylase, estrogen receptor, and 5′-AMP-activated protein kinase alpha subunit mRNA expression by ventromedial hypothalamic nucleus GABAergic neurons

The inhibitory amino acid transmitter γ-aminobutryic acid (GABA) acts within the ventromedial hypothalamus to regulate systemic glucose homeostasis, but the issue of whether this neurochemical signal originates locally or is supplied by afferent innervation remains controversial. Here, combinatory in situ immunocytochemistry/laser-catapult microdissection/single-cell multiplex qPCR techniques were used to investigate the premise that ventromedial hypothalamic nucleus ventrolateral (VMNvl) and/or dorsomedial (VMNdm) division neurons contain mRNAs that encode glutamate decarboxylase (GAD)65 or GAD67 and metabolic-sensory biomarkers, and that expression of these genes is sex-dimorphic. In male and female rats, GAD65 mRNA was elevated in VMNvl versus VMNdm GAD65/67-immunopositive (-ir) neurons, yet the female exhibited higher GAD67 transcript content in VMNdm versus VMNvl GABAergic nerve cells. Estrogen receptor (ER)-alpha transcripts were lower in female versus male GABA neurons from either VMN division; ER-beta and G-protein-coupled ER-1 mRNA expression profiles were also comparatively reduced in cells from female versus male VMNvl. VMNvl and VMNdm GAD65/67-ir-positive neurons showed equivalent levels of glucokinase and sulfonylurea receptor-1 mRNA between sexes. 5′-AMP-activated protein kinase-alpha 1 (AMPKα1) and -alpha 2 (AMPKα2) transcripts were lower in female versus male VMNdm GABAergic neurons, yet AMPKα2 mRNA levels were higher in cells acquired from female versus male VMNvl. Current studies document GAD65 and -67 gene expression in VMNvl and VMNdm GAD65/67-ir-positive neurons in each sex. Results infer that GABAergic neurons in each division may exhibit sex differences in receptiveness to estradiol. Outcomes also support the prospect that energy sensory function by this neurotransmitter cell type may predominate in the VMNvl in female versus VMNdm in the male.

Immunohistological study of the density and distribution of human penile neural tissue: gradient hypothesis.

Immunohistological patterns of density and distribution of neural tissue in the human penis, including the prepuce, are not fully characterized, and effects of circumcision (partial or total removal of the penile prepuce) on penile sexual sensation are controversial. This study analyzed extra- and intracavernosal innervation patterns on the main penile axes using formalin-fixed, paraffin-embedded human adult and fetal penile tissues, single- and double-staining immunohistochemistry and a variety of neural and non-neural markers, with a special emphasis on the prepuce and potential sexual effects of circumcision. Immunohistochemical profiles of neural structures were determined and the most detailed immunohistological characterizations to date of preputial nerve supply are provided. The penile prepuce has a highly organized, dense, afferent innervation pattern that is manifest early in fetal development. Autonomically, it receives noradrenergic sympathetic and nitrergic parasympathetic innervation. Cholinergic nerves are also present. We observed cutaneous and subcutaneous neural density distribution biases across our specimens towards the ventral prepuce, including a region corresponding in the adult anatomical position (penis erect) to the distal third of the ventral penile aspect. We also describe a concept of innervation gradients across the longitudinal and transverse penile axes. Results are discussed in relation to the specialized literature. An argument is made that neuroanatomic substrates underlying unusual permanent penile sensory disturbances post-circumcision arerelated to heightened neural levels in the distal third of the ventral penile aspect, which could potentially be compromised by deep incisions during circumcision.

Spinal Afferent Innervation of the Rat Stomach: Anterograde Tracing

Nociceptive afferents from spinal dorsal root ganglia (DRG) and vagal nodose ganglia innervate the stomach and send nociceptive signals centrally to the brain and locally to the enteric nervous system. In our other two posters (Mistareehi et al. 2022, Nguyen et al. 2022), nociceptive afferents are detected with SP and CGRP markers. To specifically study spinal afferent innervation of the stomach, we injected tracer Dextran Biotin (DB) into the left DRG (T7‐T11; 1.5 µl/each ganglion). 14 days after tracer injection, rats were perfused through the left ventricle with 40℃ PBS (1M phosphate‐buffered saline, pH = 7.4). Tissues were then fixed by perfusion with 4% paraformaldehyde in 0.1 M PBS (4℃, pH 7.4). After the perfusion, the distal esophagus and the proximal duodenum were transected, and the stomach was removed. Next, the ventral and dorsal stomach walls were separated by an incision along the lesser and greater curvatures, thus yielding two flat‐mounts per animal. The external muscle wall of the stomach was then isolated as a whole mount by removing the gastric mucosa and submucosa. Ventral stomach flat‐mounts were examined using the Neurolucida system. We found that Tracer DB‐labeled axons formed serval categories of the terminal structures. The simple type: long varicose axons without branching; The branching type: axons with multiple branches running in parallel with either circular or longitudinal muscles; The complex type: single axons with multiple branches running in various directions in both circular and longitudinal muscle layers; The ganglionic type:axons innervating myenteric ganglia; The blood vessel type: axons innervating blood vessels. Our work will provide a foundation for specific labeling of spinal afferent innervation of the stomach. In the future, we will use tracer injection of DRG and nociceptive markers (e.g., SP, CGRP, TRPV1) to examine topographical innervation of spinal nociceptive afferents in the stomach. The first two authors contributed equally to this work.

Topographical Organization and Morphology of Substance‐P Axons in the Flat‐Mounts of the Mouse Whole Stomach

More than 50 million Americans suffer from chronic pain. However, the anatomical and physiological mechanisms of peripheral nociceptive processes have not been well elucidated which has seriously impeded the progress of designing novel bioengineering manipulations/treatments for chronic pain. In this study, we performed a comprehensive topographical mapping of pain‐related neural circuitry in the flat‐mount of whole mouse stomach (male, n=8, 3‐5 months). We used Substance P (SP) as a marker for nociceptive axons and applied a combination of state‐of‐the‐art techniques, including flat‐mount tissue processing and immunohistochemistry of the whole stomach, confocal microscopy, Zeiss Imager microscopy to determine the distribution and morphology of SP‐IR axons and terminals in the whole stomach. We found that 1) SP‐IR axons formed extensive terminal networks in both the ventral and dorsal stomachs. 2) SP‐IR axons were much denser in the antrum and corpus regions than in the fundus and cardia. 3) SP‐IR varicose axons ran in parallel with the circular and longitudinal muscle layers. 4) SP‐IR axons innervated blood vessels. 5) SP‐IR varicose axons formed terminals wrapping around individual myenteric neurons. 6) There were no confirmed SP‐IR myenteric neurons. 7) SP‐IR afferent innervation of the stomach showed a similar pattern of SP‐IR axons and terminals between the ventral and dorsal stomachs. In control mice (n=8), we injected tracer DiI into the ventral and dorsal stomach muscular layers or Fluorogold injection (i.p) and found that the main extrinsic source of SP‐IR afferent axons in the stomach was from the T7‐T11 DRG and to a lesser extent the VNG, but not from the celiac ganglia and dorsal motor nucleus of vagus. Our data provide for the first time a comprehensive topographical map for SP‐IR axons and terminals in the whole stomach with single cell/axon/synapse resolution. This work will contribute to a 3D digital representation of a brain‐stomach nociceptive atlas in a stomach scaffold, thus providing a novel anatomical foundation for functional mapping of nociceptive afferent axons and their pathological remodeling in the stomach. The first two authors contributed equally to this work.