Pairwise interaction Markov model for 3D epidermal nerve fibre endings.

In this paper, the spatial arrangement and possible interactions between epidermal nerve fibre endings are investigated and modelled by using confocal microscopy data. We are especially interested in possible differences between patterns from healthy volunteers and patients suffering from mild diabetic neuropathy. The locations of the points, where nerves enter the epidermis, the first branching points and the points where the nerve fibres terminate, are regarded as realizations of spatial point processes. We propose an anisotropic point process model for the locations of the nerve fibre endings in three dimensions, where the points interact in cylindrical regions. First, the locations of end points in R 2 $\mathbb {R}^2$ are modelled as clusters around the branching points and then, the model is extended to three dimensions using a pairwise interaction Markov field model with cylindrical neighbourhood for the z-coordinates conditioned on the planar locations of the points. We fit the model to samples taken from healthy subjects and subjects suffering from diabetic neuropathy. In both groups, after a hardcore radius, there is some attraction between the end points. However, the range and strength of attraction are not the same in the two groups. Performance of the model is evaluated by using a cylindrical version of Ripley's K function due to the anisotropic nature of the data. Our findings suggest that the proposed model is able to capture the 3D spatial structure of the end points.

The presence and degradation of nerve fibers in articular cartilage of neonatal rats.

PURPOSE: To investigate the presence and change of nerve fibers and neuropeptide during early development of articular cartilage in neonatal rats. METHODS: Articular cartilage in distal-femoral epiphyses was collected from neonatal Sprague Dawley rats, which were 1-day, 5-day, and 10-day postnatal (P1, P5 and P10). Microscopy, immunofluorescence, transmission and scanning electron microscopy (TEM and SEM) were performed for detection of nerve fibers. Quantitative analysis for substance P (SP) and neuropeptide Y (NPY) was conducted using immunofluorescence and enzyme-linked immunosorbent assay (ELISA). RESULTS: TEM showed the existence of myelinated nerve fibers in the extracellular matrix of articular cartilage in both P1, P5 and P10 rats, and they formed synaptic contacts with chondrocytes. During this time, chondrocytes proceeded with their development, and the nerve fibers gradually degraded. The ELISA results showed significant increase of the sensory neuropeptide SP and the sympathetic neuropeptide NPY in the cartilage tissue. Immunofluorescence results showed the distribution of SP and NPY in the perichondrium, the cartilage canals, the plasma of chondrocytes, and extracellular matrix in the cartilage tissue. CONCLUSIONS: Nerve fibers exist in the matrix of articular cartilage during early development of knee joints in neonatal rats. Nerve fibers form synaptic contacts with chondrocytes at the early stage and then degrade gradually in the course of chondrocyte development. SP and NPY significantly increase in articular cartilage during this very period. These results indicate that the nerve fibers and the neuropeptide they secrete may exert important effect on the development of articular cartilage.

Multi-Elemental Analysis of Human Optic Chiasm-A New Perspective to Reveal the Pathomechanism of Nerve Fibers' Degeneration.

The effect of metals on the functioning of the human eye is multifactorial and includes enzyme activity modulation, trace metal metabolic pathways changes, and cytotoxic activity. Functional dysfunctions appear mostly as a result of the accumulation of toxic xenobiotic metals or disturbances of micronutrients' homeostasis. So far, the affinity of selected metals to eye tissues, i.e., the cornea, choroid, lens, and anterior chamber fluid, has been most studied. However, it is known that many eye symptoms are related to damage to the optic nerve. In order to fill this gap, the aim of the study is to perform a multi-element analysis of tissue collected postmortem from optic chiasm and optic nerves. A total of 178 samples from 107 subjects were tested. The concentrations of 51 elements were quantified by inductively coupled plasma mass spectrometry (ICP-MS) after the wet-mineralization step. In terms of elemental composition, the optic chiasm is dominated by two trace elements, i.e., iron (Fe) and zinc (Zn), besides macro-elements Ca, K, Na, P, and Mg. The subjects formed a homogeneous cluster (over 70% subjects) with the highest accumulation of aluminum (Al). The remaining two departing clusters were characterized by an increased content of most of the elements, including toxic elements such as bismuth (Bi), uranium (U), lead (Pb), chromium (Cr), and cadmium (Cd). Changes in elemental composition with age were analyzed statistically for the selected groups, i.e., females, males, and subjects with alcohol use disorder (AUD) and without AUD. A tendency of women to lose Se, Cu, Zn, Fe with age was observed, and a disturbed Ca/Mg, Na/K ratio in subjects with AUD. Although the observed trends were not statistically significant, they shed new light on the risks and possible pathologies associated with metal neurotoxicity in the visual tract.

Somatostatin immunoreactivity within the urinary bladder nerve fibers and paracervical ganglion urinary bladder projecting neurons in the female pig.

The study was designed to examine the distribution and chemical coding of somatostatin-immunoreactive (SOM-IR) nerve fibers supplying the urinary bladder wall and to establish the distribution and immunohistochemical characteristics of the subpopulation of paracervical ganglion (PCG) SOM-IR neurons projecting to this organ in female pigs. The PCG-urinary bladder projecting neurons (PCG-UBPN) were visualized with retrograde neuronal tracer Fast Blue (FB). Double-labeling immunohistochemistry performed on cryostat sections from the urinary bladder wall revealed that the greatest density of SOM-IR nerve fibers was found in the muscle layer and around blood vessels, a moderate number of these nerve terminals supplied the submucosa and only single SOM-IR axons were encountered beneath the urothelium. In all the investigated sections the vast majority of SOM-IR nerve fibers were immunopositive to vesicular acetylcholine transporter (VAChT) and many SOM-IR axons contained immunoreactivity to neuropeptide Y (NPY). Approximately 65 % of FB-positive (FB+) PCG-UBPN were immunoreactive to SOM. Moreover, PCG FB+/SOM + nerve cells were simultaneously immunoreactive to choline acetyltransferase (ChAT; 64.6 ± 0.6 %), NPY (59.7 ± 1.2 %), neuronal nitric oxide synthase (nNOS; 46.1 ± 0.7 %), vasoactive intestinal polypeptide (VIP; 29.9 ± 2.2 %), Leu5-enkephalin (L-ENK; 19.5 ± 6.3 %), dopamine ß-hydroxylase (DßH; 14.9 ± 1.9 %) or pituitary adenylate cyclase-activating polypeptide (PACAP; 14.8 ± 2.4 %). The present study reveals the extensive expression of SOM in both the nerve fibres supplying the porcine urinary bladder wall and the PCG neurons projecting to this organ, indicating an important regulatory role of SOM in the control of the urinary bladder function.

Alpha-melanocyte stimulating hormone immunoreactivity in the brain of the cichlid fish Oreochromis mossambicus.

This study reports the distribution of a pro-opiomelanocortin-derived neuropeptide α-MSH in the brain of the cichlid fish Oreochromis mossambicus. α-MSH-ir fibres were found in the granule cell layer of the olfactory bulb, the medial olfactory tract, the pallium and the subpallium, whereas in the preoptic area of the telencephalon, few large α-MSH-ir perikarya along with extensively labeled fibres were observed close to the ventricular border. Dense network of α-MSH-ir fibres were seen in the hypothalamic areas such as the nucleus preopticus pars magnocellularis, the nucleus preopticus pars parvocellularis, the suprachiasmatic nucleus, the nucleus anterior tuberis, the paraventricular organ, the subdivisions of the nucleus recessus lateralis and the nucleus recessus posterioris. In the nucleus lateralis pars medialis, some α-MSH-ir perikarya and fibres were found along the ventricular margin. In the diencephalon, numerous α-MSH-ir fibres were detected in the nucleus posterior tuberis, the nucleus of the fasciculus longitudinalis medialis and the nucleus preglomerulosus medialis, whereas in the mesencephalon, α-MSH-ir fibres were located in the optic tectum, the torus semicircularis and the tegmentum. In the rhombencephalon, α-MSH-ir fibres were confined to the medial octavolateralis nucleus and the descending octaval nucleus. In the pituitary gland, densely packed α-MSH-ir cells were observed in the pars intermedia region. The widespread distribution of α-MSH-immunoreactivity throughout the brain and the pituitary gland suggests a role for α-MSH peptide in regulation of several neuroendocrine and sensorimotor functions as well as darkening of pigmentation in the tilapia.

A PPAR-α agonist protects the non-adrenergic, non-cholinergic inhibitory system of guinea pig trachea from the effect of inhaled ammonium persulphate: a pilot study.

SUMMARY: Aim of the study. Inhaled ammonium persulphate (AP) reduces non adrenergic, non cholinergic (NANC) relaxation in the guinea pig trachea, as a part of its inflammatory effects. Peroxisome Proliferator-Activated Receptor (PPAR) stimulation has shown anti-inflammatory properties. This study aimed at evaluating whether the PPAR-α agonist WY 14643 can prevent the reduction in NANC relaxation caused by inhaled AP in the guinea pig trachea. Materials and Methods. Four groups of ten male guinea pigs were treated for three weeks with inhaled AP (10 mg/m3, 30 min per day, group A), saline (group B), AP and WY 14643 (0.36 µM/die, per os, group C), and AP, WY 14643 and the PPAR-α antagonist GW 6471 (0.36 µM/die, per os, group D). NANC relaxations to electrical field stimulation (EFS) at 3 Hz were evaluated in whole tracheal segments as intraluminal pressure changes. Results. The tracheal NANC relaxations were reduced by 90.3% in group A, as compared to group B. In group C, they were reduced by only 22.2%. In group D, they were reduced by 92.6 %. PPAR-α receptors were detected in inhibitory nerve fibers within the trachea as shown by immonohistochemical analysis. Conclusions. The PPAR-α agonist WY 14643 protects the NANC inhibitory system of the guinea pig trachea from the effect of inhaled ammonium persulphate and its protective effect is antagonized by GW 6471. PPAR-α might be exploited.

An unbiased stereological method for corneal confocal microscopy in patients with diabetic polyneuropathy.

Corneal confocal microscopy (CCM) derived corneal nerve measures are lower in diabetic sensorimotor polyneuropathy (DSPN). There are, however, methodological challenges in relation to adequate and unbiased sampling of images with objective corneal nerve quantification. Here we compare a new sampling method and adjusted area calculation with established methods of corneal nerve quantification in patients with and without DSPN and healthy controls. CCM images from 26 control subjects and 62 patients with type 1 diabetes with (n = 17) and without (n = 45) DSPN were analyzed. The images were randomly selected and corneal nerve fiber length (CNFL), corneal nerve fiber branch density (CNBD) and corneal nerve fiber density (CNFD) were determined in both a manual and automated manner. The new method generated 8-40% larger corneal nerve parameters compared to the standard procedure (p < 0.05). CNFL was significantly reduced using the new method for both manual and automated analysis; whilst CNFD and CNBD were significantly reduced using the automated method in both diabetic groups compared with controls. The new, objective method showed a reduction in corneal nerve parameters in diabetic patients with and without DSPN. We recommend using a randomized sampling method and area-dependent analysis to enable objective unbiased corneal nerve quantification.

Notch Signaling Pathway Expression in the Skin of Leprosy Patients: Association With Skin and Neural Damage.

Introduction: Leprosy is an infectious disease caused by Mycobacterium leprae, a debilitating disease that affects the skin and peripheral nerves. It is possible that tissue changes during infection with leprosy are related to alterations in the activity of the Notch signaling pathway, an innate signaling pathway in the physiology of the skin and peripheral nerves. Methods: This is a descriptive observational study. Thirty skin biopsies from leprosy patients and 15 from individuals with no history of this disease were evaluated. In these samples, gene expressions of cellular components associated with the Notch signaling pathway, Hes-1, Hey-1, Runx-1 Jagged-1, Notch-1, and Numb, were evaluated using q-PCR, and protein expression was evaluated using immunohistochemistry of Runx-1 and Hes-1. Results: Changes were observed in the transcription of Notch signaling pathway components; Hes-1 was downregulated and Runx-1 upregulated in the skin of infected patients. These results were confirmed by immunohistochemistry, where reduction of Hes-1 expression was found in the epidermis, eccrine glands, and hair follicles. Increased expression of Runx-1 was found in inflammatory cells in the dermis of infected patients; however, it is not related to tissue changes. With these results, a multivariate analysis was performed to determine the causes of transcription factor Hes-1 reduction. It was concluded that tissue inflammation was the main cause. Conclusions: The tissue changes found in the skin of infected patients could be associated with a reduction in the expression of Hes-1, a situation that would promote the survival and proliferation of M. leprae in this tissue.

Modular organization of cerebellar climbing fiber inputs during goal-directed behavior.

The cerebellum has a parasagittal modular architecture characterized by precisely organized climbing fiber (CF) projections that are congruent with alternating aldolase C/zebrin II expression. However, the behavioral relevance of CF inputs into individual modules remains poorly understood. Here, we used two-photon calcium imaging in the cerebellar hemisphere Crus II in mice performing an auditory go/no-go task to investigate the functional differences in CF inputs to modules. CF signals in medial modules show anticipatory decreases, early increases, secondary increases, and reward-related increases or decreases, which represent quick motor initiation, go cues, fast motor behavior, and positive reward outcomes. CF signals in lateral modules show early increases and reward-related decreases, which represent no-go and/or go cues and positive reward outcomes. The boundaries of CF functions broadly correspond to those of aldolase C patterning. These results indicate that spatially segregated CF inputs in different modules play distinct roles in the execution of goal-directed behavior.

Refined Quantitation of Sweat Gland Innervation.

Skin biopsies have gained increasing popularity as a tool to evaluate disorders affecting small nerve fibers. While reports on sweat gland nerve fiber density (SGNFD) to quantitate sudomotor innervation have been promising, methodologies vary significantly. Although conventional stereology is commonly used, no standard technique has been established. We sought to develop an accurate and reproducible technique to quantify SGNFD. Skin punch biopsies from healthy individuals were cut and stained. Images of sweat glands (SGs) were acquired using confocal and widefield microscopes, and optimized using deconvolution. Nerve fibers were reconstructed and nerve fiber length (NFL) was quantified using three-dimensional (3D) automated software. SGNFD was obtained by dividing NFL by SG volume. SGNFD was also assessed using stereology for comparison. Ninety-two SGs from 10 healthy subjects were analyzed by independent observers. Using confocal microscopy, the software reliably traced nerve fibers. In contrast, rendering of nerve fibers was inferior using widefield microscopy. Interobserver reliability was suboptimal using widefield images compared to confocal (ICC = 0.82 vs ICC = 0.98). Correlation between 3D-reconstruction and stereology was poor (ICC = 0.38). The newly developed technique of SGNFD quantitation using 3D reconstruction of SG innervation with confocal microscopy reliably traces nerve fibers, shows outstanding reproducibility, is almost completely unbiased, and superior to conventional stereology methods.

Neurochemical characterization of intramural nerve fibres in the porcine oesophagus.

The gastrointestinal (GI) tract is innervated by nerve processes derived from the intramural enteric neurons and neurons localized outside the digestive tract. This study analysed the neurochemical characterization of nerves in the wall of the porcine oesophagus using single immunofluorescence technique. Immunoreactivity to vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), neuronal isoform of nitric oxide synthase (nNOS), substance P (SP), leucine enkephalin (LENK), calcitonin gene-related peptide (CGRP) or dopamine beta-hydroxylase (DBH) was investigated in intramuscular and intramucosal nerves of the cervical, thoracic and abdominal oesophagus. The results indicate that all of the substances studied were present in the oesophageal nerves. The density of particular populations of fibres depended on the segment of the oesophagus. The most numerous were fibres immunoreactive to VIP in the longitudinal and circular muscle layers of the abdominal oesophagus: The number of these fibres amounted to 16.4 ± 0.8 and 18.1 ± 3.1, respectively. In turn, the least numerous were CGRP-positive fibres, which were present only in the circular muscle layer of the cervical oesophagus and mucosal layer of the abdominal oesophagus in the number of 0.3 ± 0. The obtained results show that nerves in the porcine oesophageal wall are very diverse in their neurochemical coding, and differences between particular parts of the oesophagus suggest that organization of the innervation clearly depends on the fragment of this organ.

Site-dependent differences in the composite fibers of male pelvic plexus branches: an immunohistochemical analysis of donated elderly cadavers.

BACKGROUND: Although the pelvic autonomic plexus branches are considered to be a mixture of sympathetic and parasympathetic nerves, little is known regarding the composite fibers of the pelvic plexus branches. This study aimed to investigate the immunohistochemical features of sympathetic and parasympathetic nerves in the pelvic autonomic plexus branches. METHODS: Using 10 donated elderly male cadavers, the detailed topohistology of nerve fibers at and around the bladder, seminal vesicle, prostate, and rectum was examined. Neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) were used as parasympathetic nerve markers; tyrosine hydroxylase (TH) was used as a sympathetic nerve marker. The myenteric plexus of the colon was utilized as a positive control. RESULTS: Most nerve fibers in the bladder, seminal vesicle, prostate, and rectum were both nNOS- and TH-positive. Thus, pelvic plexus branches were classified into two types: 1) triple-positive mixed nerves (nNOS+, VIP+, TH+, thick myelinated fibers + or -) and 2) double-positive mixed nerves (nNOS+, VIP-, TH+, thick myelinated fibers + or -). Notably, triple-positive nerves were localized within the posterosuperior part of the plexus (near the rectum) and travelled anteroinferiorly toward the posterolateral corner of the prostate. The posteriorly and inferiorly located nerves were predominantly composed of parasympathetic, rather than sympathetic, fibers. In contrast, nerve fibers within and along the bladder and seminal vesicle contained either no or few VIP-positive nerves. These superiorly located nerves were characterized by clear sympathetic nerve dominance. CONCLUSIONS: The nerves of the pelvic plexus branches were clearly classified into nerves around the bladder and seminal vesicle (VIP-negative) and nerves around the prostate (VIP-positive). Although nNOS- and VIP-positive nerve fibers are candidate cavernous nerves, cavernous nerve identity cannot be definitively concluded for these nerves in the periprostatic region.

Accumulation of Advanced Glycation Endproducts and Subclinical Inflammation in Deep Tissues of Adult Patients With and Without Diabetes.

OBJECTIVES: Advanced glycation endproducts (AGEs) play a key role in the development of foot complications in people with diabetes. Skin autofluorescence (AF) might noninvasively determine tissue accumulation of AGEs. This study evaluated the association between skin AF and AGE contents in the deep tissues of those with diabetes and the further consequences of such contents. METHODS: Between September 2014 and September 2015, we studied 33 patients, with and without diabetes, who had received lower-limb amputations. Skin AF was measured. Artery, nerve and skin were harvested during surgery. AGE contents were quantified using high-performance liquid chromatography mass spectrometry and were located by immunohistochemistry staining. Inflammatory cells were also located by immunohistochemistry, immunofluorescence and scanning electron microscopy. RESULTS: Values of skin AF and AGE contents in artery, nerve and skin in patients with diabetes were higher than those in healthy patients. Skin AF was strongly affected by AGE contents in these tissues. AGE contents in various tissues were strongly correlated with each other. Differing AGEs were deposited in similar manners in the same tissues and were accompanied by inflammatory cells. CONCLUSIONS: AGE contents were strongly correlated with each other and were accompanied by inflammatory cells. Skin AF measurement could provide information about the systemic accumulation of AGEs.

Angiotensinergic Innervation of the Human Right Atrium: Implications for Cardiac Reflexes.

BACKGROUND: The right atrium is densely innervated and provides sensory input to important cardiocirculatory reflexes controlling cardiac output and blood pressure. Its angiotensin (Ang) II-expressing innervation may release Ang II as a neuropeptide cotransmitter to modulate reflexes but has not yet been characterized. METHODS: Intraoperative surgical biopsies from human right atria (n = 7) were immunocytologically stained for Ang II, tyrosine hydroxylase (TH), and synaptophysin (SYN). Tissue angiotensins were extracted and quantified by radioimmunoassay. RESULTS: Angiotensinergic fibers were frequent in epicardial nerves and around vessels with variable TH co-localization (none to >50%/bundle). Fibers were also widely distributed between cardiomyocytes and in the endocardium where they were typically nonvaricose, TH/SYN-negative and usually accompanied by varicose catecholaminergic fibers. In the endocardium, some showed large varicosities and were partially TH or SYN-positive. A few endocardial regions showed scattered nonvaricose Ang fibers ending directly between endothelial cells. Occasional clusters of thin varicose terminals co-localizing SYN or TH were located underneath, or protruded into, the endothelium. Endocardial density of Ang and TH-positive fibers was 30-300 vs. 200-450/mm2. Atrial Ang II, III, and I concentrations were 67, 16, and 5 fmol/g (median) while Ang IV and V were mostly undetectable. CONCLUSIONS: The human right atrium harbors an abundant angiotensinergic innervation and a novel potential source of atrial Ang II. Most peripheral fibers were noncatecholaminergic afferents or preterminal vagal efferents and a minority was presumably sympathetic. Neuronal Ang II release from these fibers may modulate cardiac and circulatory reflexes independently from plasma and tissue Ang II sources.

Fix the problem - A practical guide to whole-mount immunohistochemistry of teased nerve fibres.

BACKGROUND: Immunohistochemical staining of entire nerve fibres allows for studying the molecular composition of functional fibre subunits and may add to the diagnostic value of nerve fibre teasing. NEW METHOD: In this study, we established a sealed-slide method for reproducible immunostaining of deep axoplasmic proteins in permanently straightened nerve fibres. RESULTS: Immunostaining of teased nerve fibres very much is facilitated by tip-fixation with biocompatible glass adhesives. Antibody penetration in fresh nerves can be achieved by thermic and chemical permeabilisation while enzymatic digestion allows for sufficient permeability after aldehyde fixation. COMPARISON WITH EXISTING METHODS: The methods recommended herein are easy to perform and represent a reliable and reproducible way to whole mount immunostaining. CONCLUSIONS: Sealed-slide immunostaining of tip-fixed and permeabilised nerve biopsies will help to validate neurophysiological abnormalities and to screen for target molecules and predictive markers of peripheral nerve disorders such as in inherited neuropathies and Guillain-Barré syndrome.

Morphological and neurochemical differences in peptidergic nerve fibers of the mouse vagina.

The vagina is innervated by a complex arrangement of sensory, sympathetic, and parasympathetic nerve fibers that contain classical transmitters plus an array of neuropeptides and enzymes known to regulate diverse processes including blood flow and nociception. The neurochemical characteristics and distributions of peptide-containing nerves in the mouse vagina are unknown. This study used multiple labeling immunohistochemistry, confocal maging and analysis to investigate the presence and colocalization of the peptides vasoactive intestinal polypeptide (VIP), calcitonin-gene related peptide (CGRP), substance P (SP), neuropeptide tyrosine (NPY), and the nitric oxide synthesizing enzyme neuronal nitric oxide synthase (nNOS) in nerve fibers of the murine vaginal wall. We compared cervical and vulvar areas of the vagina in young nullipara and older multipara C57Bl/6 mice, and identified differences including that small ganglia were restricted to cervical segments, epithelial fibers were mainly present in vulvar segments and most nerve fibers were found in the lamina propria of the cervical region of the vagina, where a higher number of fibers containing immunoreactivity for VIP, CGRP, SP, or nNOS were found. Two populations of VIP-containing fibers were identified: fibers containing CGRP and fibers containing VIP but not CGRP. Differences between young and older mice were present in multiple layers of the vaginal wall, with older mice showing overall loss of innervation of epithelium of the proximal vagina and reduced proportions of VIP, CGRP, and SP containing nerve fibers in the distal epithelium. The distal vagina also showed increased vascularization and perivascular fibers containing NPY. Immunolabeling of ganglia associated with the vagina indicated the likely origin of some peptidergic fibers. Our results reveal regional differences and age- or parity-related changes in innervation of the mouse vagina, effecting the distribution of neuropeptides with diverse roles in function of the female genital tract.

5-HT3A -driven green fluorescent protein delineates gustatory fibers innervating sour-responsive taste cells: A labeled line for sour taste?

Taste buds contain multiple cell types with each type expressing receptors and transduction components for a subset of taste qualities. The sour sensing cells, Type III cells, release serotonin (5-HT) in response to the presence of sour (acidic) tastants and this released 5-HT activates 5-HT3 receptors on the gustatory nerves. We show here, using 5-HT3A GFP mice, that 5-HT3 -expressing nerve fibers preferentially contact and receive synaptic contact from Type III taste cells. Further, these 5-HT3 -expressing nerve fibers terminate in a restricted central-lateral portion of the nucleus of the solitary tract (nTS)-the same area that shows increased c-Fos expression upon presentation of a sour tastant (30 mM citric acid). This acid stimulation also evokes c-Fos in the laterally adjacent mediodorsal spinal trigeminal nucleus (DMSp5), but this trigeminal activation is not associated with the presence of 5-HT3 -expressing nerve fibers as it is in the nTS. Rather, the neuronal activation in the trigeminal complex likely is attributable to direct depolarization of acid-sensitive trigeminal nerve fibers, for example, polymodal nociceptors, rather than through taste buds. Taken together, these findings suggest that transmission of sour taste information involves communication between Type III taste cells and 5-HT3 -expressing afferent nerve fibers that project to a restricted portion of the nTS consistent with a crude mapping of taste quality information in the primary gustatory nucleus.

Distribution and sub-types of afferent fibre in the mouse urinary bladder.

AIM: Increased afferent fibre activity contributes to pathological conditions such as the overactive bladder syndrome. Nerve fibres running near the urothelium are considered to be afferent as no efferent system has yet been described. The aim of this study was to identify sub-types of afferent nerve fibres in the mouse bladder wall based on morphological criteria and analyse regional differences. MATERIALS AND METHODS: 27 bladders of six month old C57BL/6 mice were removed and tissues were processed for immunohistochemistry. Cryostat sections were cut and stained for Protein Gene Product 9.5 (PGP), calcitonin gene related polypeptide (CGRP), neurofilament (NF), vesicular acetylcholine transporter (VAChT) and neuronal nitric oxide synthase (nNOS). RESULTS: In the sub-urothelium, different types of afferent nerve fibre were found, i.e. immunoreactive (IR) to; CGRP, NF, VAChT, and/or nNOS. At the bladder base, the sub-urothelium was more densely innervated by CGRP-IR and VAChT-IR nerve fibres, then at the lateral wall. NF- and nNOS nerves were sparsely distributed in the sub-urothelium throughout the bladder. At the lateral wall the inner muscle is densely innervated by CGRP-IR nerve fibres. NF, VAChT and nNOS nerves were evenly distributed in the different muscle layers throughout the bladder. Nerve fibre terminals expressing CGRP and NF were found within the extra-mural ganglia at the bladder base. CONCLUSIONS: Different types of afferent nerve fibres were identified in the sub-urothelium of the mouse bladder. At the bladder base the sub-urothelium is more densely innervated than the lateral wall by CGRP-IR and VAChT-IR afferent nerve fibres. CGRP and NF afferent nerve fibres in the muscle layer probably relay afferent input to external ganglia located near the bladder base. The identification of different afferent nerves in the sub-urothelium suggests a functional heterogeneity of the afferent nerve fibres in the urinary bladder.

Innervation of taste buds revealed with Brainbow-labeling in mouse.

Nerve fibers that surround and innervate the taste bud were visualized with inherent fluorescence using Brainbow transgenic mice that were generated by mating the founder line L with nestin-cre mice. Multicolor fluorescence revealed perigemmal fibers as branched within the non-taste epithelium and ending in clusters of multiple rounded swellings surrounding the taste pore. Brainbow-labeling also revealed the morphology and branching pattern of single intragemmal fibers. These taste bud fibers frequently innervated both the peripheral bud, where immature gemmal cells are located, and the central bud, where mature, differentiated cells are located. The fibers typically bore preterminal and terminal swellings, growth cones with filopodia, swellings, and rounded retraction bulbs. These results establish an anatomical substrate for taste nerve fibers to contact and remodel among receptor cells at all stages of their differentiation, an interpretation that was supported by staining with GAP-43, a marker for growing fibers and growth cones.

[Characteristics of Distribution of Blood Vessels and Nerve Fibers in the Skin Tissues of Acupoint "Taichong" (LR 3) in the Rat].

OBJECTIVE: To investigate the microstructure (blood vessels and nerve fibers) of the skin tissue in "Taichong" (LR 3) region for reveling morphological characteristics of acupoint. METHODS: Five SD rats were used in the present study. The skin tissue in the region of acupoint LR 3 was taken from the dorsum of hind foot following transcardial perfusion with 4% paraformaldehyde. Then, the skin samples were sagittally or horizontally cut into sections (20 µm or 40 µm in the thickness) to be stained with Phalloidin and calcitonin gene related peptide (CGRP) by using fluorescent histochemistry and immunohistochemistry. The labeled vascular structure and nerve fibers were observed and recorded using fluorescent microscope and laser scanning confocal microscope. RESULTS: In the skin tissue of LR 3, different types of blood vessels labeled by phalliodin, including capillaries and glomera mainly distributing in the superficial layer of the dermis, and thicker blood vessels and their sub-branches mainly existing in the deeper layer of the dermis and the subcutaneous layer, were found. In addition, CGRP positive nerve fibers were found to run parallel to the thin blood vessels or to gather around the thicker blood vessels. From the subcutaneous la-yer to the epidermis, blood vessels and nerve fibers coexisted and formed a stereo-network structure. CONCLUSIONS: In the skin of LR 3 area, there exists a stereo-network structure consisting of different types of blood vessels and nerve fibers. Although this structure is not specifically associated with acupoint area alone, this result may provide a new sight to further understand the microstructure of acupoint.