Phosphorylated Tau protein in the myenteric plexus of the ileum and colon of normothermic rats and during synthetic torpor.

Tau protein is of primary importance for neuronal homeostasis and when hyperphosphorylated (PP-Tau), it tends to aggregate in neurofibrillary tangles, as is the case with tauopathies, a class of neurodegenerative disorders. Reversible PP-Tau accumulation occurs in the brain of hibernating rodents and it was recently observed in rats (a non-hibernator) during synthetic torpor (ST), a pharmacological-induced torpor-like condition. To date, the expression of PP-Tau in the rat enteric nervous system (ENS) is still unknown. The present study immunohistochemically investigates the PP-Tau expression in the myenteric plexus of the ileum and colon of normothermic rats (CTRL) and during ST, focusing on the two major subclasses of enteric neurons, i.e., cholinergic and nitrergic.Results showed that both groups of rats expressed PP-Tau, with a significantly increased percentage of PP-Tau immunoreactive (IR) neurons in ST vs. CTRL. In all rats, the majority of PP-Tau-IR neurons were cholinergic. In ST rats, the percentage of PP-Tau-IR neurons expressing a nitrergic phenotype increased, although with no significant differences between groups. In addition, the ileum of ST rats showed a significant decrease in the percentage of nitrergic neurons. In conclusion, our findings suggest an adaptive response of ENS to very low core body temperatures, with changes involving PP-tau expression in enteric neurons, especially the ileal nitrergic subpopulation. In addition, the high presence of PP-Tau in cholinergic neurons, specifically, is very interesting and deserves further investigation. Altogether, these data strengthen the hypothesis of a common cellular mechanism triggered by ST, natural hibernation and tauopathies occurring in ENS neurons.

Water immersion vs. air insufflation in canine duodenal endoscopy: is the future underwater?

Endoscopy represents a commonly employed technique for canine enteropathies. Different trials in human intestinal endoscopy have suggested that the introduction of water for luminal distension, in place of air, improves the visualization of the mucosal texture and decreases pain. The aim of the study was to compare water immersion (WI) vs. air insufflation (AI) during duodenoscopy in anesthetized dogs in terms of mucosal visualization and nociception. Twenty-five dogs undergoing duodenoscopy were included. The same image of the descending duodenum was recorded applying WI and AI. Each pair of images was analyzed using morphological skeletonization, an image entropy evaluation, and a subjective blind evaluation by three experienced endoscopists. To evaluate differences in nociception related to the procedure applied, heart rate and arterial blood pressure were measured before, during and after WI/AI. To compare the two methods, a t-test for paired data was applied for the image analysis, Fleiss' Kappa evaluation for the subjective evaluation and a Friedman test for anesthetic parameters. No differences were found between WI and AI using morphological skeletonization and entropy. The subjective evaluation identified the WI images as qualitatively better than the AI images, indicating substantial agreement between the operators. No differences in nociception were found. The results of the study pointed out the absence of changes in pain response between WI and AI, likely due to the sufficient control of nociception by the anesthesia. Based on subjective evaluation, but not confirmed by the image analysis, WI provided better image quality than AI.

Localization of the 5-hydroxytryptamine 4 receptor in equine enteric neurons and extrinsic sensory fibers.

BACKGROUND: Serotonin plays a pivotal role in regulating gut motility, visceral sensitivity, and fluid secretion via specific receptors. Among these receptors, 5-HT4 exerts a prominent control on gut motor function. Although the prokinetic effect exerted by 5-HT4 agonists is well known, the cellular sites of 5-HT4 expression remain poorly understood in large mammals, e.g., horses. In this study, we evaluated the distribution of 5-HT4 in the horse intestine and in foals with enteric aganglionosis, reminiscent of human Hirschsprung's disease. METHODS: The intestine and spinal ganglia were obtained from three healthy horses and two foals with hereditary ileocolonic aganglionosis. Tissues were processed for immunohistochemistry using a specific antibody to 5-HT4 and a variety of neuronal markers. Myenteric and submucosal plexus 5-HT4 -immunoreactive (IR) neurons were quantified as relative percentage (mean±SD) to the total number of neurons counted. Furthermore, the density of 5-HT4 -IR nerve fibers was evaluated in the mucosa and tunica muscularis. KEY RESULTS: The 5-HT4 immunoreactivity was localized to large percentages of myenteric neurons ranging from 28±9% (descending colon) to 63±19% (ileum), and submucosal neurons ranging from 54±6% (ileum) to 68±14% (duodenum). The 5-HT4 -immunoreactivity was co-expressed by some substance P-IR (SP-IR) spinal ganglion neurons and extrinsic sensory fibers of aganglionic foals. CONCLUSIONS & INFERENCES: The presence of 5-HT4 in many enteric and extrinsic sensory neurons and nerve fibers provides solid morphological evidence of the cellular sites of 5-HT4 expression in horses. The evidence of SP-IR sensory neurons positive for 5-HT4 suggests its role in visceral sensitivity.

Morphological Study of the Persian Leopard (Panthera pardus saxicolor) Tongue.

This study described the morphological features of the Persian leopard (Panthera pardus saxicolor) tongue using light and scanning electron microscopy techniques. The keratinized filiform papillae were distributed all over the entire dorsal surface of the tongue and contained small processes. They were changed into a cylindrical shape in the body and conical shape in the root. The fungiform papillae were found on the apex and margin of the tongue. Few taste pores were observed on the dorsal surface of each papilla. The foliate papillae on the margins of the tongue were composed of several laminae and epithelial fissures. Taste buds were not seen within the non-keratinized epithelium. The vallate papillae were six in total and arranged in a "V" shape just rostral to the root. Each papilla was surrounded by a groove and pad. Taste buds were seen within their lateral walls. Lyssa was visible on the ventral surface of the tongue tip and was found as cartilaginous tissue surrounded by thin connective tissue fibres. The core of the tongue was composed of lingual glands, skeletal muscle and connective tissue. These glands were confined to the posterior portion of the tongue and were composed of many serous cells and a few mucous cells. The results of this study contributed to the knowledge of the morphological characteristics of the tongue of wild mammals and provided data for the comparison with other mammals.

Downregulation of neuronal vasoactive intestinal polypeptide in Parkinson's disease and chronic constipation.

BACKGROUND: Chronic constipation (CC) is a common and severe gastrointestinal complaint in Parkinson's disease (PD), but its pathogenesis remains poorly understood. This study evaluated functionally distinct submucosal neurons in relation to colonic motility and anorectal function in PD patients with constipation (PD/CC) vs both CC and controls. METHODS: Twenty-nine PD/CC and 10 Rome III-defined CC patients were enrolled. Twenty asymptomatic age-sex matched subjects served as controls. Colonic transit time measurement and conventional anorectal manometry were evaluated in PD/CC and CC patients. Colonoscopy was performed in all three groups. Colonic submucosal whole mounts from PD/CC, CC, and controls were processed for immunohistochemistry with antibodies for vasoactive intestinal polypeptide (VIP) and peripheral choline acetyltransferase, markers for functionally distinct submucosal neurons. The mRNA expression of VIP and its receptors were also assessed. KEY RESULTS: Four subgroups of PD/CC patients were identified: delayed colonic transit plus altered anorectal manometry (65%); delayed colonic transit (13%); altered manometric pattern (13%); and no transit and manometric impairment (9%). There were no differences in the number of neurons/ganglion between PD/CC vs CC or vs controls. A reduced number of submucosal neurons containing VIP immunoreactivity was found in PD/CC vs controls (P<.05). VIP, VIPR1, and VIPR2 mRNA expression was significantly reduced in PD/CC vs CC and controls (P<.05). CONCLUSIONS AND INFERENCES: Colonic motor and rectal sensory functions are impaired in most PD/CC patients. These abnormalities are associated with a decreased VIP expression in submucosal neurons. Both sensory-motor abnormalities and neurally mediated motor and secretory mechanisms are likely to contribute to PD/CC pathophysiology.

Quantification of nitrergic neurons in the myenteric plexus of gastric antrum and ileum of healthy and diabetic dogs.

Diabetes mellitus (DM) determines a wide array of severe clinical complications including gastrointestinal motility disorders. The present study investigates the effects of spontaneous DM on the intramural innervation and in particular on nitrergic neurons of the myenteric plexus (MP) of the canine gastric antrum and ileum. Specimens of antrum and ileum from eight control-dogs and five insulin-dependent DM-dogs were collected. MP neurons were immunohistochemically identified with the anti-HuC/HuD antibody, while nitrergic neurons were identified with the antibody anti-neuronal nitric oxide synthase (nNOS). The density of HuC/HuD-immunoreactive (IR) neurons was determined and the nitrergic neurons were quantified as a relative percentage, in consideration of the total number of HuC/HuD-IR neurons. Furthermore, the density of nitrergic fibers in the muscular layers was calculated. Data were expressed as mean±standard deviation. Compared to control-dogs, no significant differences resulted in the density of HuC/HuD-IR neurons in the antrum and ileum of DM-dogs; however, HuC/HuD-immunolabeling showed nuclear localization and fragmentation in DM-dogs. In the stomachs of control- and DM-dogs, the percentages of nitrergic neurons were 30±6% and 25±2%, respectively (P=0.112). In the ileum of the control-dogs, the percentage of nitrergic neurons was 29±5%, while in the DM-dogs, it was significantly reduced 19±5% (P=0.006). The density of nNOS-IR nervous fibers was meaningful reduced in either the tracts considered. Notably, the ganglia of DM-dogs showed also a thickening of the periganglionic connective tissue. These findings indicate that DM in dogs induce modification of the myenteric neurons and, in particular, of the nitrergic neuronal subpopulation.

Extrinsic innervation of ileum and pelvic flexure of foals with ileocolonic aganglionosis.

Equine ileocolonic aganglionosis, which is also called lethal white foal syndrome (LWFS), is a severe congenital condition characterized by the unsuccessful colonization of neural crest progenitors in the caudal part of the small intestine and the entire large intestine. LWFS, which is attributable to a mutation in the endothelin receptor B gene, is the horse equivalent of Hirschsprung's disease in humans. Affected foals suffer from aganglionosis or hypoganglionosis of the enteric ganglia resulting in intestinal akinesia and colic. In other species with aganglionosis, fibers of extrinsic origin show an abnormal distribution pattern within the gut wall, but we have no information to date regarding this occurrence in horses. Our present aim is to investigate the distribution of extrinsic sympathetic and sensory neural fibers in LWFS, focusing on ileum and the pelvic flexure of the colon of two LWFS foals compared with a control subject. The sympathetic fibers were immunohistochemically identified with the markers tyrosine hydroxylase and dopamine beta-hydroxylase. The extrinsic sensory fibers were identified with the markers Substance P (SP) and calcitonin gene-related peptide (CGRP). Since SP and CGRP are also synthesized by subclasses of horse intramural neurons, LWFS represents a good model for the selective study of extrinsic fiber distribution. Affected foals showed large bundles of extrinsic fibers, compared with the control, as observed in Hirschsprung's disease. Furthermore, altered adrenergic pathways were observed, prominently in the pelvic flexure. The numbers of SP- and CGRP-immunoreactive fibers in the muscle, a target of enteric neurons, were dramatically reduced, whereas fibers deduced to be extrinsic sensory axons persisted around submucosal blood vessels. Fiber numbers in the mucosa were reduced. Thus, extrinsic innervation, contributing to modulate enteric functions, might also be affected during LWFS.

Excitatory and inhibitory enteric innervation of horse lower esophageal sphincter.

The lower esophageal sphincter (LES) is a specialized, thickened muscle region with a high resting tone mediated by myogenic and neurogenic mechanisms. During swallowing or belching, the LES undergoes strong inhibitory innervation. In the horse, the LES seems to be organized as a "one-way" structure, enabling only the oral-anal progression of food. We characterized the esophageal and gastric pericardial inhibitory and excitatory intramural neurons immunoreactive (IR) for the enzymes neuronal nitric oxide synthase (nNOS) and choline acetyltransferase. Large percentages of myenteric plexus (MP) and submucosal (SMP) plexus nNOS-IR neurons were observed in the esophagus (72 ± 9 and 69 ± 8 %, respectively) and stomach (57 ± 17 and 45 ± 3 %, respectively). In the esophagus, cholinergic MP and SMP neurons were 29 ± 14 and 65 ± 24 vs. 36 ± 8 and 38 ± 20 % in the stomach, respectively. The high percentage of nitrergic inhibitory motor neurons observed in the caudal esophagus reinforces the role of the enteric nervous system in the horse LES relaxation. These findings might allow an evaluation of whether selective groups of enteric neurons are involved in horse neurological disorders such as megaesophagus, equine dysautonomia, and white lethal foal syndrome.

Enteric neuroplasticity in seawater-adapted European eel (Anguilla anguilla).

European eels live most of their lives in freshwater until spawning migration to the Sargasso Sea. During seawater adaptation, eels modify their physiology, and their digestive system adapts to the new environment, drinking salt water to compensate for the continuous water loss. In that period, eels stop feeding until spawning. Thus, the eel represents a unique model to understand the adaptive changes of the enteric nervous system (ENS) to modified salinity and starvation. To this purpose, we assessed and compared the enteric neuronal density in the cranial portion of the intestine of freshwater eels (control), lagoon eels captured in brackish water before their migration to the Sargasso Sea (T0), and starved seawater eels hormonally induced to sexual maturity (T18; 18 weeks of starvation and treatment with standardized carp pituitary extract). Furthermore, we analyzed the modification of intestinal neuronal density of hormonally untreated eels during prolonged starvation (10 weeks) in seawater and freshwater. The density of myenteric (MP) and submucosal plexus (SMP) HuC/D-immunoreactive (Hu-IR) neurons was assessed in wholemount preparations and cryosections. The number of MP and SMP HuC/D-IR neurons progressively increased from the freshwater to the salty water habitat (control > T0 > T18; P < 0.05). Compared with freshwater eels, the number of MP and SMP HuC/D-IR neurons significantly increased (P < 0.05) in the intestine of starved untreated salt water eels. In conclusion, high salinity evokes enteric neuroplasticity as indicated by the increasing number of HuC/D-IR MP and SMP neurons, a mechanism likely contributing to maintaining the body homeostasis of this fish in extreme conditions.

Neurochemistry of myenteric plexus neurons of bank vole (Myodes glareolus) ileum.

The neurochemistry of enteric neurons differs among species of small laboratory rodents (guinea-pig, mouse, rat). In this study we characterized the phenotype of ileal myenteric plexus (MP) neuronal cells and fibers of the bank vole (Myodes glareolus), a common rodent living in Europe and in Northern Asia which is also employed in prion experimental transmission studies. Six neuronal markers were tested: choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), calbindin (CALB), calcitonin gene-related peptide (CGRP) and substance P (SP), along with HuC/D as a pan-neuronal marker. Neurons expressing ChAT- and nNOS-immunoreactivity (IR) were 36 ± 12% and 24 ± 5%, respectively. Those expressing CGRP-, SP- and CALB-IR were 3 ± 3%, 21 ± 5% and 6 ± 2%, respectively. Therefore, bank vole MPs differ consistently from murine MPs in neurons expressing CGRP-, SP- and CALB-IR. These data may contribute to define the prion susceptibility of neuron cell populations residing within ileal MPs from bank voles, along with their morpho-functional alterations following oral experimental prion challenge.

Localization of peripheral autonomic neurons innervating the boar urinary bladder trigone and neurochemical features of the sympathetic component.

The urinary bladder trigone (UBT) is a limited area through which the majority of vessels and nerve fibers penetrate into the urinary bladder and where nerve fibers and intramural neurons are more concentrated. We localized the extramural post-ganglionic autonomic neurons supplying the porcine UBT by means of retrograde tracing (Fast Blue, FB). Moreover, we investigated the phenotype of sympathetic trunk ganglion (STG) and caudal mesenteric ganglion (CMG) neurons positive to FB (FB+) by coupling retrograde tracing and double-labeling immunofluorescence methods. A mean number of 1845.1±259.3 FB+ neurons were localized bilaterally in the L1-S3 STG, which appeared as small pericarya (465.6±82.7 µm2) mainly localized along an edge of the ganglion. A large number (4287.5±1450.6) of small (476.1±103.9 µm2) FB+ neurons were localized mainly along a border of both CMG. The largest number (4793.3±1990.8) of FB+ neurons was observed in the pelvic plexus (PP), where labeled neurons were often clustered within different microganglia and had smaller soma cross-sectional area (374.9±85.4 µm2). STG and CMG FB+ neurons were immunoreactive (IR) for tyrosine hydroxylase (TH) (66±10.1% and 52.7±8.2%, respectively), dopamine beta-hydroxylase (DßH) (62±6.2% and 52±6.2%, respectively), neuropeptide Y (NPY) (59±8.2% and 65.8±7.3%, respectively), calcitonin-gene-related peptide (CGRP) (24.1±3.3% and 22.1±3.3%, respectively), substance P (SP) (21.6±2.4% and 37.7±7.5%, respectively), vasoactive intestinal polypeptide (VIP) (18.9±2.3% and 35.4±4.4%, respectively), neuronal nitric oxide synthase (nNOS) (15.3±2% and 32.9±7.7%, respectively), vesicular acetylcholine transporter (VAChT) (15±2% and 34.7±4.5%, respectively), leu-enkephalin (LENK) (14.3±7.1% and 25.9±8.9%, respectively), and somatostatin (SOM) (12.4±3% and 31.8±7.3%, respectively). UBT-projecting neurons were also surrounded by VAChT-, CGRP-, LENK-, and nNOS-IR fibers. The possible role of these neurons and fibers in the neural pathways of the UBT is discussed.

Intrinsic innervation of the Persian squirrel (Sciurus anomalus) ileum.

Most investigations related to the characterisation of the enteric nervous system (ENS) are pivoted on the intestine of small rodents, but few studies are available on the ENS of wild or 'unconventional' rodents. Anti-PGP 9.5 and anti-Hu antibodies were utilised to recognise the distribution pattern of neuronal cell bodies and fibres of the ileum of the Persian squirrel (Sciurus anomalus) ENS. The percentages of subclasses of enteric neurones in the total neuronal population were investigated by neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP), substance P (SP), and calbindin (CALB). Myenteric plexus (MP) and submucosal plexus (SMP) neurones showing nNOS immunoreactivity (IR) were 41±4% and 11±6%, respectively, whereas cells expressing ChAT-IR were 56±9% and 74±16%, respectively. nNOS-IR was co-expressed by 21±2% and 9±4% of the MP and SMP cholinergic neurones, respectively, whereas the nNOS-IR MP and SMP neurones co-expressing ChAT-IR were 86±6% and 89±2%, respectively. CGRP-IR and SP-IR were expressed, respectively, by 13±5% and 6±3% of MP and 18±2% and 2±2% of SMP neurones. CALB-IR was expressed by 22±8% and 56±14% of MP and SMP neurones, respectively. MP and SMP cholinergic neurones co-expressed nNOS-IR (21±2% and 9±4%, respectively) and a very high percentage of nNOS-IR neurones showed ChAT-IR (86±6% and 89±2%, respectively). MP and SMP CALB-IR neurones co-expressed ChAT-IR (100% and 63±11%, respectively) and CGRP-IR (89±5% and 26±7%, respectively). Our data might contribute to the neuroanatomical knowledge of the gastrointestinal tract in exotic mammals and provide a comparison with the available data on other mammals.

Neurochemical features of boar lumbosacral dorsal root ganglion neurons and characterization of sensory neurons innervating the urinary bladder trigone.

Porcine lumbosacral dorsal root ganglion (DRG) neurons were neurochemically characterized by using six neuronal markers: calcitonin gene-related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (nNOS), neurofilament 200kDa (NF200), transient receptor potential vanilloid 1 (TRPV1), and isolectin B4 (IB4) from Griffonia simplicifolia. In addition, the phenotype and cross-sectional area of DRG neurons innervating the urinary bladder trigone (UBT) were evaluated by coupling retrograde tracer technique and immunohistochemistry. Lumbar and sacral DRG neuronal subpopulations were immunoreactive (IR) for CGRP (30 ± 3% and 29 ± 3%, respectively), SP (26 ± 8% and 27 ± 12%, respectively), nNOS (21 ± 4% and 26 ± 7%, respectively), NF200 (75 ± 14% and 81 ± 7%, respectively), and TRPV1 (48 ± 13% and 43 ± 6%, respectively), and labeled for IB4 (56 ± 6% and 43 ± 10%, respectively). UBT sensory neurons, which were distributed from L2 to Ca1 DRG, had a segmental localization, showing their highest density in L4-L5 and S2-S4 DRG. Lumbar and sacral UBT sensory neurons expressed similar percentages of NF200 immunoreactivity (64 ± 33% and 58 ± 12%, respectively) but showed a significantly different immunoreactivity for CGRP, SP, nNOS, and TRPV1 (56 ± 9%, 39 ± 15%, 17 ± 13%, 62 ± 10% vs. 16 ± 6%, 16 ± 11%, 6 ± 1%, 45 ± 24%, respectively). Lumbar and sacral UBT sensory neurons also showed different IB4 labeling (67 ± 19% and 48 ± 16, respectively). Taken together, these data indicate that the lumbar and sacral pathways probably play different roles in sensory transmission from the UBT. The findings related to cell size also reinforced this hypothesis, because lumbar UBT sensory neurons were significantly larger than sacral ones (1,112 ± 624 µm(2) vs. 716 ± 421 µm(2) ).

The topography and gross anatomy of the abdominal gastrointestinal tract of the Persian squirrel (Sciurus anomalus) / La topografía y anatomía macroscópica del tracto gastrointestinal abdominal de la ardilla persa (Sciurus anomalus)

The persian squirrel (Sciurus anomalus) is the only member of the Sciuridae family found in the Middle East. It is herbivorous, feeding mostly on pine acorns and other seeds and fruits. It is a wild animal nesting in forest trees, although it is frequently found close to city gardens and parks. As Persian squirrels are also found in homes as "companion animals", veterinarian assistance may be sometimes required; this is a good reason to gain more specific knowledge of the anatomical features of this animal. Due to the scantiness of relevant literature, we carried out this study with the aim to provide further information on the topography and gross anatomy of its abdominal gastrointestinal tract (GIT). Seven animals of this species were utilized to measure the length, content weight and area surface of the relative segments of the abdominal GIT. The stomach is unilocular, lined with glandular epithelium; the small intestine is divided into duodenum, jejunum and ileum. The cecum is situated on the right side of the abdominal cavity and lacks the vermiform appendix observed in some rodents. The ascending colon is extensive, consisting of two loops and two straight parts forming a unique topographic arrangement, closely resembling the ascending colon of the horse. The transverse colon connects the ascending and the descending colon located on the left side of the abdominal cavity, and shows a distinct sigmoid flexure before entering the pelvic cavity. Overall, the in situ examination and the relative measurements of the various parts of the abdominal GIT suggest that the ascending colon is the main fermentation chamber in the Persian Squirrel.

La ardilla es el único miembro de la familia de los Sciuridae que se encuentra en el Medio Oriente. Es un herbívoro, come semillas, principalmente de pino, y fruta. Vive mayormente en el bosque en estado salvaje, aunque a veces se puede encontrar en las ciudades, en parques y jardines. Las ardillas persianas tambien se tienen en casas como mascotas y algunas veces es necesaria la atención del veterinario. Esta es una buena razón para conocer más profundamente sus características anatómicas. Teniendo en cuenta la escasa bibliografía existente a cerca de este animal, el objetivo de este estudio fue aportar nueva información sobre la anatomía topográfica y mascroscópica del tracto abdominal gastrointestinal (GIT) de la ardilla. Siete animales de esta especie han sido utilizados para medir el largo, el peso y el área de la superficie de los segmentos del GIT. El estómago es unilocular, alineado con epitelio glandular; el pequeño intestino está dividido en duodeno, jejuno e ileo. El ciego está localizado en la parte derecha de la cavidad abdominal y le falta el apéndice vermiforme que poseen los roedores. El colon ascendente es extenso, constituido de dos partes curvas y dos rectilíneas que forman una única dispoción topográfica cerrada como el colon ascendente en el caballo. El colon trasverso, que conecta el ascendente y el descendente, está localizado en el lado izquierdo de la cavidad abdominal y muestra una clara flexión sigmoide antes de entrar en la cavidad pélvica. En resumen, el examen in situ y las relativas medidas de las varias partes del GIT sugieren que el colon ascendente es la principal "cámara" de fermentación en la ardilla persiana.

Characterization of spinal ganglion neurons in horse (Equus caballus). A morphometric, neurochemical and tracing study.

Spinal ganglion (SG) neurons have been widely described in rodents, and classified according to various criteria. On the basis of such studies, many features of rodent SG neurons have become benchmarks to classify these cells. However, these traits cannot be confirmed in all other species. In the present study, horse SG neurons were morphometrically and neurochemically characterized by detecting the neuronal markers calcitonin gene-related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (nNOS) and isolectin B4 (IB4) from Griffonia simplicifolia. Moreover, spinal cord staining and tracer studies were also performed injecting Fast Blue tracer in the ileo-cecal junction. The statistical analysis of the histograms related to the cross sectional area of dark and light SG neurons confirmed the presence of the categories of small and large neurons. The staining methods employed yielded the following results: (1) in all triple staining experiments performed, most SG stained neurons were triple-labeled; (2) SP-IR neurons showed the largest percentages of co-localization with the other markers studied; (3) CGRP-IR and IB4-labeled neurons were the SG neurons showing the largest percentages of single staining; (4) nNOS-IR neurons were more represented in horse SGs than in those from rodents; (5) IB4 was widely co-localized with both CGRP and SP. Retrograde tracer investigation combined with neurochemical evaluation showed that in horse, contrarily to rodents, IB4-labeled neurons are widely involved in visceral innervations. The results obtained from the observations of serial stained sections and from a critical analysis of triple-labeling experiments allowed us to conclude that (1) most stained SG neurons co-expressed IB4-nNOS-CGRP-SP neuronal markers, (2) IB4 is not indicated as a marker of non-peptidergic neurons in the horse, (3) horse IB4-labeled neurons are widely involved in visceral sensation, (4) differently from rodents, horse IB4-, CGRP- and SP-labeled fibers share the same spinal cord level terminations.

Sympathetic innervation of the ileocecal junction in horses.

The distribution and chemical phenotypes of sympathetic and dorsal root ganglion (DRG) neurons innervating the equine ileocecal junction (ICJ) were studied by combining retrograde tracing and immunohistochemistry. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) was investigated. Sympathetic neurons projecting to the ICJ were distributed within the celiac (CG), cranial mesenteric (CranMG), and caudal mesenteric (CaudMG) ganglia, as well as in the last ganglia of the thoracic sympathetic chain and in the splanchnic ganglia. In the CG and CranMG 91 +/- 8% and 93 +/- 12% of the neurons innervating the ICJ expressed TH- and DBH-IR, respectively. In the CaudMG 90 +/- 15% and 94 +/- 5% of ICJ innervating neurons were TH- and DBH-IR, respectively. Sympathetic (TH-IR) fibers innervated the myenteric and submucosal ganglia, ileal blood vessels, and the muscle layers. They were more concentrated at the ICJ level and were also seen encircling myenteric plexus (MP) and submucosal plexus (SMP) descending neurons that were retrogradely labeled from the ICJ. Among the few retrogradely labeled DRG neurons, nNOS-, CGRP-, and SP-IR nerve cells were observed. Dense networks of CGRP-, nNOS-, and SP-IR varicosities were seen around retrogradely labeled prevertebral ganglia neurons. The CGRP-IR fibers are probably the endings of neurons projecting from the intestine to the prevertebral ganglia. These findings indicate that this crucial region of the intestinal tract is strongly influenced by the sympathetic system and that sensory information of visceral origin influences the sympathetic control of the ICJ.

Extrinsic afferents supplying the ovine duodenum and ileum.

The study aimed at establishing the distribution of primary sensory neurons by means of retrograde tracers Diamidino Yellow (DY) and Fast Blue (FB) injected into both the sheep duodenum and ileum, respectively. Many DY-labelled cells were found in both the distal vagal ganglia (DVG) and the spinal ganglia (SG) from T9-L3; on the contrary, the majority of the FB-labelled cells were found in the SG. In the SG, a double immunofluorescence stain was used to reveal Nitric Oxide Synthase-Immunoreactivity (NOS-IR) in association with: substance P (SP), calcitonin gene-related peptide (CGRP), neurofilament 200kDa (NF) and isolectin B(4) (IB4). The labelled neurons, both DY and FB generally ranged in size from medium to large. The majority of the SG duodenal projections were NOS negative; the majority of the SG ileal afferent neurons expressed NOS-IR. Both DY and FB NOS-IR neurons often co-localized IB4, CGRP and SP, but rarely NF.

Characterization of sheep (Ovis aries) palatine tonsil innervation.

Palatine tonsils (PTs), together with ileal Peyer's patches, rank among the first colonization sites for infectious prions. After replicating in these lymphoid tissues, prions undertake the process of "neuroinvasion," which is likely mediated by the peripheral nerves connecting lymphoid tissues to the central nervous system (CNS). To study the connections between the tonsils and the CNS, we injected fluorescent tracers into the PTs of lambs; the highest number of Fast Blue (FB)-labeled neurons was found in cranial cervical ganglia (CCG), whereas a progressively decreasing number of cells were detected in proximal glossopharyngeal, proximal vagal, trigeminal, pterygopalatine, and cervicothoracic ganglia. Immunohistochemistry was carried out on tonsil and ganglia cryosections. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and calcium-binding protein S100 (S100), was observed in the fibers around and within PT lymphoid nodules. In the trigeminal, proximal glossopharyngeal and vagal ganglia the retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR. In all ganglia some retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR co-localization. It is worth noting that only 66+/-19% and 75+/-13% of retrogradely-labeled neurons in CCG showed TH- and DBH-IR, respectively. The present results allow us to attribute PT innervation mainly to the sympathetic component and to the glossopharyngeal, vagal and trigeminal cranial nerves. Furthermore, these data also provide a plausible anatomic route through which infectious agents, such as prions, may access the CNS, i.e. by traveling along several cranial and sympathetic nerves, as well as by migration via glial cells.

Ileal tract and Peyer's patch innervation in scrapie-free versus scrapie-affected ovines.

Ileal Peyer's patches (PPs) are involved early during sheep scrapie infection. This study qualitatively and semi-quantitatively evaluated ileal tract and PP innervation in 29 Sarda ovines of different age, PrP genotype and scrapie status. A prominent network of fibres was detected within PPs, mainly located in interfollicular lymphoid and stromal components. Intrafollicular fibres were rarely observed, with no apparent differences between scrapie-free and scrapie-affected animals, or among ovines carrying different PrP genotypes. In adult sheep, independent of their scrapie status, nerve fibres could be detected infrequently, close to the follicle-associated epithelium. Fibres were also detected within newly formed follicles and intrafollicular microgranulomas.