C. Sorteni

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 μm2 vs. 716 ± 421 μm2). J. Comp. Neurol. 521:342–366, 2013.

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 ganglia (STG) and caudal mesenteric ganglia (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 (DβH) (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), leuenkephalin (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 nNOSIR fibers. The possible role of these neurons and fibers in the neural pathways of the UBT is discussed.

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.

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.

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.

Expression of β2 adrenoceptors within enteric neurons of the horse ileum.

The activity of the gastrointestinal tract is regulated through the activation of adrenergic receptors (ARs). Since data concerning the distribution of ARs in the horse intestine is virtually absent, we investigated the distribution of β2-AR in the horse ileum using double-immunofluorescence. The β2-AR-immunoreactivity (IR) was observed in most (95%) neurons located in submucosal plexus (SMP) and in few (8%) neurons of the myenteric plexus (MP). Tyrosine hydroxylase (TH)-IR fibers were observed close to neurons expressing β2-AR-IR. Since β2-AR is virtually expressed in most neurons located in the horse SMP and in a lower percentage of neurons in the MP, it is reasonable to retain that this adrenergic receptor could regulate the activity of both secretomotor neurons and motor neurons innervating muscle layers and blood vessels. The high density of TH-IR fibers near β2-AR-IR enteric neurons indicates that the excitability of these cells could be directly modulated by the sympathetic system.

Role of Palatine Tonsils as a Prion Entry Site in Classical and Atypical Experimental Sheep Scrapie

ABSTRACT Atypical and classical scrapie-infected sheep brain tissue was monolaterally injected into the tonsils of lambs to investigate their role as a prion entry point. We first detected classical PrPSc within the inoculated tonsil and in the ipsilateral retropharyngeal lymph node at 3 months postinoculation (p.i.). At 7 months p.i., PrPSc colonized other lymphoid tissues bilaterally, including ileal Peyers patches. The earliest PrPSc deposition within the brain was ipsilaterally observed at 9 months p.i. in the substantia reticularis of the medulla oblongata. At 12 months p.i., PrPSc deposition was present bilaterally in the nucleus parasympathicus nervi vagi, as well as in the intermediolateral cell column of the thoracolumbar spinal cord. No PrPSc was detected in the lambs inoculated with atypical scrapie. These findings suggest that neuroinvasion may naturally occur from the tonsil after a widespread prion replication within the lymphoid tissues during classical scrapie only, thus mimicking the pathogenesis after oral ingestion.

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.

Distribution Pattern and Chemical Coding of Sympathetic Trunk Ganglia Neurons Supplying the Boar Urinary Bladder Trigone

Sympathetic trunk ganglia (STG) neurons projecting to the urinary bladder trigone of the boar were studied by coupling retrograde tracing Fast Blue (FB) and double-labeling immunofluorescence methods. FB-positive neurons were localized in the L1-S3 STG. Immunohistochemical staining revealed the catecholaminergic (tyrosine hydroxylase-/dopamine beta hydroxylase immunoreactivity) phenotype of the majority of FB-positive neurons, which also preferentially expressed neuropeptide Y. In addition, some of the FB-positive dopaminergic perikarya were immunoreactive for calcitonin gene-related peptide, substance P, vasoactive intestinal peptide, vesicular acetylcholine-transporter, neuronal nitric oxide synthase, somatostatin, and leu-enkephalin. Functional hypotheses have been formulated.