Distribution and chemical coding of neurons in intramural ganglia of the porcine urinary bladder trigone

This study presents the distribution and chemical coding of neurons in the porcine intramural ganglia of the urinary bladder trigone (IG-UBT) demonstrated using combined retrograde tracing and double-labelling immunohistochemistry. Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of both the left and right side of the bladder trigone during laparotomy performed under pentobarbital anaesthesia. Ten-microm-thick cryostat sections were processed for double-labelling immunofluorescence with antibodies against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP), substance P (SP), Leu5-enkephalin (LENK) and choline acetyltransferase (ChAT). IG-UBT neurons formed characteristic clusters (from a few to tens neuronal cells) found under visceral peritoneum or in the outer muscular layer. Immunohistochemistry revealed four main populations of IG-UBT neurons: SOM- (ca. 35%), SP- (ca. 32%), ChAT- and NPY- immunoreactive (-IR) (ca. 23%) as well as non-adrenergic non-cholinergic nerve cells (ca. 6%). This study has demonstrated a relatively large population of differently coded IG-UBT neurons, which constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.     

Coexistence of calcitonin gene-related peptide and galanin immunoreactivity in female rat pelvic and lumbosacral dorsal root ganglia.

Coexistence of immunoreactivity for calcitonin gene-related peptide (CGRP) and galanin (GAL) was examined in varicose nerve endings in female rat pelvic paracervical ganglia (PG) and in perikarya of lumbosacral dorsal root ganglia (DRG). Varicose peptide-containing nerves were closely adjacent to somata of neurons in the PG, certain somata being virtually surrounded by immunoreactive varicosities. Some nerve endings were immunoreactive for either CGRP or GAL; in others, immunoreactivity for CGRP and GAL coexisted. Likewise, many perikarya in DRG were CGRP immunoreactive, fewer were GAL immunoreactive, and in some immunoreactivity for CGRP and GAL coexisted. The results suggest there are subpopulations of neuropeptide-containing sensory nerve endings in the PG; some contain CGRP, some contain GAL, and in some CGRP and GAL coexist. These substances contained in sensory nerve endings could have important roles in pelvic ganglionic functions.     

Distribution,immunohistochemical characteristics and nerve pathways of primary sensory neurons supplying the porcine vas deferens

The present study investigated: (1) the distribution and chemical coding of primary sensory neurons supplying the vas deferens in juvenile pigs by the use of retrograde tracing combined with double-labelling immunofluorescence, (2) nerve pathways from dorsal root ganglia (DRG) to the vas deferens by means of denervation procedures involving transection of the hypogastric or pelvic nerve combined with a retrograde tracing method, and (3) possible interactions of the substance P (SP)/calcitonin gene-related peptide (CGRP)-immunoreactive varicose nerve fibres on vas deferens projecting neurons (VDPN) in the anterior pelvic ganglion (APG). The vast majority of VDPN were found mainly in the lumbar L2, L3 and sacral S2, S3 pairs of DRG and showed a clear ipsilaterally organized projection pattern. Immunohistochemistry revealed that most of these neurons contained SP and/or CGRP, occasionally coexpressed with galanin. Interestingly, pronounced differences in the expression of SP and/or CGRP were observed between the lumbar and sacral VDPN in that most of the lumbar but less than half of the sacral neurons stained for these peptides. Denervation experiments showed that the neurons located within the lumbar DRG project through the ipsilateral hypogastric nerve, whereas those found within the sacral DRG send their processes through the ipsilateral and contralateral pelvic nerve. In the nerve-lesioned animals, especially in those with the hypogastric nerve cut, a dramatic reduction in the number of SP and/or CGRP-containing nerve terminals surrounding the efferent VDPN within the APG was observed. This study has disclosed the distribution and, for the first time, chemical coding and nerve pathways of vas deferens-projecting primary sensory neurons in a mammalian species, the pig. The results obtained also provide some novel information about the possible morphological and functional relationship between vas deferens-projecting primary sensory and pelvic efferent nerve cells.     

Distribution of spinal sensory neurons supplying the adrenal gland in the pig

The present study was aimed at disclosing which spinal ganglia contribute to the innervation of the adrenal gland in the pig with special regard to the morphology and intraganglionic distribution of the primary sensory neurons within these ganglia. To this end, the animals were injected with a retrograde fluorescent neuronal tracer Fast Blue into the left adrenal gland during laparotomy. After a survival period of three weeks, labelled spinal sensory neurons were found in the ipsilateral dorsal root ganglia (approximately 99% of all retrogradely marked neurons). However, single adrenal gland-projecting perikarya were observed also in the contralateral dorsal root ganglia from Th3 to L3. The majority of the retrogradely labelled afferent neurons (above 65% of all Fast Blue-positive (FB+) perikarya) were located in two groups of spinal ganglia (at neuromeres Th6-7 and Th12-14, approximately 18% and 47% of neurons, respectively), forming two distinct multiganglionic centres of origin for this neural pathway. The morphometric evaluation of FB+ neurons revealed that the vast majority of them (approximately 81%) belonged to the class of small-sized perikarya (10-30 microm in diameter), while the medium-sized (diameter 30-80 microm) and large neurons comprised only up to 13% and 6.5% of adrenal gland-projecting neurons, respectively. Furthermore, the analysis of the intraganglionic distribution pattern of the retrogradely labelled cells revealed that the highest number of them was located in the medio-caudal domain of the dorsal root ganglia, irrespective of the neuromere studied. Thus, the present study has revealed sources and morphological characteristic of spinal afferent neurons supplying the porcine adrenal gland, simultaneously pointing out to the characteristic features of their inter- and intraganglionic distribution pattern.     

Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation

To investigate extrinsic origins of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibres in the sheep ileum, the retrograde fluorescent tracer Fast Blue (FB) was injected into the ileum wall. Sections of thoraco-lumbar dorsal root ganglia (DRG) and distal (nodose) vagal ganglia showing FB-labelled neurons were processed for CGRP immunohistochemistry. The distribution of CGRP-IR in fibres and nerve cell bodies in the ileum was also studied. CGRP-IR enteric neurons were morphometrically analysed in myenteric (MP) and submucosal plexuses (SMP) of lambs (2–4 months). Sensory neurons retrogradely labelled with FB were scattered in T5-L4 DRG but most were located at the upper lumbar levels (L1-L3); only a minor component of the extrinsic afferent innervation of the ileum was derived from nodose ganglia. In the DRG, 57% of retrogradely labelled neurons were also CGRP-IR. In cryostat sections, a dense network of CGRP-IR fibres was observed in the lamina propria beneath the epithelium, around the lacteals and lymphatic follicles (Peyer's platches), and along and around enteric blood vessels. Rare CGRP-IR fibres were also present in both muscle layers. Dense pericellular baskets of CGRP-IR fibres were observed around CGRP-negative somata. The only CGRP-IR nerve cells were well-defined Dogiel type II neurons localised in the MP and in the external and internal components of the SMP. CGRP-IR neurons in the myenteric ganglia were significantly larger than those in the submucosal ganglia (mean profile areas: about 1,400 μm² for myenteric neurons, 750 μm² for submucosal neurons). About 6% of myenteric neurons and 25% of submucosal neurons were CGRP-IR Dogiel type II neurons. The percentages of CGRP-IR neurons that were also tachykinin-IR were about 9% (MP) and 42% (SMP), whereas no CGRP-IR neurons exhibited immunoreactivity for vasoactive intestinal peptide, nitric oxide synthase or tyrosine hydroxylase in either plexus. Thus, CGRP immunoreactivity occurs in the enteric nervous system of the sheep ileum (as in human small intestine and MP of pig ileum) in only one morphologically defined type of neuron, Dogiel type II cells. These are probably intrinsic primary afferent neurons. This work was supported by grants from the Ricerca Fondamentale Orientata (RFO) and Fondazione Del Monte di Bo e Ra.     

Pituitary adenylate cyclase activating polypeptide is expressed in autonomic neurons

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide, which is present in neuronal elements of several peripheral organs, and thus a putative neurotransmitter/modulator. In the present study, the expression of PACAP in two parasympathetic ganglia (otic, sphenopalatine) and one mixed parasympathetic/sensory ganglion (jugular-nodose) in rat was characterized by use of in situ hybridization and immunocytochemistry and compared to that of VIP and calcitonin gene-related peptide (CGRP). PACAP and VIP were expressed in virtually all nerve cell bodies in the otic and sphenopalatine ganglia; PACAP and VIP were also expressed in subpopulations of nerve cell bodies in the jugular-nodose ganglion. CGRP was expressed in numerous nerve cell bodies in the jugular-nodose ganglion and in a few, scattered, nerve cell bodies in the sphenopalatine ganglion. In the otic and sphenopalatine ganglia, PACAP- and VIP-like immunoreactivities were frequently co-localized; in the jugular-nodose ganglion, PACAP-like immunoreactivity was frequently co-localized with CGRP-like immunoreactivity in presumably sensory neurons and to a lesser extent with VIP in parasympathetic neurons. Thus, PACAP is synthesized and stored in autonomic parasympathetic neurons as well as in vagal sensory neurons, which provides an anatomical basis for the diverse effects of PACAP previously described.     

Differences in the chemical coding of nerve fibres supplying major populations of neurons between the caudal mesenteric ganglion and anterior pelvic ganglion in the male pig

The present study was designed to investigate and to compare the chemical coding of nerve fibres supplying major populations of neurons in the caudal mesenteric (CaMG) and anterior pelvic (APG) ganglion in juvenile male pigs (n=5) using double-labelling immunofluorescence. The co-existence patterns of some biologically active substances including tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT) as well as vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), Leu5-enkephalin (LENK) and serotonin (5-HT) were analysed under a confocal laser scanning microscope. Profound differences in the neurochemical features of the nerve terminals between the ganglia were observed. Moreover, there were also distinct differences in the chemical coding of nerve fibres associated with the particular populations and subpopulations of neurons within the ganglia. In both ganglia, nearly all adrenergic and cholinergic neurons were supplied with VAChT-positive nerve fibres (putative preganglionic fibres). However, in the CaMG, they were more numerous and, in contrast to the APG, many of them also stained for VIP. In the APG, a great number of nerve terminals expressed immunoreactivity to SP and CGRP (putative collaterals of sensory neurons). Interestingly, they densely supplied almost exclusively adrenergic neurons. SP-positive nerve fibres were moderate in number in the CaMG, but, in addition to VAChT-IR nerve terminals, the most numerous populations of nerve fibres in this ganglion were those expressing highly colocalized immunoreactivities to CGRP and LENK, and those which stained for 5-HT (putative processes of enteric neurons). However, these fibres supplied almost exclusively larger, intensely stained for TH and clustered adrenergic neurons. This diversity of the nerve terminals reflects the complexity of nerve circuits involved in the innervation of structures supplied by neurons in the porcine CaMG and APG. It also demonstrates the importance of nerve inputs for the proper function of autonomic neurons and thus their target tissues.     

Pathway-specific patterns of the co-existence of substance P,calcitonin gene-related peptide,cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig

Summary The co-existence of immunoreactivities to substance P (SP), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and dynorphin (DYN) in neurons of the dorsal root ganglion (DRG) of guinea-pigs has been investigated with a double-labelling immunofluorescence procedure. Four main populations of neurons could be identified that contained different combinations of these peptides and had distinctive peripheral projections: (1) Neurons that contained immunoreactivity to SP, CGRP, CCK and DYN were distributed mainly to the skin. (2) Neurons with immunoreactivity to SP, CGPR and CCK, but not DYN, were distributed mainly to the small blood vessels of skeletal muscles. (3) Neurons with immunoreactivity to SP, CGRP and DYN, but not CCK, were distributed mainly to pelvic viscera and airways. (4) Neurons containing immunoreactivity to SP and CGRP, but not CCK and DYN, were distributed mainly to the heart, systemic blood vessels, blood vessels of the abdominal viscera, airways and sympathetic ganglia. Other small populations of DRG neurons containing SP, CGRP or CCK alone also were detected. Perikarya containing these combinations of neuropeptides were not found in autonomic ganglia. The peripheral axons of neurons containing immunoreactivity to at least SP and CGRP were damaged by chronic treatment with capsaicin. However, some sensory neurons containing CCK alone were not affected morphologically by capsaicin.These results clearly show that individual DRG neurons can contain many different neuropeptides. Furthermore, the combination of neuropeptides found in any particular neuron is related to its peripheral projection.     

In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.     

Formation of Neuromuscular Junctions and Synthesis of Sensory Neuropeptides in the Co-cultures of Dorsal Root Ganglion and Cardiac Myocytes

Aim The interactions between primary sensory neurons and cardiac myocytes are still unclear. In the present study, the co-culture model of dorsal root ganglion (DRG) explant and cardiac myocytes was used to characterize the morphological relationship between primary sensory nerve endings and cardiac myocytes and to investigate whether cardiac myocytes could induce substance P (SP) and calcitonin gene-related peptide (CGRP) synthesis in DRG neurons and release from DRG neurons in the neuromuscular co-cultures. Methods The formation of neuromuscular junctions was observed with scanning electron microscopy (SEM). SP and CGRP expression were detected by immunocytochemistry. Basal SP and CGRP release and capsaicin-evoked SP and CGRP release were analyzed by radioimmunoassay (RIA). Results In this study, neuromuscular junctions were observed in the co-cultures of DRG explant and cardiac myocytes. SP-immunoreactive (IR) and CGRP-IR neurons were detected in both neuromuscular co-cultures and DRG explant cultures, but the number of SP-IR and CGRP-IR neurons migrating from DRG explant was significantly increased in neuromuscular co-cultures. Capsaicin-evoked SP and CGRP release but not basal SP and CGRP release in neuromuscular co-cultures increased significantly as compared with that in the cultures of DRG explant alone. Conclusions The results implicated that the morphological relationship between sensory nerve terminal and cardiac myocyte is much more close in vitro than it is in vivo. Cardiac myocytes may induce sensory neuropeptide synthesis and capsaicin-evoked neuropeptide release in neuromuscular co-cultures. Further experiment needs to be performed about the significance of neuropeptide synthesis and capsaicin-evoked neuropeptide release induced by target cardiac myocytes. Zhen Liu and Huaxiang Liu contributed equally to this article.     

Immunohistochemical analysis of intracardiac ganglia of the rat heart

The neurochemistry of intracardiac neurons in whole-mount preparations of the intrinsic ganglia was investigated. This technique allowed the study of the morphology of the ganglionated nerve plexus found within the atria as well as of individual neurons. Intracardiac ganglia formed a ring-like plexus around the entry of the pulmonary veins and were interconnected by a series of fine nerve fibres. All intracardiac neurons contained immunoreactivity to PGP-9.5, choline acetyl transferase (ChAT) and neuropeptide Y (NPY). Two smaller subpopulations were immunoreactive to calbindin or nitric oxide synthase. Furthermore, a subpopulation (approximately 6%) of PGP-9.5/ChAT/NPY-immunoreactive cells lacking both calbindin and nitric oxide synthase (NOS) was surrounded by pericellular baskets immunoreactive to ChAT and calbindin. Vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activated peptide (PACAP), substance P and tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibres within the ganglion, but never in neuronal somata. Furthermore, immunoreactivity for NPY was not observed in pericellular baskets surrounding intracardiac neurons, despite being present in all intrinsic neuronal cell bodies. Taken together, the results of this study indicate a moderate level of chemical diversity within the intracardiac neurons of the rat. Such chemical diversity may reflect functional specialisation of neurons in the intracardiac ganglia.This work was supported by a grant-in-aid (G00M0670) from the National Heart Foundation of Australia     

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.     

Endothelin as a putative sensory neuropeptide in the guinea-pig: different properties in comparison with calcitonin gene-related peptide

Both endothelin-(ET) and calcitonin gene-related peptide- (CGRP) like immunoreactivity (-LI) were present in a variety of organs and neuronal tissue of the guinea-pig as determined by radioimmunoassay (RIA). Neuronal tissues like dorsal root ganglia (DRG) contained by far the highest levels of both ET- (65 +/- 11 pmol/g) and CGRP-LI (34 +/- 5 pmol/g). The tissue levels of ET-LI remained unchanged after 6-hydroxydopamine and capsaicin-pretreatment, while CGRP-LI was markedly reduced after capsaicin. Chromatographic characterization revealed that the main portion of ET-LI in the DRG, right atrium and lung corresponded to synthetic ET-1. Immunohistochemical studies showed the presence of ET-LI in a few neurons of intact DRG and many neurons in DRG cell-cultures, partly co-existing with CGRP-LI. In the neuronal cells of DRG cultures the ratio between the ET- and CGRP-LI was 1:27 compared to 2:1 in intact DRG. 24 h after ligation of the sciatic or vagal nerves no accumulation of ET-LI was observed above the ligation, while CGRP-LI was increased 4-5-fold. Transection (10 days) of the sciatic nerve caused a 85-95% depletion of CGRP-LI in the distal skin, gastrocnemius muscle and trunk below the transection site, while in the proximal portion of the nerve CGRP-LI increased. No effects on ET-LI in these tissues were observed after sciatic nerve transfection. In release experiments on DRG cell cultures. Langendorff heart preparations or perfused guinea-pig lungs, potassium (60 mM), capsaicin or antidromic nerve stimulation evoked a clear-cut increase in the supernatant levels of CGRP-LI, suggesting release, while no effect on the ET-LI concentration was observed in the effluent. Furthermore, anoxia failed to influence the outflow of ET-LI from the heart and lung. It is concluded that ET-1-LI is present in high levels in spinal ganglia and ET-LI occurs in afferent cell-bodies, but in comparison with CGRP, ET shows remarkable inertness upon various experimental conditions including no evidence for axonal transport, loss after denervation or release. The neuronal ET-LI seems to increase under culture conditions, however. The possible function for the high content of ET-LI in the intact guinea-pig peripheral nervous system remains to be elucidated and may mainly be related to a non-neuronal pool considering the relatively low content of ET-LI compared to CGRP in cultured DRG cells.     

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ～25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.     

Sources of porcine longissimus dorsi muscle (LDM) innervation as revealed by retrograde neuronal tract-tracing

The aim of the present study was to establish the origin of the motor, autonomic and sensory innervation of the L1-L2 segment of the porcine longissimus dorsi muscle (LDM), in order to provide morphological basis for further studies focusing on this neural pathway under experimental conditions, e.g. phototerapy and/or lateral electrical surface stimulation. To reach the goal of the study, multiple injections of the fluorescent neuronal tracer Fast Blue (FB) were made into the LDM region between the spinal processes of the vertebrae L1 and L2. The spinal cord (Th13-S1 segments) as well as the sensory and autonomic ganglia of interest, i.e., dorsal root (DRG) and sympathetic chain ganglia from corresponding spinal cord levels were collected three weeks later. FB-positive (FB+) motoneurons were observed exclusively within the nucleus ventromedialis at L1 and L2 spinal cord level, forming the most ventro-medially arranged cell column within this nucleus. Primary sensory and sympathetic chain neurons were found in appropriate ipsilateral ganglia at Th15-L3 levels. The vast majority of retrogradely traced neurons (virtually all motoneurons, approximately 76% of sensory and 99.4% of sympathetic chain ganglia neurons) was found at the L1 and L2 levels. The morphometric evaluation of FB-labeled DRG neurons showed that the majority of them (approximately 66%) belonged to the class of small-diameter perikarya (10-30 microm in diameter), whereas those of medium size (30-80 microm in diameter) and of large diameter (more than 80 microm) constituted 22.6% and 11.5% of all DRG neurons, respectively. The results of the present study demonstrated that the nerve terminals supplying porcine LDM originated from different levels of the spinal cord, dorsal root and sympathetic chain ganglia. Thus, the study has revealed sources and morphological characteristic of somatic, autonomic and spinal afferent neurons supplying porcine LDM, simultaneously pointing out the characteristic features of their distribution pattern.     

Effect of intestinal inflammation on capsaicin-sensitive afferents in the ileum of<Emphasis Type="Italic"> Schistosoma mansoni</Emphasis>-infected mice

In the present study, the effect of intestinal schistosomiasis on the extrinsic sensory innervation of the murine ileum was investigated. Immunocytochemical techniques to localize calcitonin gene-related peptide (CGRP), substance P (SP), and vanilloid receptor 1 (VR1) were combined with retrograde tracing techniques and capsaicin treatment. Neurochemical characterization of extrinsic primary afferent neurons (EPANs) in normal and capsaicin-treated mice, revealed that CGRP and VR1, but not SP, were expressed in extrinsic afferents. Immunocytochemical analysis using the above-mentioned antibodies yielded three different populations of neurons in both dorsal root and nodose ganglia, namely CGRP/--, SP/--, and CGRP/SP-expressing neurons. Retrograde tracing revealed that only CGRP/--expressing neurons projected to the ileum. Intestinal schistosomiasis resulted in an upregulation of the number of CGRP-immunoreactive (ir) nerve fibers in the lamina propria of the villi, coinciding with an increase in mucosal mast cells in acutely and chronically infected animals. In infected animals, mucosal mast cells were found closely associated with a dense mucosal CGRP-ir fiber network. Neonatal capsaicin treatment led to a 70% reduction in the number of mucosal mast cells. In conclusion, the present study provides evidence that CGRP is a valid marker for EPANs in the mouse ileum, which are involved in the recruitment of mucosal mast cells. Morphological evidence is provided of a neuroimmune interaction between mucosal mast cells and EPANs in schistosoma-infected mice.     

Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-β-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and α-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.     

Distribution and ontogeny of SP, CGRP, SOM, and VIP in chick sensory and sympathetic ganglia

The distribution and ontogeny of four neuropeptides in developing chick lumbosacral sensory and sympathetic ganglia were studied using immunohistochemical techniques. Antibodies to two of these peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), stained small neurons in the medial part of the dorsal root ganglia from embryonic Day 5 and Day 10, respectively, whereas neurons in the lateral part of the ganglia were negative; this distribution persisted throughout development. Both sets of neurons apparently send fibers to the dorsal horn of the spinal cord: SP to laminae I and II, and CGRP to lamina I, suggesting that the SP- and CGRP-positive sensory neurons are nociceptive or thermoreceptive. This correlation between the presence of SP or CGRP in a neuron and a particular functional modality thus provides evidence for a functional distinction between the mediodorsal and ventrolateral zones that are apparent during the development of chick dorsal root ganglia. Moreover, this study suggests that the type of neuron that develops within the dorsal root ganglion correlates with its position within the ganglion. In contrast to SP and CGRP, somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) immunoreactivities were not seen in the lumbosacral sensory ganglia at any stage during development. However, both were present in sympathetic ganglia: SOM from embryonic Day 4.5 and VIP from embryonic Day 10. VIP immunoreactivity persisted throughout development in a large number of sympathetic neurons, but the number of cells with SOM immunoreactivity decreased from embryonic Day 10 onward. SOM therefore appears to be present only transiently in most chick lumbosacral sympathetic cells.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.