Immunohistochemical properties of motoneurons supplying the porcine trapezius muscle.

The trapezius muscle (TRAP) belongs to the scapulothoracic group of muscles, which play a crucial role in the integrity and strength of the upper limb, trunk, head, and neck movements and, thus, in maintaining balance. Combined retrograde tracing (using fluorescent tracer Fast Blue, FB) and double-labelling immunohistochemistry were applied to investigate the chemical coding of motoneurons projecting to the porcine TRAP. FB-positive (FB+) motoneurons supplying the cervical (c-TRAP) and thoracic part (th-TRAP) of the right (injected with the tracer) TRAP were located within the IX-th Rexed lamina in the ipsilateral ventral horn of the grey matter of the spinal medulla. Immunohistochemistry revealed that nearly all the neurons were cholinergic in nature [choline acetyltransferase (CHAT)- or vesicular acetylcholine transporter (VACHT)-positive]. Many retrogradelly labelled neurons displayed also immunoreactivity to calcitonin gene-related peptide (CGRP; approximately 68% of FB+ neurons). The smaller number of nerve cells (5%, 3%, 2% or 1%, respectively) stained for nitric oxide synthase (n-NOS), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and substance P (SP). The retrogradely labelled neurons were closely apposed by nerve fibres expressing immunoreactivity to CHAT, VACHT, CGRP, SP, DßH, VIP, n-NOS, NPY, GAL, Leu-Enk and Met-Enk. Taking into account the clinical relevance of TRAP, the present results may be useful in designing further research aimed at the management of various dysfunctions of the muscle.

Enteric neuronal development in canine small intestine - an immunohistochemical study.

The development of the enteric nervous system (ENS) is still a valid and intensely studied issue. However, literature in the field has no data on this topic in the dog. The present investigations were performed in three groups of fetuses from mongrel dogs - from the third, sixth- -seventh, and ninth week of pregnancy - and in 3-5-day-old puppies (3 specimens for each age group). The tissues (the medial parts of the duodenum, jejunum, and ileum with the cecum and a small portion of the adjacent ascending colon) were cut using a cryostat and the sections were processed for single- and double-labeling immunohistochemistry using antisera against acetylated tubulin (AcTub), vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), galanin (GAL), neuropeptide Y (NPY), substance P (SP), and calcitonin gene-related peptide (CGRP). In the 3-week-old fetuses, some oval cells invading the gut wall were found. From the seventh week of pregnancy onwards, two different enteric ganglia were present: submucosal and myenteric. The estimated number of nerve elements in the 9-week-old fetuses was much higher than that observed in the 6-7-week-old individuals. There was no significant difference in the estimated number of nerve structures between the 9-week-old fetuses and the 3-5-day-old puppies. The colonization pattern and the development of the ENS in the canine small intestine are very similar to those observed in other mam- mals. However, a few exceptions have been confirmed, regarding the time of appearance of the VIP-, GAL-, and CGRP-immunoreactive neurons, and their distribution in different portions of the canine bowel during development.

Chemical Coding of Sensory Neurons Supplying the Hip Joint Capsule in the Sheep.

Immunohistochemical properties of nerve fibres supplying the joint capsule were previously described in many mammalian species, but the localization of sensory neurons supplying this structure was studied only in laboratory animals, the rat and rabbit. However, there is no comprehensive data on the chemical coding of sensory neurons projecting to the hip joint capsule (HJC). The aim of this study was to establish immunohistochemical properties of sensory neurons supplying HJC in the sheep. The study was carried out on 10 sheep, weighing about 30-40 kg. The animals were injected with a retrograde neural tracer Fast Blue (FB) into HJC. Sections of the spinal ganglia (SpG) with FB-positive (FB+) neurons were stained using antibodies against calcitonin gene-related peptide (CGRP) substance P (SP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (n-NOS), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), Leu-5-enkephalin (Leu-Enk), galanin (GAL) and vesicular acetylcholine transporter (VACHT). The vast majority of FB+ neurons supplying HJC was found in the ganglia from the 5th lumbar to the 2nd sacral. Immunohistochemistry revealed that most of these neurons were immunoreactive to CGRP or SP (80.7 ± 8.0% or 56.4 ± 4.8%, respectively) and many of them stained for PACAP or GAL (52.9 ± 2.9% or 50.6 ± 19.7%, respectively). Other populations of FB+ neurons were those immunoreactive to n-NOS (37.8 ± 9.7%), NPY (34.6 ± 6.7%), VIP (28.7 ± 4.8%), Leu-Enk (27.1 ± 14.6) and VACHT (16.7 ± 9.6).

Pituitary adenylate cyclase-activating peptide-27 (PACAP-27) is co-stored with galanin, substance P and corticotropin releasing factor (CRF) in intrapancreatic ganglia of the sheep.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide existing in two variant forms (of either 27 or 38 residues), widely present in numerous organs and evoking multiple effects both in the central and peripheral nervous systems. The present study was undertaken to evaluate the distribution pattern of PACAP-27 expression in the ovine pancreas. Using double immunohistochemical stainings co-localizations of PACAP-27 with galanin, SP or CRF were studied in intrapancreatic neurons. In intrapancreatic ganglia, immunoreactivty to PACAP-27 was found in 87.6 ± 5.4% of PGP 9.5-positive intrapancreatic neurons but not in intraganglionic nerve fibres. Numerous PACAP-27-immunoreactive nerve terminals were also observed between pancreatic acini and around small arterioles. No immunoreactivity to PACAP-27 was found in the endocrine pancreas. In 42.9 ± 6.2% of PACAP-27-immunoreactive intrapancreatic neurons the expression of galanin was also found. Statistically lower subpopulation (12.4 ± 4.0%) of intrapancreatic neurons exhibited simultaneously the immunoreactivity to PACAP-27 and SP. The expression of CRF was detected in the relatively smallest group (3.2 ± 1.4%) of PACAP-27-positive intrapancreatic neurons. The present results suggest that in the ovine pancreas PACAP-27 may play an important role as mediator of pancreatic functions. In PACAP-related pancreatic activities, a modulatory role of galanin, SP and to a lower extend of CRF is also likely.

Morphology and immunohistochemical characteristics of the pterygopalatine ganglion in the chinchilla (Chinchilla laniger, Molina).

Histological and histochemical investigations revealed that the pterygopalatine ganglion (PPG) in the chinchilla is a structure closely connected with the maxillary nerve. Macro-morphological observations disclosed two different forms of the ganglion: an elongated stripe representing single agglomeration of nerve cells, and a ganglionated plexus comprising smaller aggregations of neurocytes connected with nerve fibres. Immunohistochemistry revealed that nearly 80% of neuronal cell bodies in PPG stained for acetylcholine transferase (CHAT) but only about 50% contained immunoreactivity to vesicular acetylcholine transporter (VACHT). Many neurons (40%) were vasoactive intestinal polypeptide (VIP)-positive. Double-staining demonstrated that approximately 20% of the VIP-immunoreactive neurons were VACHT-negative. Some neurons (10%) in PPG were simultaneously VACHT/nitric oxide synthase (NOS)- or Met-enkephaline (Met-ENK)/CHAT-positive, respectively. A small number of the perikarya stained for somatostatin (SOM) and solitary nerve cell bodies expressed Leu-ENK- and galanin-immunoreactivity. Interestingly about 5-8% of PPG neurons exhibited immunoreactivity to tyrosine hydroxylase (TH). Intraganglionic nerve fibres containing immunoreactivity to VACHT-, VIP- and Met-ENK- were numerous, those stained for calcitonin gene related peptide (CGRP)- and substance P (SP)- were scarce, and single nerve terminals were TH-, GAL-, VIP- and NOS-positive.

Immunohistochemical properties of motoneurons supplying the trapezius muscle in the rat.

Combined retrograde tracing (using fluorescent tracer Fast blue) and double-labelling immunofluorescence were used to study the distribution and immunohistochemical characteristics of neurons projecting to the trapezius muscle in mature male rats (n = 9). As revealed by retrograde tracing, Fast blue-positive (FB+) neurons were located within the ambiguous nucleus and accessory nucleus of the grey matter of the spinal cord. Immunohistochemistry revealed that nearly all the neurons were cholinergic in nature [choline acetyltransferase (ChAT)-positive]. Retrogradely labelled neurons displayed also immunoreactivities to calcitonin gene-related peptide (CGRP; approximately 60% of FB+ neurons), nitric oxide synthase (NOS; 50%), substance P (SP; 35%), Leu5-Enkephalin (LEnk; 10%) and vasoactive intestinal polypeptide (VIP; 5%). The analysis of double-stained tissue sections revealed that all CGRP-, VIP- and LEnk-immunoreactive FB+ perikarya were simultaneously ChAT-positive. The vast majority of the neurons expressing SP- or NOS-immunoreactivity were also cholinergic in nature; however, solitary somata were ChAT-negative. FB+ perikarya were surrounded by numerous varicose nerve fibres (often forming basket-like structures) immunoreactive to LEnk or SP. They were also associated with some CGRP-, NOS- and neuropeptide Y-positive nerve terminals.

Characterization of autonomic nerve markers and lymphocyte subsets in the ileal Peyer's patch of pigs infected experimentally with Brachyspira hyodysenteriae.

The aim of the present study was to investigate potential interrelationships between immune and neural elements of Peyer's patches in normal pigs (n=8) and in pigs infected experimentally with Brachyspira hyodysenteriae and suffering from swine dysentery (n=8). Assessment of tissue concentration of neuropeptides by enzyme linked immunosorbent assay revealed increased levels of galanin (GAL) and substance P (SP) in samples from the infected animals. In contrast, concentrations of vasoactive intestinal polypeptide (VIP) and somatostatin (SOM) were similar in both groups. Immunohistochemistry demonstrated reactivity of nerve fibres with antibodies specific for dopamine ß hydroxylase, vesicular acetylcholine transporter, SOM, GAL, VIP and SP in the interfollicular region and peripheral areas of the Peyer's patch lymphoid follicles. In the dysenteric pigs, the GAL-positive nerve fibres were more numerous and more intensely labelled than those in the normal animals. Flow cytometry revealed a decreased percentage of CD21(+) lymphocytes and lymphocytes expressing T-cell receptor (TCR)-γ, with or without CD8 (TCR-γ(+)CD8(-) and TCR-γ(+)CD8(+)), in the dysenteric pigs as compared with the normal animals. Percentages of other lymphocyte subsets (CD2(+), CD4(+), CD5(+), CD8(+), CD5(-)CD8(+)) were comparable between the groups. Immunohistochemical investigations generally correlated with results obtained by flow cytometry related to lymphocyte subpopulations. Swine dysentery can therefore affect neuroimmunomodulatory processes in the ileal Peyer's patch, in addition to the large intestine. GAL and SP may play a specific role in this neuroimmune cross-talk.

Porcine hypothalamic aromatase cytochrome P450: isoform characterization, sex-dependent activity, regional expression, and regulation by enzyme inhibition in neonatal boars.

Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the "gonadal/hypothalamic" porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function.

Distribution and chemical coding of intramural neurons in the porcine ileum during proliferative enteropathy.

Enteric neurons are highly adaptive in their response to various pathological processes including inflammation, so the aim of this study was to describe the chemical coding of neurons in the ileal intramural ganglia in porcine proliferative enteropathy (PPE). Accordingly, juvenile Large White Polish pigs with clinically diagnosed Lawsonia intracellularis infection (PPE; n=3) and a group of uninfected controls (C; n=3) were studied. Ileal tissue from each animal was processed for dual-labelling immunofluorescence using antiserum specific for protein gene product 9.5 (PGP 9.5) in combination with antiserum to one of: vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptide Y (NPY) or galanin (GAL). In infected pigs, enteric neurons were found in ganglia located within three intramural plexuses: inner submucosal (ISP), outer submucosal (OSP) and myenteric (MP). Immunofluorescence labelling revealed increases in the number of neurons containing GAL, SOM, VIP and CGRP in pigs with PPE. Neuropeptides may therefore have an important role in the function of porcine enteric local nerve circuits under pathological conditions, when the nervous system is stressed, challenged or afflicted by disease such as PPE. However, further studies are required to determine the exact physiological relevance of the observed adaptive changes.

The distribution and chemical coding of intramural neurons supplying the porcine stomach - the study on normal pigs and on animals suffering from swine dysentery.

The present study was designed to investigate the expression of biologically active substances by intramural neurons supplying the stomach in normal (control) pigs and in pigs suffering from dysentery. Eight juvenile female pigs were used. Both dysenteric (n = 4; inoculated with Brachyspira hyodysenteriae) and control (n = 4) animals were deeply anaesthetized, transcardially perfused with buffered paraformalehyde, and tissue samples comprising all layers of the wall of the ventricular fundus were collected. The cryostat sections were processed for double-labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene-product 9.5) and their chemical coding using antibodies against vesicular acetylcholine (ACh) transporter (VAChT), nitric oxide synthase (NOS), galanin (GAL), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), Leu(5)-enkephalin (LENK), substance P (SP) and calcitonin gene-related peptide (CGRP). In both inner and outer submucosal plexuses of the control pigs, the majority of neurons were SP (55% and 58%, respectively)- or VAChT (54%)-positive. Many neurons stained also for CGRP (43 and 45%) or GAL (20% and 18%) and solitary perikarya were NOS-, SOM- or VIP-positive. The myenteric plexus neurons stained for NOS (20%), VAChT (15%), GAL (10%), VIP (7%), SP (6%) or CGRP (solitary neurons), but they were SOM-negative. No intramural neurons immunoreactive to LENK were found. The most remarkable difference in the chemical coding of enteric neurons between the control and dysenteric pigs was a very increased number of GAL- and VAChT-positive nerve cells (up to 61% and 85%, respectively) in submucosal plexuses of the infected animals. The present results suggest that GAL and ACh have a specific role in local neural circuits of the inflamed porcine stomach in the course of swine dysentery.

Distribution and gene expression of neuropeptides during brain development.

Neuropeptides and catecholamines are biologically active substances which play the roles of neurotransmitters, neuromodulators and cotransmitters in the central nervous system (CNS). These substances are known to regulate and influence a wide spectrum of functions; such as food and water intake, thermoregulation, growth and maturation, sexual behaviour, reproduction, function of the hypothalamic-hypophysial-organ axes, and many others. This large family of neuropeptides is comprised of around 40 fundamental polypeptides, which does not include their derivatives and related peptides. Localization and development of several of them was intensively studied in the brain of a wide range of species, including the rat and the pig. In this present paper, data regarding distribution of catecholamine synthesising enzymes and some neuropeptides, as well as their gene expression during development in the rodent and porcine brain are summarised and related to development of LHRH-containing structures.

Influence of active immunization against GnRH on VIP- and NPY-positive innervation of the porcine testis.

The influence of an anti-GnRH vaccine on VIP- and NPY-positive innervation of testes was studied in the pig. The immunization prevented the occurrence of changes in the pattern of VIP- and NPY-positive testicular innervation associated with the sexual maturation: it maintained the density of innervation at the high level characteristic for sexually immature animals. The effect was dependent on the method of immunization: the application of two doses of the vaccine was more efficient than application of only one dose, and vaccination with adjuvant was more efficient than vaccination with the plain vaccine. The studies on VIP and NPY concentration in the testicular tissue with radioimmunoassay (RIA) revealed immunization-dependent changes in the peptide concentration, however, some discrepancies between morphological changes and peptide levels were observed.

Has active immunization against gonadotrophin-releasing hormone any effect on testis innervation in the pig? An immunohistochemical study.

The innervation of porcine testes was studied in intact animals and in boars undergoing active immunization against gonadotrophin-releasing hormone (GnRH) by means of immunohistochemistry using antibodies to tyrosine hydroxylase (TH), dopamine beta-hydroxylase (D beta H), vasoactive intestinal polypolypeptide (VIP), neuropeptide Y (NPY), synaptosome-associated protein of 25 kDa (SNAP-25) and protein gene product 9.5 (PGP 9.5). Moreover, the distribution of luteinizing hormone (LH) receptors in clusters of Leydig cells was also investigated. To identify these cells easily, either the NADPH-diaphorase histochemical technique or the Mayer counter-staining procedure was applied. Differences in the distribution pattern and relative density of particular subsets of intratesticular nerve fibres were observed in immunized boars as compared to those found in the intact animals. In the testes of non-treated animals, only single TH-immunoreactive (TH-IR) nerve fibres were observed. However, many D beta H-IR nerve terminals surrounded blood vessels in the tunica albuginea and parenchyma. Very scarce VIP-IR nerves occurred only in the tunica albuginea, mainly in close vicinity to blood vessels. Immunoreactivity to NPY occurred in single nerve fibres. Immunoreactivity to SNAP-25 and PGP 9.5 was found in single nerve fibres distributed mainly in the tunica albuginea. The interstitial cells were heavily stained for LH-receptors and NADPH-diaphorase. In the testes of immunized animals, only single TH-IR nerve fibres, scattered mainly in the tunica albuginea, were observed. Some TH-IR nerve terminals were also encountered in the parenchyma of the organ, where they were always associated with blood vessels. D beta H-IR nerve fibres formed a dense network distributed throughout the testis in association with the capsule, vasculature and interstitium. Some fibres were observed to run between seminiferous tubules. VIP-IR nerve fibres were located in the neighbourhood of blood vessels in the tunica albuginea and parenchyma. Only single VIP-IR nerves were found between seminiferous tubules. Numerous NPY-IR nerve fibres occurred in the tunica albuginea and parenchyma of the organ. SNAP-25-IR and PGP 9.5-IR nerve terminals formed a dense network distributed throughout the testis and many fibres were observed between seminiferous tubules. Interstitial cells were very weakly stained for LH receptors or NADPH-diaphorase.

Distribution of catecholamine-synthesizing enzymes and some neuropeptides in the median eminence-arcuate nucleus complex (MEARC) of the immature female pig.

The presence of the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (D beta H) and some neuropeptides, including neuropeptide Y (NPY), Leu5-enkephalin (LENK), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL) and somatostatin (SOM) was investigated in nerve fibres and perikarya of the median eminence-arcuate nucleus complex (MEARC) of the sexually immature female pigs by means of the immunohistochemical avidin-biotin complex method. Although immunoreactivities to all the studied substances were found in nerve fibres of the porcine MEARC, there were differences in the distribution and density of particular subsets of nerve fibres within the complex. While loose D beta H-immunoreactive (D beta H-IR) and dense TH-, NPY- and VIP-IR nerve meshworks occurred predominantly in the internal layer of the MEARC, nerve fibres immunoreactive to TH, CGRP, SOM, SP and LENK were more numerous in the external than in the internal layer of the median eminence (ME). Numerous TH-, D beta H-, NPY-, VIP-, SP- and CGRP-IR perivascular nerve fibres were also observed within both layers of the median eminence. There were also differences in the distribution of a particular subset of neurons within the porcine MEARC: NPY-, VIP-, GAL-, SP- and TH-IR (but not D beta H-IR) perikarya were found in the arcuate nucleus, while in the median eminence only subpopulations of NPY-, VIP and GAL-IR neurons were observed.

Neuroendocrine cells in the female urogenital tract of the pig, and their immunohistochemical characterization.

A systematic search for neuroendocrine (NE) cells in the urogenital organs of the pig was carried out by means of Linder's argyrophil method and immunohistochemical techniques. The occurrence, distribution and immunohistochemical character of NE cells (paraneurons) were studied in the vaginal vestibulum, vagina, uterus, oviduct, ovary, urethra, urinary bladder and ureter. In the vestibular glands paraneurons were found to be the most numerous, while a moderate number of these cells occurred in the uterine horn and in the urethra. A distinctly smaller number of paraneurons was present in the oviduct and only occasional NE cells were observed in the urinary bladder. Immunohistochemistry was performed by using the peroxidase-antiperoxidase procedure. Different subpopulations of paraneurons were distinguishable. Chromogranin A-positive paraneurons were found in the vestibular glands, uterine horns, oviducts, urethra and urinary bladder. Somatostatin positivity was observed in NE cells of the vestibular gland, uterine horn, oviduct and urethra. The subpopulation of serotonin-positive paraneurons was present in the vestibular gland and urethra. Bombesin, vasoactive intestinal polypeptide, cholecystokinin, substance P, nitric oxide synthase, beta-endorphin, insulin, adrenocorticotropic hormone, oxytocin and thyroid-stimulating hormone antibodies gave negative reactions in the studied NE cells.

Peptidergic innervation of the porcine oviduct studied by double-labelling immunohistochemistry.

Neurochemical coding of nerve fibres supplying the porcine oviduct was studied by means of double-labelling immunofluorescence. Immunoreactivities to rate-limiting enzymes of catecholamine synthesis, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DbetaH), were found in numerous oviductal nerve fibres. All TH-immunoreactive (TH-IR) nerve processes were simultaneously DbetaH-IR. This subset of nerves was considered to be sympathetic noradrenergic. In addition to noradrenaline, many axons additionally exhibited immunoreactivity to neuropeptide Y (NPY), or leu5-enkephalin (Leu-ENK). Small numbers of somatostatin- (SOM-) or vasoactive intestinal polypeptide-immunoreactive (VIP-IR) fibres, sometimes coexpressing TH/DbetaH-immunoreactivity, supplied the porcine oviduct. Substance P- (SP- ) and/or calcitonin gene-related peptide-immunoreactive (CGRP-IR) nerve fibres were only sporadically found. Although these nerves did not contain TH/DbetaH-immunoreactivity, they often ran in close vicinity to TH/DbetaH-IR axons, forming together thin nerve bundles. All the above mentioned subpopulations of nerve fibres were found throughout the entire length of the oviduct being mainly related to the vascular and non-vascular smooth myocytes. However, some of the putative afferent (i.e. SP- or CGRP-IR) or parasympathetic efferent (i.e. VIP- or NPY-IR but TH/DbetaH-immunonegative) axons were located beneath the epithelium. Such distribution implies these nerve fibres to be involved in the regulation of the oviductal blood flow, non-vascular smooth myocyte tonus, transmission of sensory information and control of the epithelial secretion.

Immunohistochemical localization of galanin in bovine reproductive organs.

The vagina, uterus and oviduct were shown to receive galanin immunoreactive (GAL-IR) nerve fibres, the number of which varied between particular organs. In the ovary, GAL-IR nerves were absent. A small number of these nerves were located in the layers of the oviduct. A moderate number of GAL-IR nerves were situated in the body and uterine horns, whereas the uterine cervix and vagina wall contained a large number of GAL-IR nerve fibres, evenly distributed throughout particular membranes of the organs. GAL-IR nerves were found to contain, simultaneously, either vasoactive intestinal polypeptide (VIP), substance P (SP) or Leu5-enkephalin (ENK). Many of the GAL-IR nerves contained tyrosine hydroxylase (TH). A group of GAL-IR nerves that did not possess immunoreactivity to VIP, SP, ENK or TH was also observed.

The distribution and co-localization of immunoreactivity to nitric oxide synthase, vasoactive intestinal polypeptide and substance P within nerve fibres supplying bovine and porcine female genital organs.

The distribution of nitric oxide synthase-immunoreactive (NOS-IR) axons and their relationship to structures immunoreactive to vasoactive intestinal polypeptide (VIP), substance P (SP) and tyrosine hydroxylase (TH) were studied by means of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) technique or double-labelling immunofluorescence in the genital organs of cow and pig. Relevant neurons were also investigated in the pig. NOS-containing neural structures were TH-immunonegative in bovine or porcine genital organs, or in the studied ganglia. In the bovine ovary, NOS-IR nerves were neither VIP-IR nor SP-IR, whereas in the pig, most NOS-containing axons were also VIP-IR. The oviduct was supplied by single NOS/VIP- or NOS/SP-containing nerves, whereas in the uterus, NOS-IR axons were moderate in number, often being immunoreactive for VIP or SP. Numerous NOS/VIP-IR and NOS/SP-IR nerves were found in the vagina of both species. In all tissues studied, NOS-IR axons were mainly related to vascular smooth muscle. Most of the neurons of the paracervical ganglia and some neurons in dorsal root ganglia exhibited strong NOS activity. Only single neurons in sympathetic ganglia were NADPH-d-positive. Most nitrergic neurons in the autonomic ganglia were VIP-IR but SP-immunonegative. The sensory neurons were mostly NOS/SP-IR, whereas only single neurons co-expressed NOS and VIP immunoreactivity.

Existence and co-existence of vasoactive substances in nerve fibres supplying the abdomino-pelvic arterial tree of the female pig and cow.

The occurrence and co-localization of several presumed vasoactive neuropeptides, serotonin, and catecholamine-synthesising enzymes--tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (D beta H) and phenylethanolamine-N-methyltransferase (PNMT)--were investigated in perivascular nerves supplying the systemic and distributing arteries of the abdomino-pelvic arterial tree of the female pig and certain arteries supplying female reproductive organs in the cow. As revealed by single immunofluorescence, perivascular axons immunoreactive for TH, D beta H, neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP) and Leu-enkephalin (LENK) occurred in both species examined, whereas galanin-immunoreactive (GAL-IR) nerve fibres were found exclusively in the pig. PNMT-, serotonin-, dynorphin A-, alpha-neoendorphin-, bombesin- or cholecystokinin-IR nerve terminals were not observed. The following classes of perivascular nerve fibres might be distinguished in the present study: 1) noradrenergic (i.e. TH/D beta H-IR), 2) NPY-, 3) GAL- (pig only), 4) LENK-, 5) VIP-, 6) SP-, 7) VIP/NPY-, 8) SP/CGRP-, 9) SP/GAL- (pig only), 10) SP/VIP- (cow only), 11) TH/D beta H/NPY- and 12) TH/D beta H/NPY/LENK-IR. Distinct differences in the distribution of LENK- and SP-IR axons around particular parts of the studied arterial tree in individual species were also observed. The present data indicate that the abdomino-pelvic arterial tree of the pig and cow receive perivascular nerve fibres that exhibit diverse chemical codes, and that different chemical coding of perivascular nerve fibres in individual species may depend on the target organ of the particular artery.

Occurrence and coexistence of some neuropeptides in nerve fibers supplying the bovine ovary and its extrinsic blood vessels.

Single- and double-labelling immunofluorescence was applied to investigate the occurrence and coexistence of vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P (SP) in nerve fibers supplying the bovine ovary as well as its extrinsic blood vessels. We also studied the coexistence of these neuropeptides with tyrosine hydroxylase (TH) and neuropeptide Y (NPY) to determine features of "chemical coding" of nerve fibers innervating this organ. The bovine ovary was found to be supplied by VIP- and SP-immunoreactive (VIP- and SP-IR) nerve fibers. No intraovarian CGRP-IR nerve terminals were detected. Immunoreactivity to VIP was shown in a moderate number of nerve fibers occurring in all ovarian regions - ovarian hilus, cortex and medulla. VIP-IR nerves were found to innervate blood vessels and ovarian follicles, especially primordial and primary ones. Immunoreactivity to SP was detected only in solitary intraovarian nerve fibers which were also encountered in all ovarian regions, but with special preference to ovarian medulla, where they innervated blood vessels. SP-IR nerve fibres were sometimes observed in close vicinity to primordial and primary follicles. Double-labelling immunofluorescence revealed that some VIP-IR nerve fibres supplying intraovarian blood vessels were simultaneously TH- or NPY-IR. Small numbers of VIP-IR fibres contained immunoreactivity to SP. All SP-IR nerve terminals were TH-or NPY-negative. The ovarian artery as well as arteries of the paraovarian plexus were moderately supplied with VIP-, or by single CGRP- or SP-IR nerve fibres. Veins were innervated by only solitary nerve terminals containing these neuropeptides. The following patterns of coexistence were determined in nerve fibres supplying the investigated blood vessels: VIP, VIP/CGRP, VIP/SP, CGRP/SP, VIP/NPY. No distinct differences in the innervation of the ovary and its extrinsic blood vessels between juvenile and adult cows were found.