Presence and co-localization of vasoactive intestinal polypeptide with neuronal nitric oxide synthase in cells and nerve fibers within guinea pig intrinsic cardiac ganglia and cardiac tissue

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VIP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia. This work was supported by NIH grant HL65481 (R.L.P.) and HL54633 (D.B.H.). Use of the DeltaVision Restoration microscope was provided through the Imaging/Physiology Core supported by NIH Grant P20 RR16435 from the COBRE program of the National Center for Research Resources     

Nitric oxide synthase-containing nerve fibers and neurons in the genital tract of the female mouse

Nitric oxide (NO) is generated intracellularly from L-arginine by the action of the enzyme nitric oxide synthase (NOS). The present investigation demonstrates immunoreactivity against NOS and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in nerve cells and fibers of the reproductive system of the female mouse. The density of nerve fibers staining for NOS varied among different genital organs. The ovary and Fallopian tube were devoid of NOS-positive nerves. The uterine horns received sparse innervation by NOS-containing nerve fibers. The most abundant NOergic innervation was found in the uterine cervix and vagina, where the nerve fibers ran parallel to the smooth muscle bundles and beneath the epithelium; they also accompanied intramural blood vessels. The vaginal muscular wall contained single or groups of NOS-reactive nerve cells. Clusters of NOS-containing neurons were located in Frankenhäuser's ganglion at the cervico-vaginal junction. NO may therefore act as a transmitter in the nervous control of the female reproductive tract.     

Dopamine-β-hydroxylase-, neurotensin-, substance P-, vasoactive intestinal polypeptide- and enkephalin-immunohistochemistry of paravertebral and prevertebral ganglia in the cat

Summary Para and prevertebral ganglia of the cat were investigated for immunoreactivity (IR) against neurotensin (NT), vasoactive intestinal polypeptide (VIP), substance P (SP) and enkephalin (ENK). Dopamine--hydroxylase- (DBH)-IR was studied in consecutive sections to correlate the distribution of noradrenergic/adrenergic neurons with that of peptidergic nerve fibres and cells.In paravertebral (cervical and thoracic) ganglia, NT-IR or ENK-IR nerve fibres were seen in areas in which DBH-IR fibre networks also occurred. NT-IR varicosities were often in close contact with perikarya of principal ganglionic cells on which DBH-IR varicosities also terminated. Such an association was rarely seen between ENK-IR and DBH-IR fibre baskets. NT-IR and ENK-IR fibre baskets were not found to occur around the same principal ganglionic cell. The distribution of VIP-IR and SP-IR nerve fibres did not coincide with that of DBH-IR fibres.In prevertebral ganglia (celiac-superior mesenteric and inferior mesenteric) DBH-IR or VIP-IR varicosities surrounded the majority of principal ganglionic neurons. ENK-IR or SP-IR fibres were closely associated with only a minority of the neurons; NT-IR networks were rather sparse. Some principal neurons were approached by DBH-IR fibres and by different peptide-IR fibres.In paravertebral ganglia some principal ganglionic cells contained VIP-IR, a few of which were also surrounded by NT-IR varicosities. VIP-IR perikarya in prevertebral ganglia were extremely rare. No NT-IR, SP-IR or ENK-IR principal ganglionic cells were found.Glomus-like paraganglionic cell clusters in paravertebral and prevertebral ganglia exhibited DBH-IR cell bodies. Moreover, the clusters also contained ENK-IR or SP-IR cells. NT-IR varicosities were observed adjacent to clustered paraganglionic cells. Only few singly located paraganglionic cells were NT-IR or ENK-IR.The differential distribution of peptide-IR nerve endings in the investigated ganglia suggests a regulation of impulse transmission that seems to be related to the target organs.Fellow of the Heisenberg foundationSupported by the DFG, grants He 919/5, Re 520/1-2, and SFB 90 Carvas, Heidelberg     

Neurohormonal peptides, serotonin, and nitric oxide synthase in the enteric nervous system and endocrine cells of the gastrointestinal tract of neotenic and thyroid hormone-treated axolotls (Ambystoma mexicanum)

Immunoreactivity against vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), somatostatin (SOM), serotonin (SER), and nitric oxide synthase (NOS) was investigated in the gastrointestinal tract of the urodele Ambystoma mexicanum, the axolotl, by the use of immunohistochemical techniques. The study also compares the distribution patterns and frequencies of the neurohormones, and NOS in neotenic and thyroxine-treated (metamorphosed) individuals. GAS/CCK, SP, NT, SOM, and SER immunoreactivities occurred in endocrine mucosal cells and VIP, SP, CGRP, NTSER, SER, and NOS immunoreactivities in the enteric nervous system. The GAS/CCK-immunoreactive (-IR) cells were restricted to the upper small intestine. NT-IR and SP-IR endocrine cells were found in the entire gastrointestinal tract and were most prominent in the distal large intestine. The density of the SOM-IR cells decreased from the stomach toward the large intestine. SER-IR endocrine cells were found throughout the gastrointestinal tract, with particularly high densities in the stomach and distal large intestine. The VIP-IR enteric nerve fibers were the most prominent ones, present in all layers of the entire gastrointestinal tract, and supplied the smooth muscle and the vasculature. The SER-IR fibers exhibited similar distribution patterns but were less numerous. Very few NT-IR but many SP-IR fibers were found in the muscle and submucosal layers. The NT-IR fibers mainly supplied blood vessels, while the SP-IR fibers were also in contact with the smooth muscle. In the muscle and submucosal layers, CGRP-IR fibers were associated to the vasculature; CGRP immunoreactivity occurred also in a minority of SP-IR fibers. NOS-IR nerve fibers were in contact with submucosal arteries but were the least frequent . After metamorphosis provoked by exogenous thyroxine, the number of SOM-IR endocrine cells in the stomach mucosa was increased as well as the density of VIP-IR, SER-IR, and SP-IR nerve fibers in the gastrointestinal tract. It is proposed that the observed increases may reflect refinements of the neurohormonal system after metamorphosis.     

Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse

Immunohistochemical techniques were used to examine the presence and co-localisation of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse. Distinct sub-populations of myenteric neurons were identified, based on the combinations of substances they contained and the distribution of their fibres. In the small intestine, there were two major classes of circular muscle motor neurons; one class was characterised by the presence of nitric oxide synthase, vasoactive intestinal peptide plus neuropeptide Y (NOS/VIP/NPY), and the second class contained calretinin plus substance P (CalR/SP). There were seven classes of neurons that innervated myenteric ganglia; these contained NOS, VIP, NOS/VIP, NPY, CalR/calbindin (CalB), SP or 5-HT. In the large intestine, there were five major classes of motor neurons that contained NOS, NOS/VIP, GABA, SP, or CalR/SP, and seven major classes of neurons that innervated myenteric ganglia and contained NOS, VIP, CalR/CalB, CalR, SP, GABA or 5-HT. Although some aspects of the patterns of co-localisation are similar to those in other species, this study re-inforces recent analyses that indicate significant species differences in neurochemical patterns in the enteric neurons of different species. Received: 28 August 1995 / Accepted: 30 November 1995     

The longitudinal smooth muscle layer of the pig small intestine is innervated by both myenteric and submucous neurons

Originally, intestinal motility was thought to be exclusively regulated by myenteric neurons. Some years ago, however, it was demonstrated in large mammals that submucous neurons also participate in the innervation of the circular smooth muscle layer. To date, no information is available about the submucous innervation of the longitudinal smooth muscle layer (LM). This study provides evidence that in the small intestine of large mammals, the LM is innervated not only by the myenteric plexus, but also by the inner and outer submucous plexuses (ISP and OSP). In the porcine small intestine, the involved neurons can be subdivided into the following neurochemically distinct populations: leu-enkephalin (ENK)- and/or substance P (SP)-IR neurons and nitric oxide synthase (NOS)- and/or vasoactive intestinal polypeptide (VIP)-IR neurons. In the myenteric plexus, the majority of VIP- and/or NOS-IR neurons and ENK(+)/SP(-)-IR neurons exhibit descending projections, whereas ENK(+)/SP(+)-IR neurons preferentially have ascending projections. The ENK(-)/SP(+)-IR neurons do not show a polarized pattern. In the OSP, only ENK(+)/SP(-)- and VIP(+)/NOS(-)-IR neurons display a polarized (descending) projection pattern, whereas no polarization can be noted in the ISP. Morphological analysis of the traced neurons revealed that, in general, myenteric descending LM motor neurons have larger cell bodies than ascending ones and, in addition, myenteric descending VIP- and/or NOS-IR neurons have longer projections than ENK and/or SP-IR neurons. In conclusion, the present study demonstrates the involvement of not only myenteric, but also submucous neurons in the innervation of the LM. The two major populations are descending nitrergic neurons and ascending tachykinergic motor neurons, but also other subpopulations with specific projection patterns and neurochemical features have been identified.     

An immunohistochemical study on the innervation of SP-IR nerves in the cerebral arteries of the bent-winged bat

The overall distribution of substance P (SP) immunoreactive (IR) nerves surrounding the cerebral arteries of the bent-winged bat were investigated immunohistochemically. In this microchiropteran species, the walls from the vertebral artery to the caudal part of the basilar artery have considerably well-developed plexuses of SP-IR nerves, whereas no demonstrable SP-IR fibers were found in the rostral part of the basilar artery, and in more rostrally located arteries the nerve supply was very sparse or occasionally lacking. This innervation pattern has not yet been established for the cerebral arterial systems of other mammals that have been studied under normal conditions, but it is very similar to the pattern of SP-IR innervation observed in the guinea pig and cat of which the trigeminal ganglia have been destroyed. From the combination of this and other immunohistochemical findings, it is suggested that SP-IR nerves innervating the vertebral and basilar arteries of the bent-winged bat originate from the upper cervical dorsal root ganglia (DRG) and enter the cranial cavity along the vertebral artery and through the meninges.     

Distribution of nitric oxide synthase and vasoactive intestinal polypeptide immunoreactivity in the sphincter of Oddi and duodenum of the possum

The nitrergic innervation of the sphincter of Oddi (SO) and duodenum in the Australian brush-tailed possum and the possible association of this innervation with the neuropeptide vasoactive intestinal polypeptide (VIP) were investigated by using immunohistochemical localisation of nitric oxide synthase (NOS) and VIP, together with the general neuronal marker, protein gene product 9.5 (PGP9.5). Whole-mount preparations of the duodenum and attached SO without the mucosa, submucosa and circular muscle (n=12) were double- and triple-labelled. The density of myenteric nerve cell bodies of the SO in the more distal region (duodenal end) was significantly higher than that in the more proximal region. In the SO, approximately 50% of all cells were NOS-immunoreactive (IR), with 27% of the NOS-IR cells being VIP-IR. Within the duodenal myenteric plexus, NOS immunoreactivity was present in about 25% of all neurons, with 27% of these NOS-IR neurons also being VIP-IR, a similar proportion to that in the SO. Varicose nerve fibres with NOS and VIP immunoreactivity were present within the myenteric and submucous plexuses of the SO and duodenum, and in the circular and longitudinal muscle layers. The NOS-positive cells within both the SO and duodenum were unipolar, displaying a typical Dogiel type I morphology. The myenteric plexuses of the SO and duodenum were in direct continuity, with many interconnecting nerve trunks, some of which showed NOS and VIP immunoreactivity. Thus, the possum possesses an extensive NOS innervation of the SO and duodenum, with a significantly higher proportion of NOS-IR neurons within the SO, a subset of which contains VIP.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

An immunohistochemical study on the innervation of SP-IR nerves in the cerebral arteries of the bent-winged bat

Summary The overall distribution of substance P (SP) immunoreactive (IR) nerves surrounding the cerebral arteries of the bent-winged bat were investigated immunohistochemically. In this microchiropteran species, the walls from the vertebral artery to the caudal part of the basilar artery have considerably well-developed plexuses of SP-IR nerves, whereas no demonstrable SP-IR fibers were found in the crostral part of the basilar artery, and in more rostrally located arteries the nerve supply was very sparse or occasionally lacking. This innervation pattern has not yet been established for the cerebral arterial systems of other mammals that have been studied under normal conditions, but it is very similar to the pattern of SP-IR innervation observed in the guinea pig and cat of which the trigeminal ganglia have been destroyed. From the combination of this and other immunohistochemical findings, it is suggested that SP-IR nerves innervating the vertebral and basilar arteries of the bent-winged bat originate from the upper cervical dorsal root ganglia (DRG) and enter the cranial cavity along the vertebral artery and through the meninges.Abbreviations BA basilar artery - CSN cervical spinal nerves - ICS internal carotid system - SCG superior cervical ganglion - SNB sympathetic nerve bundle - VA vertebral artery - VBS vertebro-basilar system     

Co-existence of peptide HI (PHI) and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man

By immunohistochemistry it was found that PHI- and VIP-like immunoreactivity (-IR) occurred in the same autonomic neurons in the upper respiratory tract, tongue and salivary glands with associated ganglia in rat, guinea-pig, cat, pig and man. VIP- and PHI-like immunoreactivity was also found in similar locations in the human heart. The N-terminally directed, but not the C-terminally directed, PHI antiserum or the VIP antiserum stained endocrine cells in the pig duodenum. This suggests the existence of an additional PHI-like peptide. Ligation of nerves acutely caused marked overlapping axonal accumulations of PHI- and VIP-IR central to the lesion. Two weeks after transection of the nerves, both types of immunoreactivities were still observed in accumulations both in the axons as well as in the corresponding cell bodies. The levels of PHI- and VIP-IR in normal tissues from the cat were around 10-50 pmol/g with a molar ratio of about 1 to 2. Systemic administrations of PHI and VIP induced hypotension, probably due to peripheral vasodilation in both guinea-pig and cat. Furthermore, both PHI and VIP caused an inhibition of the vagally induced increase in respiratory insufflation pressure in guinea-pig. PHI and VIP relaxed the guinea-pig trachea in vitro, suggesting a direct action on tracheobronchial smooth muscle. VIP was about 5-10 times more potent than PHI with regard to hypotensive effects and 2-3-fold, considering respiratory smooth muscle-relaxant effects in the guinea-pig. PHI was about 50-fold less potent to induce hypotension in the cat than in the guinea-pig. Although species differences seem to exist as regards biological potency, PHI should also be considered when examining the role of VIP as an autonomic neurotransmitter.     

Innervation of the pancreas by vasoactive intestinal polypeptide (VIP) immunoreactive nerves

The vasoactive intestinal polypeptide (VIP) has been shown to exert effects on endocrine and exocrine pancreatic secretion. Immunocytochemistry reveals that VIP immunoreactive nerves occur in the porcine, canine, feline and avian pancreas. In the pancreas of pig and cat VIP nerves are abundant around non-immunoreactive nerve cell bodies of the intrapancreatic ganglia but scarce in the islets and in the exocrine parenchyma. In the dog pancreas, however, the intrapancreatic ganglia contain strongly immunoreactive VIP nerve cell bodies which give off axons that seem to heavily innervate vessels as well as endocrine and exocrine cells. We suggest that in the pig and cat the pancreatic VIP nerves mainly affect the activity of a second type of intrapancreatic neuron, whose transmitter is unknown, whereas in the dog pancreas VIP nerves directly contact their putative effector structures.     

Tissue distribution and innervation pattern of peptide immunoreactivities in the rat pancreas.

The distribution of calcitonin gene-related peptide (CGRP), substance P/tachykinin (SP/TK), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and gastrin-releasing peptide (GRP) immunreactivities (IR) in the rat pancreas was investigated using radioimmunoassay and immunohistochemistry. CGRP, NPY and VIP tissue contents are much higher than GRP and SP/TK concentrations. Peptide-containing nerves are distributed to both the exocrine and endocrine pancreas. However, differences exist in terms of density and targets of innervation for each peptidergic system. In the acini and through the stroma, fibers IR for CGRP, NPY and VIP are greater than GRP- and SP/TK-containing processes. The vasculature is supplied by a prominent NPY, CGRP and, to a lesser extent, SP/TK innervation. VIP-IR is found occasionally, and GRP-IR is never detected, in fibers associated with blood vessels. Around ducts, CGRP- and NPY-positive neurites are greater than SP/TK- greater than or equal to VIP-IR fibers, whereas GRP-containing nerves are not visualized. In the islets, the density of peptidergic nerves is: VIP-, GRP- greater than or equal to CGRP-IR greater than NPY or SP/TK. In intrapancreatic ganglia. VIP- and, to a lesser extent, NPY-IRs are found in numerous neuronal cell bodies and in nerve fibers; GRP-IR is present in numerous nerve processes and in few cell bodies; CGRP- and SP/TK-IRs are detected only in fibers wrapping around unlabeled ganglion cells. The majority of CGRP-IR fibers contain SP/TK-IR. The existence of differential patterns of peptidergic nerves suggests that peptides exert their effects on pancreatic functions via different pathways.     

Neuropeptide Y: presence in sympathetic and parasympathetic innervation of the nasal mucosa

Summary The occurrence of neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in the sympathetic and parasympathetic innervation of the nasal mucosa was studied in various species including man. A dense network of NPY-immunoreactive (IR) fibres was present around arteries and arterioles in the nasal mucosa of all species studied. NPY was also located in nerves around seromucous glands in pig and guinea-pig, but not in rat, cat and man. The NPY-IR glandular innervation corresponded to about 20% of the NPY content of the nasal mucosa as revealed by remaining NPY content determined by radioimmunoassay after sympathectomy. These periglandular NPY-positive fibres had a distribution similar to the VIP-IR and PHI-IR nerves but not to the noradrenergic markers tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH). The NPY nerves around glands and some perivascular fibres were not influenced by sympathectomy and probably originated in the sphenopalatine ganglion where NPY-IR and VIP-IR ganglion cells were present. The venous sinusoids were innervated by NPY-positive fibres in all species except the cat. Dense NPY and DBH-positive innervation was seen around thick-walled vessels in the pig nasal mucosa; the latter may represent arterio-venous shunts. Double-labelling experiments using TH and DBH, and surgical sympathectomy revealed that the majority of NPY-IR fibres around blood vessels were probably noradrenergic. The NPY-positive perivascular nerves that remained after sympathectomy in the pig nasal mucosa also contained VIP/PHI-IR. The major nasal blood vessels, i.e. sphenopalatine artery and vein, were also densely innervated by NPY-IR fibres of sympathetic origin. Perivascular VIP-IR fibres were present around small arteries, arterioles, venous sinusoids and arterio-venous shunt vessels of the nasal mucosa whereas major nasal vessels received only single VIP-positive nerves. The trigeminal ganglion of the species studied contained only single TH-IR or VIP-IR but no NPY-positive ganglion cells. It is concluded that NPY in the nasal mucosa is mainly present in perivascular nerves of sympathetic origin. In some species, such as pig, glandular and perivascular parasympathetic nerves, probably of VIP/PHI nature, also contain NPY.     

An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals.

The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuron-specific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6-hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY-immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetylcholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immunoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholinergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Summary Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetycholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immnoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholingergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

Biochemical Characterization and Histochemical Localization of Nitric Oxide Synthase in the Nervous System of the Snail, Helix pomatia

Abstract: Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde-sensitive and cytosolic paraformaldehyde-insensitive NADPH-diaphorase. The cytosolic NADPH-diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca²⁺ and NADPH and was inhibited by N ^G-nitro- l -arginine ( l -NNA). Proteins purified by 2',5'-ADP affinity chromatography were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60-kDa protein. Characterization of the cDNA of the corresponding 60-kDa NOS-immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca²⁺- and NADPH-dependent NOS activity, which was partially inhibited by EGTA and l -NNA. Histochemistry showed NADPH-diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH-diaphorase-positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH-diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS-associated/regulating protein in mollusk nervous tissue.     

Neurotrophin-3 and neurotrophin receptor immunoreactivity in peptidergic enteric neurons

In the rat small intestine, neurotrophin-3 immunoreactivity was identified in ganglion cells and in processes mostly innervating the mucosa and occasionally the muscle layer and vasculature. The vast majority of neurotrophin-3 immunoreactive neurons contained vasoactive intestinal polypeptide (VIP), but not substance P or related tachykinin (SP/TK). Neurotrophin receptors visualized by pan-trk immunoreactivity were found in numerous ganglion cells of both plexuses and in nerve processes in the intestinal wall. Pan-trk submucosal neurons contained VIP (36%) or SP/TK-IR (47%). Pan-trk myenteric neurons contained VIP-IR (57%) or SP/TK (27%). Our data suggest that neurotrophin-3 and neurotrophin receptors may be involved in the maintenance of enteric neuronal circuits, transmission and phenotypic expression.     

Distribution and changes with age of nitric oxide synthase-immunoreactive nerves of the rat urinary bladder, ureter and in lumbosacral sensory neurons

In the distal parts of the urinary tract, nerves containing nitric oxide (NO) are either postganglionic parasympathetic nerves, with cell bodies in the major pelvic ganglia, or sensory nerves with cell bodies in the lumbosacral dorsal root ganglia. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive for neuronal nitric oxide synthase (NOS) in the urinary bladder, distal ureter and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made of NOS fibres innervating the dome, body and base of the urinary bladder and distal ureter. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for NOS. The dome and the body regions, in both age groups, contained no NOS-immunoreactive axons. The bladder base and distal ureter in young adults showed sparse to moderate numbers of fibres immunoreactive to NOS within the urothelium and in the subepithelium and muscle coat. In the aged rat there were slight reductions in the densities of NOS-immunoreactive nerves in all three regions. In the lumbosacral dorsal root ganglia, the percentage of NOS-immunoreactive neuronal profiles showed a significant reduction from 4.6 +/- 0.2% in young adult to 2.7 +/- 0.2% (means +/- S.E.M) in aged rats. These findings suggest that the effects of NO on the bladder and distal ureteric musculature and also its expression in dorsal root ganglion neurons are affected in aged rats and that the micturition reflex may be perturbed as a result.