Substance P- and vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers in relation to ovarian postganglionic perikarya in para- and prevertebral ganglia: Evidence from combined retrograde tracing and immunocytochemistry

Summary Retrograde neuronal tracing with the fluorescent dye True Blue and immunocytochemistry were utilized to examine postganglionic sympathetic neurons in para- and prevertebral ganglia projecting to the rat ovary. Perikarya in both ganglia were labeled with True Blue after application of the tracer to either the superior ovarian or ovarian plexus nerve. After application of True Blue to the superior ovarian nerve, 17% of the labeled cells in paravertebral ganglia were immunoreactive for vasoactive intestinal polypeptide. In contrast, after application of True Blue to the ovarian plexus nerve, approximately 1 % of the labeled cells in paravertebral ganglia were immunoreactive for the same polypeptide. Some vasoactive intestinal polypeptide-immunoreactive perikarya in paravertebral ganglia were not labeled with True Blue. In some cases, substance P- and vasoactive intestinal polypeptide-immunoreactive fibers were closely apposed to True Blue-labeled perikarya in para-and prevertebral ganglia. Paravertebral vasoactive intestinal polypeptide-immunoreactive perikarya projecting to the ovary presumably participate directly in the control of various ovarian functions. Substance P- and vasoactive intestinal polypeptide-immunoreactive fibers closely apposed to perikarya projecting to the ovary may participate indirectly in the control of various ovarian functions by affecting the activity of ovarian postganglionic neurons.     

A neurochemical characterisation of the golden hamster myenteric plexus

The neurochemical composition of nerve fibres and cell bodies in the myenteric plexus of the proventriculus, stomach and small and large intestines of the golden hamster was investigated by using immunohistochemical and histochemical techniques. In addition, the procedures for localising nitric-oxide-utilising neurones by histochemical (NADPH-diaphorase) and immunohistochemical (nitric oxide synthase) methods were compared. The co-localisation of vasoactive intestinal polypeptide and nitric oxide synthase in the myenteric plexus of all regions of the gut was also assessed. The results demonstrated the presence of nerve fibres and nerve cell bodies immunoreactive to protein gene product, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, tyrosine hydroxylase, 5-hydroxytryptamine and nitric oxide synthase in all regions of the gastrointestinal tract examined. The pattern of distribution of immunoreactive nerve fibres and nerve cell bodies containing the above markers was found to vary in different regions of the gut. Myenteric neurones and nerve fibres containing immunoreactivity to nitric oxide synthase and NADPH-diaphorase reactivity, however, were shown to have an identical distribution throughout the gut. In contrast to some studies on the guinea-pig and rat, the co-existence of vasoactive intestinal polypeptide and nitric oxide synthase was seen in only a small population of myenteric neurones.     

Differences in the distribution and chemical coding between neurons in the inferior mesenteric ganglion supplying the colon and rectum in the pig

The distribution and chemical coding of neurons in the porcine left and right inferior mesenteric ganglion projecting to the ascending colon and rectum have been investigated by using combined retrograde tracing and double-labelling immunohistochemistry. The ganglion contained many neurons supplying both gut regions. The colon-projecting neurons (CPN) occurred exclusively in the cranial part of the ganglia where they formed a large cluster distributed along the dorso-lateral ganglionic border and a smaller cluster located close to the caudal colonic nerve output. The rectum-projecting neurons (RPN) formed a long stripe along the entire length of the lateral ganglionic border and, within the right ganglion only, a small cluster located close to the caudal colonic nerve output. Immunohistochemistry revealed that the vast majority of the CPN and RPN were noradrenergic (tyrosine-hydroxylase-positive). Many noradrenergic neurons supplying the colon contained somatostatin or, less frequently, neuropeptide Y. In contrast, a significant subpopulation of the noradrenergic RPN expressed neuropeptide Y, whereas only a small proportion contained somatostatin. A small number of the non-adrenergic RPN were cholinergic (choline-acetyltransferase-positive) and a much larger subpopulation of the nerve cells supplying both the colon and rectum were non-adrenergic and non-cholinergic. Many cholinergic neurons contained neuropeptide Y. The non-adrenergic non-cholinergic neurons expressed mostly somatostatin or neuropeptide Y and some of those projecting to the rectum contained nitric oxide synthase, galanin or vasoactive intestinal polypeptide. Many of both the CPN and RPN were supplied with varicose nerve fibres exhibiting immunoreactivity against Leu5-enkephalin, somatostatin, choline-acetyltransferase, vasoactive intestinal polypeptide or nitric oxide synthase The somatotopic and neurochemical organization of this relatively large population of differently coded inferior mesenteric ganglion neurons projecting to the large bowel indicates that these cells are probably involved in intestino-intestinal reflexes controlling peristaltic and secretory activities.     

Nitric oxide synthase-containing neurons in rat parasympathetic, sympathetic and sensory ganglia: a comparative study

Summary In rats, the distribution of nerve structures staining for NADPH-diaphorase, and showing immunoreactivities for nitric oxide synthase (NOS), tyrosine hydroxylase and various neuropeptides was studied in sensory ganglia (dorsal root, nodose and trigeminal ganglia), in sympathetic ganglia (superior cervical, stellate, coeliac-superior and inferior mesenteric ganglia), parasympathetic ganglia (sphenopalatine, submandibular, sublingual and otic ganglia), and in the mixed parasympathetic/ sympathetic ganglia (major pelvic ganglia). The coincidence of neuronal cell bodies with strong NOS-immunoreactivity and strong NADPH diaphorase reactivity was almost total. The relative proportions of NOS-immunoreactive nerve cell bodies were largest in parasympathetic ganglia and major pelvic ganglia followed by sensory ganglia. In sympathetic ganglia no NOS-immunoreactive neuronal cell bodies could be detected. In parasympathetic and major pelvic ganglia, there was a very significant neuronal co-localization of immunoreactivities for NOS and vasoactive intestinal polypeptide (VIP). This was almost total in major pelvic ganglia, in which NOS-/VIP-immunoreactive nerve cell bodies were separate from sympathetic (tyrosine hydroxylase-/neuropeptide Y-immunoreactive), suggesting that NOS-/VIP-immuno-reactive neurons might also be parasympathetic.     

Nitric oxide synthase-containing nerves and ganglia in the dog prostate: A comparison with other transmitters

Summary The distribution of nitric oxide synthase immunoreactive nerves in the dog prostate was compared to the total innervation (as estimated by protein gene product 9.5 immunoreactivity), and to that of adrenergic (tyrosine hydroxylase-immunoreactive), cholinergic (acetylcholinesterase-positive), and some peptidergic nerves immunoreactive towards vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and helospectin. Clusters of ganglia with cell bodies containing acetylcholinesterase, or one of these six immunoreactive components, were found in the dorsal capsule. Coarse nerve trunks expressing these immunoreactive components extended from the ganglia, and divided into varicose terminals in the capsule and intraglandular smooth muscle strands, and gave off further branches, which surrounded acini and accompanied ducts. The labelling for nitric oxide synthase generally coincided with that for vasoactive intestinal peptide within cell bodies and nerves of various types. Cell bodies, nerve trunks and varicose terminals showing labelling for pituitary adenylate cyclase-activating peptide and helospectin were generally also labelled for vasoactive intestinal peptide. The innervation pattern suggests that nitric oxide may act in concert with vasoactive intestinal peptide and related peptides in the control of prostatic smooth muscle activity and secretion.     

The dependence on gp130 cytokines of axotomy induced neuropeptide expression in adult sympathetic neurons

Adult peripheral neurons exhibit dramatic changes in gene expression after axonal injury, including changes in neuropeptide phenotype. For example, sympathetic neurons in the superior cervical ganglion (SCG) begin to express vasoactive intestinal peptide (VIP), galanin, pituitary adenylate cyclase activating polypeptide (PACAP), and cholecystokinin after axotomy. Before these changes, nonneuronal cells in the SCG begin to express leukemia inhibitory factor (LIF). When the effects of axotomy were compared in LIF?/? and wild‐type mice, the increases in VIP and galanin expression were less in the former, though significant increases still occurred. LIF belongs to a family of cytokines with overlapping physiological effects and multimeric receptors containing the subunit gp130. Real‐time PCR revealed large increases in the SCG after axotomy in mRNA for three members of this cytokine family, interleukin (IL)‐6, IL‐11, and LIF, with modest increases in oncostatin M, no changes in ciliary neurotrophic factor, and decreases in cardiotrophin‐1. To explore the role of these cytokines, animals with selective elimination of the gp130 receptor in noradrenergic neurons were studied. No significant changes in mRNA levels for VIP, galanin, and PACAP were seen in axotomized ganglia from these mutant mice, while the increase in cholecystokinin was as large as that seen in wild‐type mice. The data indicate that the inductions of VIP, galanin, and PACAP after axotomy are completely dependent on gp130 cytokines and that a second cytokine, in addition to LIF, is involved. The increase in cholecystokinin after axotomy, however, does not require the action of these cytokines. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Enteric neural pathways mediate the anti-inflammatory actions of glucagon-like peptide 2

Glucagon-like peptide-2 (GLP-2) is an important regulator of nutritional absorptive capacity with anti-inflammatory actions. We hypothesized that GLP-2 reduces intestinal mucosal inflammation by activation of vasoactive intestinal polypeptide (VIP) neurons of the submucosal plexus. Ileitis or colitis was induced in rats by injection of trinitrobenzene sulfonic acid (TNBS), or colitis was induced by administration of dextran sodium sulfate (DSS) in drinking water. Subsets of animals received (1-33)-GLP-2 (50 mug/kg sc bid) either immediately or 2 days after the establishment of inflammation and were followed for 3-5 days. The involvement of VIP neurons was assessed by concomitant administration of GLP-2 and the VIP antagonist [Lys(1)-Pro(2,5)-Arg(3,4)-Tyr(6)]VIP and by immunohistochemical labeling of GLP-2-activated neurons. In all models, GLP-2 treatment, whether given immediately or delayed until inflammation was established, resulted in significant improvements in animal weights, mucosal inflammation indices (myeloperoxidase levels, histological mucosal scores), and reduced levels of inflammatory cytokines (IFN-gamma, TNF-alpha, IL-1beta) and inducible nitric oxide synthase, with increased levels of IL-10 in TNBS ileitis and DSS colitis. Reduced rates of crypt cell proliferation and of apoptosis within crypts in inflamed tissues were also noted with GLP-2 treatment. These effects were abolished with coadministration of GLP-2 and the VIP antagonist. GLP-2 was shown to activate neurons and to increase the number of cells expressing VIP in the submucosal plexus of the ileum. These findings suggest that GLP-2 acts as an anti-inflammatory agent through activation of enteric VIP neurons, independent of proliferative effects. They support further studies to examine the role of neural signaling in the regulation of intestinal inflammation.     

Colocalization of NADPH-diaphorase activity and certain neuropeptides in the esophagus of opossum (Didelphis virginiana)

Nitric oxide and various neuropeptides in the myenteric plexus regulate esophageal motility. We sought colocalization of nitric oxide synthase and neuropeptides in frozen sections of mid-portion of smoothmuscled opossum esophagus using NADPH-diaphorase activity to mark the synthase and immunoreactivity to detect peptides. The peptides, all with demonstrated physiological activity in this organ, were calcitonin generelated peptide, galanin, neuropeptide Y, substance P, and vasoactive intestinal polypeptide. The ExtrAvidin Peroxidase immunostain for each peptide was carried up to the final peroxidase reaction with 3-amino-9-ethylcarbazole. The NADPH-diaphorase reaction was applied with short incubation to provide light staining just before the peroxidase reaction was performed. We examined sections for the proportions of singly and dually labeled nerve cells in the myenteric plexus. NADPH-diaphorase activity was highly colocalized with calcitonin gene-related peptide (59%), galanin (54%), and vasoactive intestinal polypeptide (53%). It showed little colocalization with neuropeptide Y (10%) and substance P (8%). The proportions of all nerve cells containing each of the substances were: NADPH-diaphorase-33%, calcitonin gene-related peptide-30%, galanin-55%, neuropeptide Y-16%, substance P-35%, and vasoactive intestinal polypeptide-58%. We conclude that the nerves responsible for peristalsis in the esophagus may act by releasing nitric oxide along with other inhibitory substances, calcitonin gene-related peptide, galanin, and vasoactive intestinal polypeptide, but not excitatory substances, neuropeptide Y and substance P.     

Intrinsic choroidal neurons in the duck eye receive sympathetic input: anatomical evidence for adrenergic modulation of nitrergic functions in the choroid

Intrinsic choroidal neurons (ICN) in the duck eye form an intramural ganglionic plexus that may subserve complex integrative functions. A key feature of such ganglia is an innervation by sympathetic postganglionic neurons. The present study was thus aimed at determining the sympathetic postganglionic innervation of ICN. Choroids were processed for double immunofluorescence labelling with the following markers: tyrosine-hydroxylase (TH)/nitric oxide synthase (nNOS), TH/galanin (GAL), dopamine-beta-hydroxylase (DBH)/vasoactive intestinal polypeptide (VIP), TH/DBH and DBH/alpha-smooth-muscle actin (alphaSMA), and for triple immunofluorescence labelling with VIP/DBH/TH. Epifluorescence and confocal laser scanning microscopy were used for evaluation. Immunoperoxidase staining for TH or DBH in combination with NADPH-diaphorase histochemistry was applied for electron microscopy. ICN spread over the entire choroid but were concentrated in an equatorial zone passing obliquely from naso-cranial to temporocaudal. More than 80% of nNOS-positive ICN showed close appositions of TH/DBH-immunoreactive varicose nerve fibres at the light-microscopic level, as could be confirmed by confocal laser scanning microscopy. Ultrastructurally, these appositions could be defined as both synapses or close contacts without synaptic specialisation. Vascular and non-vascular smooth muscle fibres also received TH/DBH-immunopositive innervation. Our findings suggest that most ICN receive a sympathetic input that might modulate their nitrergic effects upon vascular and non-vascular smooth muscle fibres in the choroid and that they may have more complex functions than merely being a simple parasympathetic relay.     

Coexistence of NADPH-diaphorase and vasoactive intestinal polypeptide in the enteric nervous system of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias)

The distribution of NADPH (nicotinamide adenine dinucleotide phosphate)-diaphorase in nerve cells in the gastrointestinal tract has been investigated and compared in three fish species representing different evolutionary branches. In mammals, NADPH-diphorase is identical to nitric oxide synthase (NOS) and can, in the presence of NADPH, reduce the dye nitroblue tetrazolium, resulting in a blue product. Using this method, we have found numerous NADPH-diaphorase-containing nerve cells in the myenteric plexus of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias) but none in the hagfish (Myxine glutinosa). In the cod, nerve fibres were sparsely stained, whereas in the dogfish, they formed a dense pattern of fibre bundles. Double-staining for NADPH-diaphorase and the neuropolypeptides VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase activating peptide) revealed three separate populations designated VIP/NADPH, VIP/- and NADPH/-. The majority but not all of the NADPH-diaphorase-positive cells also showed VIP or PACAP immunoreactivity and vice versa. The presence of NADPH-diaphorase in neurons and the distribution of these neurons in the gastrointestinal tract of the two species indicate a physiological role for nitric oxide in the control of gut motility.     

Double labelling immunohistochemical characterization of autonomic sympathetic neurons innervating the sow retractor clitoridis muscle

Retrograde neuronal tracing and immunohistochemical methods were used to define the neurochemical content of sympathetic neurons projecting to the sow retractor clitoridis muscle (RCM). Differently from the other smooth muscles of genital organs, the RCM is an isolated muscle that is tonically contracted in the rest phase and relaxed in the active phase. This peculiarity makes it an interesting experimental model. The fluorescent tracer fast blue was injected into the RCM of three 50 kg subjects. After a one-week survival period, the ipsilateral paravertebral ganglion S1, that in a preliminary study showed the greatest number of cells projecting to the muscle, was collected from each animal. The co-existence of tyrosine hydroxylase with choline acetyltransferase, neuronal nitric oxide synthase, calcitonin gene-related peptide, leu-enkephalin, neuropeptide Y, substance P and vasoactive intestinal polypeptide was studied under a fluorescent microscope on cryostat sections. Tyrosine hydroxylase was present in about 58% of the neurons projecting to the muscle and was found to be co-localized with each of the other tested substances. Within fast blue-labelled cells negative to the adrenergic marker, small populations of neurons singularly containing each of the other enzymatic markers or peptides were also observed. The present study documents the complexity of the neurochemical interactions that regulate the activity of the smooth myocytes of the RCM and their vascular components.     

Peptide-immunoreactive nerves in the mammalian female genital tract

Summary Vasoactive intestinal polypeptide, substance P, neuropeptide Y and peptide histidine isoleucine immunoreactivities have been demonstrated in the female genitalia of rat, cat, mouse and guinea-pig using immunocytochemistry and radioimmunoassay. They were localized to nerves. Each type of immunoreactive nerve showed a distinct pattern of distribution, though all were associated to some degree with blood vessels and smooth muscle. Vasoactive intestinal polypeptide-immunoreactive and neuropeptide Y-immunoreactive nerves were the most abundant. Higher concentrations of peptides were detected in the female genitalia of the mouse than those of the other species studied. Vasoactive intestinal polypeptide-immunoreactive nerves were particularly concentrated in the cervix (89.1±17.2 pmol/g, mean±S.E.M.) and the uterus (57.4±14.8 pmol/g) of the mouse, while neuropeptide Y immunoreactivity was more abundant in the Fallopian tube of the mouse (31.6±11.8 pmol/g) and the vagina of the rat (38.6±4.8 pmol/g) than in other regions. Separate populations of ganglion cells in the paracervical ganglia were found to contain vasoactive intestinal polypeptide and neuropeptide Y immunoreactivities. Peptide histidine isoleucine-immunoreactive and vasoactive intestinal polypeptide-immunoreactive nerves were similarly distributed, but the former were much less frequent. Substance P-immunoreactive nerves were seen mainly beneath the epithelium of the vagina and were, in general, more numerous in the guinea-pig than in other species. The significance of these peptide-immunoreactive nerves in the female genital organ remains to be determined.Dr. Wang is on leave from The Institute of Acupuncture, The Academy of Chinese Traditional Medicine, Peking, China.     

Chemical sympathectomy and postganglionic nerve transection produce similar increases in galanin and VIP mRNA but differ in their effects on peptide content

Large changes in neuronal gene expression occur in adult peripheral neurons after axonal transection. In the rat superior cervical ganglion, for example, neurons that do not normally express vasoactive intestinal peptide (VIP) or galanin do so after postganglionic nerve transection. These effects of axotomy could result from a number of aspects of the surgical procedure. To test the idea that the important variable might be the disconnection of axotomized neuronal cell bodies from their target tissues, we examined the effects of producing such a disconnection by means of the compound 6-hydroxydopamine (6-OHDA), a neurotoxin that causes degeneration of sympathetic varicosities and avoids many of the complications of surgery. Two days after 6-OHDA treatment, VIP and galanin immunoreactivities had increased two- and 40-fold, respectively. Nevertheless, these increases were substantially smaller than the 30- and 300-fold changes seen after surgical axotomy. When expression of VIP and galanin was examined at the mRNA level, however, comparable increases were found after either procedure. The results indicate that chemical destruction of sympathetic varicosities produces an equivalent signal for increasing VIP and galanin mRNA as does axonal transection. The differences in the neuropeptide levels achieved suggests that peptide expression after nerve transection is regulated both at the mRNA and protein levels. © 1996 John Wiley & Sons, Inc.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

Innervation of the anococcygeus muscle of the rat

Summary The innervation of the anococcygeus muscle of the rat was investigated with regard to the histochemical features of nerve fibers within the muscle and to the location of the postganglionic autonomic neurons which are the source of these fibers. Acetylcholinesterase-positive fibers and catecholaminergic fibers are abundant in the anococcygeus as well as the related retractor penis muscle. Neuronal somata, either between muscle bundles of the anococcygeus or in the connective tissue sheath, are also acetylcholinesterase-positive. Nerve fibers and a minority of the ganglion cells in the anococcygeus and retractor penis muscles are immunoreactive for vasoactive intestinal polypeptide. Injection of the retrogradely transported dye Fluorogold into the anococcygeus muscle filled neurons in the abdominopelvic sympathetic chain, pelvic plexus and a small number of neurons in the inferior mesenteric ganglion. In the pelvic plexus, some neurons were located in the major pelvic ganglion but most were found along the main penile nerve and its branches to the anococcygeus muscle. Immunocytochemistry of these identified neurons indicates that about one half of them are positive for vasoactive intestinal polypeptice. These results raise the possibility that both acetylcholine and vasoactive intestinal polypeptide are important neurotransmitters in autonomic nerves to the anococcygeus muscle.     

Bone morphogenetic protein down-regulation of neuronal pituitary adenylate cyclase-activating polypeptide and reciprocal effects on vasoactive intestinal peptide expression

Among bone morphogenetic proteins (BMPs), the decapentaplegic (Dpp; BMP2, BMP4) and glass bottom boat (Gbb/60A; BMP5, BMP6, BMP7) subgroups have well-described functions guiding autonomic and sensory neuronal development, fiber formation and neurophenotypic identities. Evaluation of rat superior cervical ganglia (SCG) post-ganglionic sympathetic neuron developmental regulators identified that selected BMPs of the transforming growth factor beta superfamily have reciprocal effects on neuronal pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) expression. Dpp and Gbb/60A BMPs rapidly down-regulated PACAP expression, while up-regulating other sympathetic neuropeptides, including PACAP-related VIP. The suppressive effects of BMP on PACAP mRNA and peptide expression were potent, efficacious and phosphorylated mothers against decapentaplegic homolog (Smad) signaling-dependent. Axotomy of SCG dramatically increases PACAP expression, and the possibility that abrogation of inhibitory retrograde target tissue BMP signaling may contribute to this up-regulation of sympathetic neuron PACAP was investigated. Replacement of BMP6 to SCG explant preparations significantly blunted the injury-induced elevated PACAP expression, with a concomitant decrease in sympathetic PACAP-immunoreactive neuron numbers. These studies suggested that BMPs modulate neuropeptide identity and diversity by stimulating or restricting the expression of specific peptidergic systems. Furthermore, the liberation of SCG neurons from target-derived BMP inhibition following axotomy may be one participating mechanism associated with injury-induced neuropeptidergic plasticity.     

Nitric oxide synthase in guinea pig sympathetic ganglia: Correlation with tyrosine hydroxylase and neuropeptides

Nitric oxide synthase (NOS) has previously been reported in a small population of postganglionic sympathetic neurons in the guinea pig. The present study of paravertebral ganglia and the inferior mesenteric ganglion aimed to classify these neurons according to their content of neuropeptides (calcitonin gene-related peptide, neuropeptide Y, vasoactive intestinal peptide) and the rate-limiting enzyme of catecholamine synthesis, tyrosine hydroxylase, by means of immunohistochemical and histochemical double-labelling techniques. NOS-containing neurons belonged to the non-catecholaminergic population of postganglionic neurons, and partial coexistence was found with neuropeptide Y and vasoactive intestinal peptide immunoreactivities but not with calcitonin gene-related peptide. However, most of the NOS-containing neurons contained none of the neuropeptides, thus representing a hitherto unrecognized population of postganglionic neurons. The findings show that NOS is localized to small but neurochemically highly specific populations of postganglionic neurons, which most likely reflects an association with target- and function-specific pathways.     

Vasoactive intestinal polypeptide-immunoreactive nerves in the pulmonary vasculature of the aquatic file snake Acrochordus granulatus

Summary The indirect immunofluorescence technique was used to determine the distribution of vasoactive intestinal polypeptide-immunoreactive and somatostatin-immunoreactive axons in the pulmonary vasculature of the aquatic file snake Acrochordus granulatus. A dense distribution of vasoactive intestinal polypeptide-immunoreactive axons was found on the common pulmonary artery, the anterior and posterior pulmonary arteries, and the smaller arteries branching to the lung. The density of these axons appeared greater in arterial preparations taken from more distal regions of the lung. The densest distribution of vasoactive intestinal polypeptide-immunoreactive axons was observed on the larger pulmonary veins in all regions of the lung. These axons were observed on the larger veins within the lung parenchyma but not on the smaller veins. Axons and cell bodies were observed in the vagal nerve trunks which run parallel to the pulmonary arteries and veins. In contrast, no somatostatin-immunoreactive axons were observed in any region of the pulmonary vasculature. It is proposed that the perivascular plexus of vasoactive intestinal polypeptide-immunoreactive axons may represent part or all of the vagal postganglionic innervation of the pulmonary vasculature.     

Different biological activities in conditioned media control the expression of a variety of neuropeptides in cultured sympathetic neurons

An intriguing question regarding neuronal development is how neurons choose which neurotransmitter and/or peptide to express among over 40 candidates. We find that heart cell conditioned medium (CM) induces a number of neuropeptides and/or their precursor mRNAs, as well as acetylcholine, in cultured rat sympathetic neurons: substance P, somatostatin, vasoactive intestinal polypeptide, enkephalin derivatives, and cholecystokinin, but not neuropeptide Y. Different patterns of peptide induction were observed for CMs from primary cultures of heart, gut, and skin. Acetylcholine and substance P were induced most effectively by serum-free heart cell CM; enkephalin derivatives were induced most effectively by skin cell CM; and somatostatin and vasoactive intestinal polypeptide were induced equally well by all of the CMs. These observations suggest the possibility that many distinct, diffusible factors can influence the choice of transmitter and/or peptide phenotype in developing neurons.