Neuronal pathways in the guinea-pig lumbar sympathetic ganglia as revealed by immunohistochemistry

Tyrosine hydroxylase (TH)- and peptide-immunoreactivity of postganglionic neurons and of nerve fibres in guinea pig lumbar paravertebral sympathetic ganglia 2-4 after transection of the communicating rami and the visceral branches, respectively, were investigated by single- and double-labelling techniques. Six subpopulations of postganglionic neurons were discriminated immunohistochemically: two cell types, which were immunoreactive to only one of the applied antisera - TH, and vasoactive intestinal polypeptide (VIP); and four cell types in which immunoreactivity was colocalized - TH/neuropeptide Y (NPY), NPY/VIP, dynorphin/alpha-neoendorphin and dynorphin (alpha-neoendorphin)/NPY. Small intensely fluorescent (SIF) cells dependent on their location exhibited differential immunobehaviour to NPY-/dynorphin-(alpha-neoendorphin-) and TH-antisera. Immunoreactivity to substance P (SP), calcitonin gene-related peptide (CGRP), met-enkephalin-arg-phe (MEAP) and leu-enkephalin was present in nerve fibres but not in postganglionic neurons with frequent colocalization of SP/CGRP- and MEAP/leu-enkephalin- and, sometimes leu-enkephalin/SP- and dynorphin/SP-immunoreactivity. TH-immunoreactive intraganglionic nerve fibres were numerically more increased after cutting the visceral branches, than after transection of the communicating rami. Vice versa, NPY-, VIP-, dynorphin- and alpha-neoendorphin-immunoreactive nerve fibres were particularly increased in number after cutting the communicating rami. Many but not all of the nerve fibres exhibited colocalization of two of these peptides. SP-, CGRP-, and enkephalin-immunoreactive nerve fibres were not visibly affected by cutting the visceral branches but virtually disappeared after lesioning the communicating rami.     

The peptidergic innervation of the guinea pig uterine artery in pregnancy

The influence of pregnancy on the density and pattern of the peptidergic innervation of the guinea pig uterine artery was studied. Whole mount stretch preparations of the uterine artery from estrus and late pregnant guinea pigs were processed for the immunohistochemical demonstration of neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)-, calcitonin gene-related peptide (CGRP)- and substance P (SP)- immunoreactive nerve fibres. In late pregnancy the density of NPY- and CGRP- containing nerve fibres was remarkably decreased, while that of VIP- and SP- immunoreactive nerves showed a moderate reduction. The meaning and the possible physiological relevance of the decreased density of peptide-immunoreactive nerves in the uterine artery in late pregnancy are discussed.     

Catecholaminergic and peptidergic nerve components of intramural ganglia in the rat heart

Summary Immunohistochemical properties of the terminal nerve network in the rat heart were assessed by use of the elution-restaining method. The colocalization of the enzymes involved in catecholamine synthesis (tyrosine hydroxylase — TH, dopamine--hydroxylase — DBH) as well as the respective distributions of the neuropeptides associated with the adrenergic nervous system (neuropeptide tyrosine — NPY, C-terminal flanking peptide of neuropeptide Y — C-PON) were studied in series of serial sections throughout the interatrial septum and the atrioventricular junction. Our data suggest that ganglion cells of sulcus terminalis as well as the epicardial ganglia enclosed between the superior vena cava and ascending aorta are VIP- and TH-negative, but neuropeptide Y- and DBH-immunoreactive. They give rise to three intraseptal nerves directed towards the specialised structures of the atrioventricular junction. These nerve fascicles contain abundant, thick TH-immunoreactive nerve fibres and scarce, thin NPY- and DBH-immunoreactive fibres. The cell bodies of the intramural ganglion cells localized between the right and left branches of the bundle of His (Moravec and Moravec 1984) are strongly TH- and DBH-immunoreactive. They are innervated by thick nerve fibres having the same immunohistochemical properties (NPY- and DBH-immunoreactivities) as those of a subpopulation of the epicardial ganglion cells and seem to supply some of the TH-immunoreactive nerve fibres directed via the intraseptal nerves to the epicardial ganglia. The existence of a multicomponent nerve network, characterized by a reciprocal innervation of the sinus node and atrioventricular node areas, is suggested by our immunohistochemical data.     

Innervation of lower airways and neuropeptide effects on bronchial and vascular tone in the pig

Summary The occurrence and distribution of peptide-containing nerve fibres [substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), neuropeptide Y (NPY)] and noradrenergic nerve fibres [tyrosine hydroxylase (TH)- and dopamine beta hydroxylase (DBH)-positive] in the airways of the pig were studied by means of immunohistochemistry. SP- and CGRP-immunoreactive (-IR) nerve fibres were present close to and within the lining respiratory epithelium, around blood vessels, within the tracheobronchial smooth muscle layer and around local tracheobronchial ganglion cells. The content of CGRP- and neurokinin A (NKA)-like immunoreactivity (-LI) measured by radioimmunoassay (RIA) was twice as high in the trachea compared to that in the peripheral bronchi. SP was a more potent constrictor agent than NKA on pig bronchi in vitro. CGRP had a relaxant effect on precontracted pig bronchi. On blood vessels CGRP exerted a relaxant effect that was more pronounced on pulmonary arteries than on bronchial arteries. VIP/PHI-IR fibres were seen in association with exocrine glands and in the tracheobronchial smooth muscle layer. VIP-positive nerve fibres were abundant around blood vessels in the trachea but sparse or absent around blood vessels in the peripheral bronchi. This histological finding was supported by RIA; it was shown that the content of peptides displaying VIP-like immunoreactivity (-LI) was 18 times higher in the trachea compared to peripheral bronchi. VIP was equally potent as CGRP in relaxing precontracted pig bronchi in vitro. Both bronchial and pulmonary arteries were relaxed by VIP. NPY was colocalized with VIP in tracheal periglandular nerve fibres and in nerve fibres within the tracheobronchial smooth muscle layer. NPY was also present in noradrenergic (DBH-positive) vascular nerve fibres. The content of NPY was much higher (15-fold) in the trachea compared to small bronchi. NPY caused a contraction of both pulmonary and bronchial arteries. The bronchial smooth muscle contraction to field stimulation in vitro was purely cholinergic. A non-cholinergic relaxatory effect following field stimulation was observed after bronchial precontraction. Capsaicin had no effect on pig bronchi in vitro.     

Neuronal subpopulations in autonomic ganglia associated with the chicken ureter: an immunohistochemical study

The neurochemical coding of neurones located in ganglia of the nerve trunk accompanying the chicken ureter was analysed and quantified using NADPH-diaphorase reactivity and immunohistochemistry against tyrosine hydroxylase (TH), nitric oxide synthase (NOS), calbindin (CAL), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and calcitonin gene-related peptide (CGRP) in untreated or colchicine-treated preparation. Almost all neurones were either positive for TH (38%) or for SOM (60%). Only 4% of the neurones were both TH- and SOM-positive and 3% of the neurones exhibited neither TH nor SOM immunoreactivity. The relative numbers of NPY-, NOS-, CAL- and VIP-positive neurones were 57%, 28%, 14% and 7%, respectively. No SP- or CGRP-positive neurones were observed. All NADPH-diaphorase-positive neurones expressed NOS immunoreactivity. Only in some TH-positive neurones was NPY and/or NOS found. Four major subpopulations were found in the ureteric ganglia. The SOM-positive neurones were subdivided into SOM/NPY/NOS- (28% of all neurones), SOM/NPY- (18%) and SOM/CAL/NPY-positive neurones (14%). A subpopulation of these peptid- ergic neurones also contained VIP. About 35% of the neurones contained TH only. Neurones of all subpopulations (72% of the neurones), except most of the CAL-positive neurones, were encircled by dense plexus of varicose SP/CGRP-positive, presumably sensory nerve fibres. Dense plexus of VIP-positive fibres were observed around 89% of the neurones. The chemical coding of the neuronal subpopulations identified in the ganglia accompanying the chicken ureter resembled that observed in the ganglia of Remak’s nerve but was remarkably different from that of the autonomic neurones described in mammalian species.     

Immunohistochemical localization of opioid peptides in the brain of the leech Theromyzon tessulatum

Summary By use of antisera directed against met-enkephalin, leu-enkephalin, dynorphin or -neoendorphin, immunoreactive structures were visualized in the central nervous system and proboscis of the leech Theromyzon tessulatum. Their distribution in the various compartments of the supra- and subesophageal ganglia was mapped. No correspondence could be established between the neurons containing met- or leu-enkephalin-like substances and the different types of neurosecretory cells classically described in Hirudinea. Successive localization of leu- and met-enkephalin on the same section revealed that these two peptides occur in different neurons. Only one cell located in compartment 6 of the supraesophageal ganglion was both dynorphin- and leu-enkephalin-positive. The other dynorphinimmunoreactive cells were not stained with the anti-leuenkephalin serum. The -neoendorphin-immunopositive cells were leu-enkephalin immunonegative and vice versa.     

Annual variations of the NPYergic innervation of the pineal gland in the European hamster (Cricetus cricetus): a quantitative immunohistochemical study

A prominent innervation of the pineal gland of the European hamster with nerve fibres containing neuropeptide Y (NPY) and tyrosine hydroxylase (TH) was demonstrated by means of immunohistochemistry. Nearly all the TH- and NPY-immunoreactive nerve fibres in the superficial pineal gland disappeared after bilateral superior cervical ganglionectomy, showing that the majority of NPY- and TH-immunoreactive nerve fibres belonged to the sympathetic nervous system. Since, in the European hamster, preliminary studies of the NPY-fibre density in the pineal gland had indicated seasonal changes, the density of NPY-immunoreactive nerve fibre profiles was ascertained in the superficial pineal gland in a series of animals between the first part of November and late April. The highest density of NPY-immunoreactive nerve fibre profiles was observed during midwinter. On the other hand, during the same period of the year, the number of sympathetic TH-immunoreactive sympathetic nerve fibre profiles did not exhibit seasonal variation, nor did substitution of testosterone, during the sexually inactive period, affect the density of NPY-containing nerve fibres in the gland. Our results show the presence of a testosterone-independent annual variation in the content of NPY in the sympathetic nerve fibres innervating the pineal gland of the European hamster. This variation can be correlated with the changes in the daily pattern of melatonin production observed by others in the same species at this period of the year.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Catecholamine-synthesizing enzymes and neuropeptides in rat heart epicardial ganglia; an immunohistochemical study

Summary The subepicardial atrial ganglia of rat hearts were examined using immunohistochemical techniques and antibodies against the catecholamine-synthetic enzymes tyrosine hydroxylase (TH) and dopamine--hydroxylase (DBH), and the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and met-5-enkephalin (ENK). Some of the ganglion cells present in the ganglia exhibited DBH-like immunoreactivity (LI) and NPY-LI, whilst these cells nerver exhibited TH-, VIP-, CGRP-, SP- or ENK-LI. Groups of small cells exhibiting an intense TH-LI, corresponding to cells referred to as catecholamine-containing cells and sometimes small intensely fluorescent cells in the literature, were observed in the ganglia. A subpopulation of these cells exhibited immunoreactivity to one of the neuropeptides tested, namely SP. Only a few of the cells showing TH-LI displayed DBH-LI. Nerve fibres showing SP-, CGRP-, DBH- and TH-LI were present in the ganglia; some of these fibres being closely associated with the ganglion cells or with the cells showing TH-LI. The observations provide new information on the catecholaminesynthetic enzyme/neuropeptide expression of the ganglion and catecholamine-containing cells and of the associated nerve fibres of rat heart subepicardial ganglia.     

A study of pre- and postganglionic fibres in the intestinal nerve (Remak's nerve) of the chicken

Summary The mean peak CV's of two electrophysiologically defined groups of fibres in the intestinal nerve of the chicken have been determined.One group of fibres is constituted by the processes of enteric cholinergic neurones which project along the side branches of the intestinal nerve and synapse within the nerve trunk. These preganglionic fibres have a mean peak CV (at 40 °C) of 0.31 m·s^–1.The other group is made up of fibres of postganglionic neurones which project orally along the nerve trunk. The results suggest that some postganglionic neurones project only as far as the next ganglion whilst others project beyond the next two ganglia for distances greater than 5 mm. The postganglionic fibres have a mean peak CV (at 40 °C) of 0.71 m·s^–1.These figures demonstrate that both pre- and postganglionic fibres are unmyelinated. The temperature coefficient (Q₁₀) for the CV of unmyelinated fibres in the intestinal nerve was 1.57.Abbreviations CAP compound action potential - CV conduction velocity - Q ₁₀ temperature coefficient     

Are opioid peptides co-localized with vasopressin or oxytocin in the neural lobe of the rat?

Summary The content of vasopressin, oxytocin, neurophysin, leucine-enkephalin, methionine-enkephalin, dynorphin-(1–13), and -neoendorphin in the rat neurohypophysis was measured after different periods of dehydration and after depolarisation of isolated neural lobes and of neurosecretory nerve endings. The rates at which the amount of neurohypophysial hormone and opioid peptides decreased, and the changes in the ratios between the amount of vasopressin or oxytocin and opioid peptide in the neurohypophysis after dehydration and in the incubation medium after depolarization in vitro cast some doubt on, and can be explained by mechanisms other than co-localisation of the different peptides.     

Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced during pregnancy in parallel with noradrenergic nerves

Summary Neuropeptide Y (NPY) is a recently discovered neuropeptide with vasoconstrictor effects when given in vivo. It occurs in many sympathetic neurons, where it appears to coexist with noradrenaline (NA). It is wellknown that profound changes in the levels of uterine NA occur in many species during pregnancy. Therefore we have investigated the distribution of catecholamine neurons and NPY by immunohistochemistry in the pregnant and nonpregnant guinea pig uterus. In the virgin uterus NPY-like immunoreactivity was present in nerve fibres and terminals in the smooth muscle layers of the uterine horns and around blood vessels. The distribution of NPY fibres was very similar to that of noradrenergic nerves visualized with antibodies against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). In the pregnant uterus, NPY- and TH-like immunoreactivity disappeared almost completely. In the cervix, a slight decrease of immunoreactivity was observed, whereas in the ovaries no changes were noted between the pregnant and nonpregnant condition. The results indicate that NPY and catecholamines coexists in the adrenergic neurons of the guinea pig uterus, cervix and ovary and that they vary together in the myometrium during pregnancy. We suggest that NPY may be of functional importance for the pregnant uterus.     

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

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

Rapid anterograde and retrograde tracing from mesenteric nerve trunks to the guinea-pig small intestine in vitro

A novel technique for rapid anterograde labelling of cut axons in vitro was used to visualise the peripheral branches of mesenteric nerve trunks supplying the guinea-pig small intestine. Biotinamide, dissolved in an artificial intracellular solution, was applied to the cut ends of the mesenteric nerves and the tissue was maintained in organ culture overnight. Labelled nerve fibres were visualised by fluorescein isothiocyanate (FITC)-conjugated streptavidin. Intense staining of nerve fibres and terminal varicosities in the ganglia and internodal strands of the myenteric plexus was achieved up to 15 mm from the application site. Filled fibres formed baskets around some myenteric nerve cell bodies, suggesting target-specific neurotransmission. When combined with multiple-labelling immunohistochemistry for tyrosine hydroxylase (TH), calcitonin gene-related protein (CGRP) or choline acetyltransferase (ChAT), most anterogradely labelled nerve fibres, and many pericellular baskets, were found to be TH immunoreactive, indicating their postganglionic sympathetic origin. Double-labelling immunohistochemistry revealed that the postganglionic sympathetic pericellular baskets preferentially surrounded 5-hydroxytryptamine (5-HT)-handling myenteric neurons. Some biotinamide-filled fibres were CGRP immunoreactive, and are likely to originate from spinal sensory neurons. We describe for the first time many pericellular baskets labelled from the mesenteric nerves which were ChAT immunoreactive. Retrogradely filled intestinofugal nerve cell bodies were also observed, all of which had a single axon arising from a small nerve cell body with short filamentous or lamellar dendrites. Many of these cells were ChAT immunoreactive. This in vitro technique is effective in identifying the fine arrangement of nerve terminals arising from nerve trunks in the periphery.     

Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced during pregnancy in parallel with noradrenergic nerves

Neuropeptide Y (NPY) is a recently discovered neuropeptide with vasoconstrictor effects when given in vivo. It occurs in many sympathetic neurons, where it appears to coexist with noradrenaline (NA). It is wellknown that profound changes in the levels of uterine NA occur in many species during pregnancy. Therefore we have investigated the distribution of catecholamine neurons and NPY by immunohistochemistry in the pregnant and nonpregnant guinea pig uterus. In the virgin uterus NPY-like immunoreactivity was present in nerve fibres and terminals in the smooth muscle layers of the uterine horns and around blood vessels. The distribution of NPY fibres was very similar to that of noradrenergic nerves visualized with antibodies against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). In the pregnant uterus, NPY- and TH-like immunoreactivity disappeared almost completely. In the cervix, a slight decrease of immunoreactivity was observed, whereas in the ovaries no changes were noted between the pregnant and nonpregnant condition. The results indicate that NPY and catecholamines coexists in the adrenergic neurons of the guinea pig uterus, cervix and ovary and that they vary together in the myometrium during pregnancy. We suggest that NPY may be of functional importance for the pregnant uterus.     

Distribution patterns and coexistence of neurohormonal peptides (ANP,BNP, NPY,SP, CGRP,enkephalins) in chromaffin cells and nerve fibers of the anuran adrenal organ

Summary Immunohistochemical techniques were used to study the adrenal organs of the anuran species Rana esculenta, Caldula pulchra and Bufo marinus with respect to the distribution and coexistence of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), Leu-enkephalin (Leu-ENK). Met-enkephalin-Arg-Phe (MEAP) and dynorphin A 1–17 (DYN). Antisera against enzymes involved in catecholamine synthesis, i.e., dopamine--hydroxylase (DBH) and tyrosine hydroxylase (TH), were used for the identification of chromaffin cells. ANP-immunoreactive (-IR) cells occurred in high densities (30%–70% of the total cell population) in all species investigated. In C. pulchra and B. marinus, BNP-IR cells constituted a population of non-DBH-IR and non-TH-IR cells that were different from the ANP-IR cells. A large proportion of the adrenal cells (10%–55%) were immunoreactive to Leu-ENK, and a minority (2%–5%) showed MEAP-immunoreactivity. DYN-immunoreactivity was not observed. The anurans studied exhibited small numbers of SP-IR, CGRP-IR and NPY-IR cells. Immunoreactivities for ANP+Leu-ENK and Leu-ENK+ MEAP were shown to coexist. In C. pulchra and B. marinus, immunoreactions for ANP+NPY, ANP+SP and SP+CGRP were also colocalized. Except for DYN, all neurohormonal peptides also occurred in intra-adrenal nerve fibers. SP-IR fibers also displayed CGRP-immunoreactivity and some Leu-ENK-IR fibers contained MEAP-immunoreactivity. In C. pulchra, NPY-IR fibers were found that also showed ANP-immunoreactivity.Some results of this investigation have been presented in abstract form (Reinecke et al. 1991).     

TH and NPY in sympathetic neurovascular cultures: role of LIF and NT-3

The sympathetic nervous system is an important determinant of vascular function. The effects of the sympathetic nervous system are mediated via release of neurotransmitters and neuropeptides from postganglionic sympathetic neurons. The present study tests the hypothesis that vascular smooth muscle cells (VSM) maintain adrenergic neurotransmitter/neuropeptide expression in the postganglionic sympathetic neurons that innervate them. The effects of rat aortic and tail artery VSM (AVSM and TAVSM, respectively) on neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were assessed in cultures of dissociated sympathetic neurons. AVSM decreased TH (39 +/- 12% of control) but did not affect NPY. TAVSM decreased TH (76 +/- 10% of control) but increased NPY (153 +/- 20% of control). VSM expressed leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3), which are known to modulate NPY and TH expression. Sympathetic neurons innervating blood vessels expressed LIF and NT-3 receptors. Inhibition of LIF inhibited the effect of AVSM on TH. Inhibition of neurotrophin-3 (NT-3) decreased TH and NPY in neurons grown in the presence of TAVSM. These data suggest that vascular-derived LIF decreases TH and vascular-derived NT-3 increases or maintains NPY and TH expression in postganglionic sympathetic neurons. NPY and TH in vascular sympathetic nerves are likely to modulate NPY and/or norepinephrine release from these nerves and are thus likely to affect blood flow and blood pressure. The present studies suggest a novel mechanism whereby VSM would modulate sympathetic control of vascular function.     

Studies on colocalization of neuropeptide Y,vasoactive intestinal polypeptide,catecholamine-synthesizing enzymes and acetylcholinesterase in the larynx of the rat

Summary In the present immunohistochemical study, the distribution of nerve fibers containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in the larynx was examined and compared with that of fibers containing tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (BDH), and with that of acetylcholinesterase (AChE)-positive nerve fibers, in intact and vagotomized rats and in rats subjected to removal of the superior cervical ganglion (SCG). Fibers showing TH/DBH-like immunoreactivity (LI) were only found in the walls of arteries and arterioles, whereas AChE-positive nerve fibers were located close to the acini and ducts of the glands, in blood vessel walls, in the perichondrium and in the lamina propria. NPY-LI and VIP-LI coexisted in local AChE-positive ganglionic cells and in a subpopulation of the AChE-positive fibers, NPY-LI also being present in some periarterial fibers showing TH/DBH-LI. Unilateral removal of the SCG eliminated the TH/DBH-innervation in the upper but not the lower parts of the larynx ipsilaterally, whereas the NPY-innervation of the arteries in the upper parts only partly disappeared and the NPY-innervation of the other structures remained unchanged. The distribution of VIP-innervation was unchanged after vagotomy and removal of the SCG. The results suggest that VIP is present in the postganglionic parasympathetic innervation, whereas NPY is present in both the postganglionic parasympathetic and sympathetic innervation of the rat larynx.     

Patterns of co-existence of peptides and differences of nerve fibre types associated with noradrenergic and non-noradrenergic (putative cholinergic) neurons in the major pelvic ganglion of the male rat

Summary The pelvic ganglia supply cholinergic and noradrenergic nerve pathways to many organs. Other possible transmitters are also present in these nerves, including peptides. Multiple labelling immunofluorescence techniques were used in this study of the male rat major pelvic ganglion (MPG) to examine: (1) the peptides present in noradrenergic (tyrosine hydroxylase (TH)-positive) and non-noradrenergic (putative cholinergic) neurons, and (2) the types of peptide-containing nerve fibres closely associated with these two groups of neurons. The distribution of the peptide galanin (GAL) within the MPG was also investigated. All of the TH-neurons contained neuropeptide Y (NPY), but none of the other tested peptides. However, many NPY neurons did not contain TH and may have been cholinergic. TH-negative neurons also displayed vasoactive intestinal peptide (VIP), enkephalin (ENK) or GAL. VIP and NPY formed the most common types of putative cholinergic pelvic neurons, but few cells contained both peptides. Many ENK neurons exhibited VIP, NPY or GAL. Varicose nerve terminals surrounding ganglion cells contained ENK, GAL, somatostatin (SOM) and cholecystokinin (CCK). These peptide-immunoreactive fibres were more often associated with the non-noradrenergic (putative cholinergic) than the noradrenergic neurons; two types (SOM and CCK) were preferentially associated with the non-noradrenergic NPY neurons. GAL was distributed throughout the MPG, in small neurons, scattered small, intensely fluorescent (SIF) cells, and both varicose and non-varicose nerve fibres. The nerve fibres were concentrated near the pelvic and penile nerves; most of the varicose fibres formed baskets surrounding individual GAL-negative somata.     

Distribution of Peptide-Containing Neurons in the Developing Rat Right Atrium,Studied Using Immunofluorescence and Confocal Laser Scanning

The developmental pattern and distribution of peptide-containing neurons in the rat heart right atrium has been studied by indirect immunofluorescence. Antibodies against neuropeptide Y (NPY), substance P (SP), and vasoactive intestinal polypeptide (VIP) were applied to whole-mount stretch preparations of the right atria from hearts of newborn to 40-day-old animals. NPY-like immunoreactivity (LI) was compared with the synaptic vesicle marker SV2 in double immunoincubation studies. The distribution of immunofluorescence was studied by confocal laser scanning microscopy. NPY-LI and SP-LI were present throughout the atria already at birth, in contrast to VIP-LI that was observed at day 10. The postnatal changes of innervation were basically quantitative, with an increase in density of nerve fibres and number of varicosities, while the basic pattern of innervation was essentially established during the first 1–10 days. NPY- and SP-positive bundles of fibres appeared to enter the right atrium along the superior caval vein, having extrinsic origins. Nerve fibres with NPY-LI colocalized in most nerve terminals with SV2-LI, and showed a developmental pattern similar to that observed for adrenergic neurons earlier. These NPY/SV2 positive fibres probably represent the extrinsic NPY innervation. In addition, NPY-LI was identified in large intrinsic nerve cells bodies located near the atrioventricular (AV) region. Most of the VIP-LI was observed in short nerve fibres originating in intrinsic VIP-positive cell bodies, but a few apparently extrinsic VIP-positive fibres were found, probably representing preganglionic parasympathetic neurons. SP in the atria was probably of extrinsic (sensory) origin and no nerve cell bodies with SP-LI were detected. The results show that the peptidergic innervation in the developing rat right atrium involves both extrinsic and intrinsic peptidergic neurons which may participate in the regulation of neurotransmission in local neuronal circuits.