CGRP immunoreactivity and NADPH-diaphorase in afferent nerves of the rat penis

Calcitonin gene-related peptide (CGRP)-immunoreactive afferent nerve fibers are abundant in the rat penis. In addition, NADPH-diaphorase, which stains for nitric oxide synthase, has been localized within both autonomic and sensory dorsal root ganglia (DRG) and may be part of an important biochemical pathway involved in penile tumescence. The purpose of this study was: 1) to examine the circuitry of afferent nerves that are CGRP immunoreactive from the L6 DRG, 2) to examine the possibility that there are NADPH-diaphorase-positive afferent fibers from the L6 DRG to the rat penis, and 3) to examine the localization and colocalization of CGRP and NADPH-diaphorase within L6 DRG afferent perikarya. Calcitonin gene-related peptide immunostaining in the penis was eliminated following a bilateral transection of the pudendal nerves, but was unchanged following a bilateral transection of the pelvic splanchnic or hypogastric nerves. The NADPH-diaphorase staining was not altered by any of the nerve transections. Injection of the retrograde axonal tracer fluorogold (FG) into the dorsum penis labeled perikarya in the L6 DRG. Although the majority of FG-labeled perikarya contained neither CGRP nor NADPH-diaphorase, small subpopulations of perikarya contained either CGRP immunoreactivity, NADPH-diaphorase, or both. A unilateral pudendal nerve transection virtually eliminated (>99%) FG labeling in the ipsilateral L6 DRG. These data suggest that NADPH-diaphorase and CGRP are present, either together or separately, within a subpopulation of penile afferent perikarya. In addition, CGRP-immunoreactive afferent nerve fibers reach the penis primarily via the pudendal nerves. Finally, NADPH-diaphorase-positive penile afferents may be another important source of nitric oxide (NO) for penile tumescence.     

Nonvagal origin of galanin-containing nerve terminals innervating striated muscle fibers of the rat esophagus

We investigated the origin of galanin-positive nerve fibers on motor endplates in rat esophagus using anterograde 1,1′-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine methane sulfonate (DiI) tracing from the nucleus ambiguus combined with galanin immunocytochemistry and calcitonin gene-related peptide immunocytochemistry. To demonstrate spatial relationships of galanin-positive nerve fibers to vagal and enteric nerve fibers on motor endplates, we combined galanin immunocytochemistry with calcitonin gene-related peptide immunostaining for labeling of vagal terminals, and vasoactive intestinal peptide immunoreactivity and NADPH-diaphorase histochemistry for demonstration of enteric nerve fibers. Within fine varicose nerve fibers, galanin was colocalized with vasoactive intestinal peptide and NADPH-diaphorase to a high degree and turned out to be completely separated from calcitonin gene-related peptide-positive or anterogradely DiI-labeled vagal motor terminals. These results indicate that the enteric nervous system is the most important and possibly the only source of galanin-positive nerve terminals on motor endplates in rat esophagus. Galanin may be, in addition to nitric oxide and vasoactive intestinal peptide, a mediator of the enteric coinnervation of striated muscle in this organ.     

Distribution of NADPH-diaphorase activity in the pineal gland of the Turkey

NADPH-diaphorase activity was histochemically demonstrated in the nerve fibers, neuronal-like cell bodies and in the endothelial cells of the vasculature in the pineal gland of the turkey. The nerve fibers were localized in the choroid plexus, connecting the pineal gland with the diencephalon as well as inside the pineal gland, where they formed basket-like structures around the pineal follicles. A group of neuronal-like cell bodies was observed in the proximal part of the gland. The positive staining was not observed in the pinealocytes of rudimentary-photoreceptor type and in the supporting cells.     

NADPH-diaphorase-positive nerve fibers associated with motor endplates in the rat esophagus: new evidence for co-innervation of striated muscle by enteric neurons

NADPH-diaphorase histochemistry was combined with demonstration of acetylcholinesterase and immunocytochemistry for calcitonin gene-related peptide to study esophageal innervation in the rat. Most of the myenteric neurons stained positively for NADPH-diaphorase, as did numerous varicose nerve fibers in the myenteric plexus, among striated muscle fibers, around arterial blood vessels, and in the muscularis mucosae. A majority of motor endplates (as demonstrated by acetylcholinesterase histochemistry or calcitonin gene-related peptide immunocytochemistry) were associated with fine varicose NADPH-diaphorase-positive nerve fibers. Analysis of brainstem nuclei, sensory vagal, spinal, and sympathetic ganglia in normal and neonatally capsaicin-treated rats, and comparison with anterogradely labeled vagal branchiomotor, preganglionic and sensory fibers led to the conclusion that NADPH-diaphorase-positive fibers on motor endplates originate in esophageal myenteric neurons. No association of NADPH-diaphorasepositive nerve fibers with motor endplates was found in other organs containing striated muscle. These results suggest extensive, presumably nitrergic, co-innervation of esophageal striated muscle fibers by enteric neurons. Thus, control of peristalsis in the esophagus of the rat may be more complex than hitherto assumed.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat,guinea pig and pig female genital organs

Summary The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig. CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig ang pig, CGRP-and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP-and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct. CGRP-and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP-and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat, guinea pig and pig female genital organs

The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig, CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig and pig, CGRP- and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP- and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct, CGRP- and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP- and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Immunohistochemical localization of substance P in the pineal gland of the domestic pig

An immunohistochemical study of the pig pineal gland was carried out using polyclonal rabbit antiserum raised against substance P (SP). The pineal glands were taken from the newborn, 21-day- and 7-month-old female pigs. Immunoreactive nerve fibers were observed in the pineal gland as well as in the posterior commissure and habenular areas. The bundles of SP-immunoreactive fibers were also seen in the subependymal layer of the pineal tissue. The single SP-immunoreactive nerve fibers and few small bundles of nerve fibers were located with equal density throughout the pineal gland, in the connective tissue septa and in the parenchyma. SP-immunoreactive cell bodies were observed in the medial habenular nucleus. The obtained results point to this nucleus as one of the central sources of SP innervation in the pig pineal gland. The study did not show any differences in the distribution and the density of SP-immunoreactive nerve fibers between newborn, 21-day- and 7 month-old pigs.     

河北环毛蚓神经系统 一氧化氮合酶的组织化学定位

用依赖还原型辅酶Ⅱ的黄酶组织化学方法,研究了环节动物门寡毛纲种类河北环毛蚓（Pheretima tschiliensis)神经系统k 一氧化氮合酶(NOS)阳性细胞及阳性纤维的分布,结果表明,河北环毛蚓神经系统中脑神经节背侧有大量细胞呈现NO强阳性反应,胞体和突起染色明显。咽下神经中偶尔能见少数染色较浅的神经元。在脑神经节腹内侧、围咽神经、 咽下神经节外侧部及腹神经链中都有一氧化氮合酶阳性纤维存在脸染色很深,实验结果表明,在环节动物中作为信息分子的一氧化氮已广泛存在于神经系统中。     

Changes in cholinergic and noradrenergic nerves in the pregnant and postpartum uterus of the albino rat and guinea pig

The present study was undertaken to investigate the structural changes in both cholinesterase (ChE)-positive nerve fibers and adrenergic nerves with formaldehyde-induced fluorescence in pregnant and postpartum uteri of both the albino rat and guinea pig. Particular attention was directed to the relationship between these changes and the local factors associated with the growing fetus. ChE reaction was absent in the control and pregnant uterus of the guinea pig. In the albino rat, there were signs of degeneration in pregnancy. These were evidenced by vacuolation of large nerve trunks and the presence of focal segments with very faint reaction along the course of the nerve bundles. Myometrial segments from fetus-containing horns showed some fragmented nerve fibers, but at the same time some other normal ones. Most of the fine nerve bundles gave a weak reaction. Three weeks after delivery, multiple ChE fibers were found in the uterus of the albino rat. The normal appearance was, however, not regained and some nerve fibers were still fragmented. Noradrenergic (NA) nerve fibers were disintegrated and markedly reduced in number in the myometrium of the pregnant uterus of both the guinea pig and albino rat, particularly in the uterine horns that were distended by fetuses. The number of NA fibers was not significantly reduced in the tubal ends of the albino rat uterus. Three weeks after delivery, normal NA fibers were seen in the myometrium of both the albino rat and guinea pig uterus. Nerves with reduced fluorescence reaction were observed less frequently.     

Nitroxidergic elements in the digestive system of Mactra chinensis and Spisula sachalinensis (Mollusca: Bivalvia: Mactridae)

Localization and morphology of NO-ergic elements in the digestive system of bivalve molluscs Mactra chinensis and Spisula sachalinensis were studied using histochemical technique [1] for detection of NADPH-diaphorase (EC 1.6.99.1) [1]. The NO-producing elements were revealed in all parts of the digestive system of the studied animals. NADPH-diaphorase was found in cells of several morphological types as well as in nerve plexuses. The most abundant in the digestive tract parts of the studied molluscs were intraepithelial nerve cells of the “open” type, whose thin apical process is directed towards the gut lumen. Subepithelial NO-ergic neurons were revealed in stomach and crystalline style sac of Mactra chinensis. Besides, diformazan granules are present in brush-border epitheliocytes of the major and secondary ducts of the digestive gland as well as in cells of digestive tubules of this gland. All studied parts were found to contain basiepithelial and subepithelial NO-ergic nerve fibers forming networks of various densities from, most commonly, loose to dense plexuses particularly developed in labia, esophagus, and gut of the studied molluscs.     

Leu-enkephalin immunoreactivity in the pig pineal gland

Leu-enkephalin-positive structures in the pig pineal gland were demonstrated immunohistochemically using mouse monoclonal antibody. The pineal glands were obtained from the newborn, 21-day and 7-month old female pig. The immunopositive nerve fibers were observed in the pineal gland as well as in the epithalamic areas. The leu-enkephalin-immunoreactive nerve fibers (single or forming small bundles) were localized mainly in the proximal part of the pineal and they were scarce in other parts. The localization of the fibers points to a central source of this innervation. The study did not show any age-dependent differences in the distribution and density of leu-ekephalin-positive nerve fibers.     

Distribution of peptidergic nerves in the choroid plexus, focusing on coexistence of neuropeptide Y, vasoactive intestinal polypeptide and peptide histidine isoleucine

Choroid plexus from rat, guinea-pig, rabbit and pig was investigated by light-microscopic immunohistochemistry and by radioimmunoassay for the presence of neuropeptides. A moderately dense supply of nerve fibers containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), respectively, was found around blood vessels and in close relation to the secretory epithelium in both pig and rabbit, while lower densities of nerve fibers were found in rat and guinea-pig. Peptide concentrations ranged from 10-40 pmolequivalents/g (pmoleqv/g) for NPY and 0.5-6 pmoleqv/g for VIP in all four species. Peptide histidine isoleucine (PHI) immunoreactive nerve fibers were present in pig choroid plexus at a lower density than NPY and VIP but with a similar distribution. Low concentrations of substance P (0.3-3 pmoleqv/g) and calcitonin gene-related peptide (0.1-3 pmoleqv/g) were found to a varying degree in choroid plexus tissue from the different species, while immunohistochemical investigation was unable to detect any immunoreactive nerve fibers. NPY was often found to coexist with VIP and PHI in pig choroid plexus, while a lesser amount of nerve fibers showed coexistence of NPY and the noradrenaline synthetizing enzyme, dopamine-beta-hydroxylase. Surgical sympathetic denervation by excision of the superior cervical ganglion in the rabbit abolished NPY-containing nerve fibers, as revealed by immunohistochemistry, but only decreased NPY levels by one third, which may be due to different identity of the peptide being detected by the two techniques. It is concluded that NPY-containing nerve fibers have a dual origin in the choroid plexus and coexist with either noradrenaline or VIP/PHI.     

Demonstration of nerve fibers immunoreactive to peptide N-terminal histidine C-terminal isoleucine (PHI) and vasoactive intestinal peptide (VIP) in the pig pineal gland

The pineal functions are modulated by some neuropeptides including PHI and VIP. The presence of PHI-immunoreactive and VIP-immunoreactive nerve fibers in the pineal gland has been shown in several mammalian species. Both peptides influence the pineal serotonin N-acetyltransferase activity and melatonin synthesis. The aim of the present study was to examine the localization of PHI- and VIP-immunoreactive nerve fibers in the pig pineal gland. Four three-month old female pigs housed in natural light conditions, with free access to food and water, were used in the study. The pineals were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. An immunohistochemical ABC streptavidin-biotin-complex method was used for the demonstration of PHI and VIP. PHI- and VIP-immunopositive nerve fibers were found in the pineal gland as well as in the habenular and posterior commissural areas. In the pineal gland, the density of PHI-immunoreactive nerve fibers was considerably higher than that of the fibers containing VIP. PHI- and VIP-immunopositive nerve fibers were more abundant in the cortical than in the medullary part of the gland. The nerve fibers formed bundles in the pineal capsule, from where they penetrated to the connective tissue septa and formed a dense meshwork surrounding blood vessels. In the parenchyma, PHI- and VIP-immunoreactive nerve terminals created baskets around clusters of pinealocytes. No PHI- or VIP-immunopositive cells were found in the pig pineal gland.     

Distribution, origin and sensitivity to capsaicin of primary afferent substance P-immunoreactive nerves in the heart

This report is intended as an overview of the distribution, origin and sensitivity to capsaicin of substance P-immunoreactive (SP-I) primary afferent cardiac nerves. Immunohistochemical and physiological methods were employed to compare the presence and density of these nerve fibers in the guinea pig and rat hearts. SP-I fibers are numerous in the guinea pig heart including the parietal pericardium, atria, ventricles, valves, coronary arteries and around intrinsic cardiac ganglion cells. The rat heart contains few SP-I fibers. Vagotomy does not influence the number of intensity of immunoreactive fibers in the guinea pig heart. By stimulating the atrium or ventricle and recording from the second or third thoracic dorsal roots Ad1, Ad2 and C fibers were demonstrated in the atria, but only Ad fibers in the guinea pig ventricle; in addition, only Ad fibers were recorded from the vagus nerves. Only Ad1 fibers were demonstrated in the rat heart. Treatment with capsaicin depletes the SP-I and decreases the conduction velocity of C-fibers and some Ad2 fibers in the guinea pig heart. We suggest that SP-I primary afferent nerve fibers are unmyelinated (C-type) or small myelinated (Ad2-type) nerves in the guinea pig heart and that their cell bodies of origin are predominantly in dorsal root ganglia.     

Role of extrinsic innervation in modulating nitrergic transmission in the canine ileocolonic region

The human colon can dilate, often to life-threatening proportions. Our aim was to explore nitrergic mechanisms underlying colonic dilation in conscious dogs with enterically isolated ileocolonic loops either extrinsically innervated (n = 4) or extrinsically denervated (n = 4). We recorded phasic pressures in ileum and ileocolonic sphincter (ICS), colonic tone, compliance, and relaxation during ileal distension. By NADPH-diaphorase histochemistry, we assessed effects of extrinsic denervation and enteric isolation on nitrergic fibers. Extrinsic denervation increased phasic pressures in ileum, ICS, and colon and abolished ICS and colonic relaxation in response to ileal distension. The nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NNA) increased phasic pressures at all sites and ICS tone but did not abolish colonic relaxation during ileal distension in innervated loops. L-NNA reduced compliance and induced colonic high-amplitude propagated contractions in denervated loops. The NOS substrate donor L-arginine reversed effects of L-NNA. The number of NADPH-diaphorase fibers increased in both enterically isolated preparations. Nonnitrergic extrinsic nerve pathways mediate reflex colonic relaxation during ileal distension. Enteric isolation augments the number of NOS fibers, an effect not modified by extrinsic denervation.     

Vasopressinergic innervation of the pig pineal gland

An immunohistochemical study of the pineal gland of the domestic pig was carried out using the antisera raised against vasopressin (VP). The pineal glands were taken from the newborn, 21-day- and 7-month old female pigs. The pig pineal gland is moderately innervated by VP-immunoreactive nerve fibers. They run from the habenular commissure into the connective tissue septa and further into the pineal parenchyma. In the subependymal tissue as well as in the connective tissue septa, the fibers are smooth or with small varicosities and in the parenchyma with large ones. The obtained results point to extrapineal and extraepithalamic source of the fibers. The density of VP-immunoreactive fibers in the pineal gland of 7-month old pigs is higher than in the younger animals.     

Pituitary adenylate cyclase-activating polypeptide: localization and differential influence on isolated hearts from rats and guinea pigs

This study was done to determine if pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive nerve fibers occur in cardiac muscle as well as intracardiac ganglia of rats and guinea pigs and to clarify the chronotropic actions of PACAP27 in the same species using isolated heart preparations. PACAP nerve fibers were not detected in atrial or ventricular muscle of rat or guinea pig but a few stained nerve fibers occurred in the atrioventricular bundle of the guinea pig. Stained nerve fibers were prominent in intracardiac ganglia of both species. PACAP27 caused a dose-dependent tachycardia in isolated rat hearts (+39 +/- 3 beats/min with 1 nmol, n = 6). Positive and/or negative chronotropic responses were evoked by PACAP27 in guinea pig heart, depending on dose and prior exposure to the peptide. PACAP27 also caused arrhythmias in several guinea pig hearts. Treatment with atropine eliminated or prevented PACAP-evoked bradycardia and arrhythmias, implicating cholinergic neurons in these responses. Positive chronotropic responses to PACAP were unaffected by beta-adrenergic receptor blockade in either species, suggesting that tachycardia resulted from a direct action on the heart. These observations support the conclusion that endogenous PACAP could have a role in regulating parasympathetic input to the heart but through different mechanisms in rats versus guinea pigs. A direct positive chronotropic influence of endogenous PACAP is unlikely since atrial muscle lacks PACAP-immunoreactive nerve fibers.     

甘肃鼢鼠胃肠道肌间神经丛一氧化氮合酶阳性神经元的分布

用NADPH-黄递酶组织化学法及整装铺片技术,对甘肃鼢鼠(Myospalax cansus)胃肠道肌间神经丛NOS阳性神经元的分布进行研究.结果显示,甘肃鼢鼠胃肠道肌间神经丛NOS阳性神经元分布广泛,形态多样,神经元大小不同,阳性神经节与阳性神经纤维束形成网络;胃肠道不同部位NOS阳性神经元密度有差异,结肠最高,直肠次之.从十二指肠至回肠段,NOS阳性神经元密度整体呈上升趋势;胃与空肠、十二指肠与盲肠间NOS阳性神经元密度无显著差异,其他各段之间差异显著.甘肃鼢鼠胃肠道肌间神经丛NOS阳性神经元分布与其他已研究动物的分布模式基本一致.     

Neuropeptides in guinea pig trachea: Distribution and evidence for the release of CGRP into tracheal lumen

The airways of the guinea pig are richly innervated by peptide-containing nerve fibers. Among the most abundant neuropeptides are calcitonin gene-related peptide (CGRP) and substance P (SP), which are stored in nerve fibers located predominantly within and beneath the epithelium, and vasoactive intestinal peptide (VIP), which is located in fibers running mainly among smooth muscle bundles and seromucous glands. Sensory denervation (capsaicin treatment) of adult guinea pigs caused an almost total disappearance of CGRP- and SP-containing nerve fibers, while the density of VIP-containing nerve fibers located in smooth muscle seemed to increase. In the isolated trachea, perfused luminally, CGRP was found to appear in the intraluminal fluid after exposure to capsaicin but not after electrical vagal stimulation. CGRP concentrations in the tracheal wall did not change significantly. Luminally applied CGRP did not affect smooth muscle tension, measured as intraluminal volume changes.