Immunoreactive Nerve Research Articles

The presence of a galanin-like peptide in the gut neuroendocrine system of Lampetra fluviatilis and Acipenser transmontanus: an immunohistochemical study

Galanin is a brain-gut neuropeptide present in the central and peripheral nervous systems of vertebrates. In the present survey, the galaninergic and the diffuse endocrine systems of the alimentary canal of the river lamprey, Lampetra fluviatilis, and the white sturgeon, Acipenser transmontanus, were studied by immunohistochemistry. The results show the presence of galanin-like immunoreactive endocrine cells in the gut of L. fluviatilis. In addition, a galanin-like immunoreactivity was detected in enteric intramural neurons of both species. It is conceivable that the galaninergic system plays in both species a role in the regulation of the gut muscle contractility and in the modulation of mucosal secretive/absorptive processes. In A. transmontanus, the presence of galanin-like immunoreactive nerve fibres associated with components of the gut associated-lymphoid tissue is possibly correlated with a control of the defensive events at this site. The presence of a galanin-like immunoreactivity in the neuroendocrine system of these two ancient fishes confirms the hypothesis on the early occurrence of this regulative molecule in the gastro-enteric system of vertebrates.

Somatostatin in the brain of the cave salamander, Hydromantes genei (Amphibia, Plethodontidae): immunohistochemical localization and biochemical characterization.

The distribution of somatostatin-like immunoreactivity in the brain of the cave salamander Hydromantes genei (Amphibia, Plethodontidae) was investigated by using two distinct antisera raised against somatostatin-14. Most somatostatin-positive cells were detected in the ependymal cell layer surrounding the ventricles. These cells possessed the typical morphological characteristics of tanycytes or radial glial cells. Double-labeling with an antiserum against somatostatin and a monoclonal antibody against glial fibrillary acidic protein showed that somatostatin-immunoreactive cells lining the ventricles also exhibited GFAP-like immunoreactivity. Injection of the neurotracer biocytin into the lateral ventricle revealed that neurons lining the ventricles did not contain somatostatin-like immunoreactivity. In the telencephalon, somatostatin-like immunoreactivity was confined to radial glial cells. In the diencephalon, in addition to somatostatin-immunoreactive cells in the ependyma, positive cell bodies were also found in the periventricular preoptic nucleus, the infundibular nucleus, the epiphysis, and the subcommissural organ. In the metencephalon, positive cell bodies were found in the auricula cerebelli, whereas in the rhombencephalon numerous somatostatin-immunoreactive cells were seen lining the ventricular cavity. Immunoreactive nerve fibers were observed in the hypothalamus-median eminence complex. In the pituitary, a discrete group of somatostatin-positive cells was found in the pars distalis. High-performance liquid chromatography analysis of brain extracts revealed that the immunoreactive material coeluted with somatostatin-14. The present results show that the somatostatin peptidergic system in the brain of the cave salamander has a more simple organization than those described in the brain of frog and other vertebrates. This feature is probably related to the expression of high pedomorphic characters in plethodontids. The distribution of somatostatin-like immunoreactivity suggests that, in the cave salamander, somatostatin may act as a neurotransmitter and/or neuromodulator, a central regulator of fluid homeostasis, and a hypophysiotropic neurohormone.

Immunohistochemical analysis of the different neuropeptide containing nerve fibres and immunocompetent cells in the gastritis

Background aims: Changes of the density of different neuropeptide-containing nerve fibres might be important in the different diseases. Therefore our aim was to investigate the precise localization and distribution of the different immunoreactive (IR) nerve fibres in the mucosa of the stomach and compare with the data from the biopsy of patients with gastritis.

Down-regulated expression of agouti-related protein (AGRP) mRNA in the hypothalamic arcuate nucleus of hyperphagic and obese tub/tub mice

A mutation in the mouse tub gene causes a phenotype characterized by maturity-onset obesity, blindness and deafness. The role of the intact tubby protein and the pathogenesis resulting in the phenotype of tub/ tub mice remain largely unknown. In this study, we have investigated whether obese tub/ tub mice exhibit altered expression levels for agouti-related protein (AGRP) or glutamic acid decarboxylase-65 (GAD65) in body weight-regulating neurons of the hypothalamic arcuate nucleus. In situ hybridization revealed that AGRP, but not GAD65 mRNA levels, were significantly lower in obese tub/ tub mice as compared to tub/+ mice. The lower levels of AGRP mRNA in the arcuate nucleus of tub/ tub mice were paralleled by lower fluorescence intensity and numbers of AGRP- and neuropeptide Y (NPY)-immunoreactive (ir) nerve fibers and terminals in the arcuate, ventromedial, dorsomedial hypothalamic nuclei and perifornical and lateral hypothalamic areas. No obvious differences in GAD65-ir nerve fibers and terminals could be detected. Measurements of daily food intake revealed that tub/ tub mice displayed progressively higher food consumption as compared to lean tub/+ littermates over a 15-day observation period. When moved to an unfamiliar environment, e.g. a novel cage, daily food intake was initially lower in tub/ tub mice than in tub/+ mice suggesting that tub/ tub mice may be more sensitive to psychogenic stress. The results together show that tub/ tub mice are hyperphagic and exhibit, within the hypothalamic arcuate nucleus, a depressed expression of neuropeptides involved in the regulation of feeding behavior.

Morphological basis of sensory neuropathy and neuroimmunomodulation in minor salivary glands of patients with Sjögren’s syndrome

Objective: A predominance of sensory neuropathy was earlier described in Sjögren’s syndrome (SS), which might precede the presence of sicca symptoms. The mechanism of sensory neuropathy in SS is unknown. Therefore, the aim of this study was to determine the quantitative changes of the different neuropeptide containing nerve terminals and the immunocompetent cells in labial salivary glands of primary SS. Design: Immunohisto- and immunocytochemical methods were used for the detection of immunoreactive (IR) elements and the data were compared with the healthy controls. Results: All of the investigated IR nerve fibres were found in different quantity and localisation in both of control and SS glands. The density of them was changed variously in SS. The number of the substance P (SP), neuropeptide Y (NPY) ( P<0.05), galanin (GAL) IR nerve terminals was decreased, however, the number of vasoactive intestinal polypeptide (VIP) and tyrosine β-hydroxylase (TH) IR nerve fibres ( P<0.05) was increased compared to the control. There were no IR immunocompetent cells in the control materials, however, a large number of them showed IR for SP (46.2%) and NPY (34.4%) in the SS. The IR was demonstrated mainly in the mast cells, plasma cells and some of the lymphocytes. Conclusions: These neuropeptides might have a role in the sensory neuropathy; they might activate nociceptive and sympathetic pathways. Some neuropeptides (SP, NPY) are endogenous in the immunosystem and produced in certain conditions, e.g. inflammation and chronic autoimmune disorders such as SS, so they might participate in the neuroimmunomodulation and contribute to the atrophy, apoptosis and necrosis.

Immunohistochemical distribution of growth-associated protein 43 (GAP-43) in developing rat nasoincisor papilla.

We employed immunohistochemistry of growth-associated protein 43 (GAP-43) to trace the early development of gustatory nerves and alpha-gustducin to demonstrate mature taste buds in the rat nasoincisor papilla (NP). The sequential changes of gustatory structures revealed eight characteristic stages. One, at embryonic day 16 (E16), GAP-43-immunoreactive (IR) nerve fibers were observed in close relation with presumptive taste buds in the lateral apical epithelium on each side of NP; meanwhile, no immunoreactivity could be observed in the papillary epithelium. Two, at E17, fine GAP-43-IR nerve fibers first invaded the apical epithelium of the papilla. Three, at E19, GAP-43-IR nerve fibers were extensive in apical epithelium and colonized in immature taste buds. Four, at E20, GAP-43-IR nerve fibers were first observed in ductal epithelium (lining the medial wall of nasoincisor ducts). Five, at postnatal day 1 (P1), immunoreactive nerve fibers first coincided with immature taste buds in the ductal epithelium. Six, at P3, alpha-gustducin-IR cells identical for mature taste buds were simultaneously demonstrated in both apical and ductal epithelium. Seven, at P14, progressive taste bud proliferation and maturation as well as neural invasion were demonstrated in all regions of the epithelium. Eight, during advanced stage in adult animals, extensive innervation was traced especially in close relation with taste buds. The sequential topographic patterns of NP gustatory structures seem very specific as compared to those in other locations of mammalian gustatory system. The present study reveals that gustatory nerves preceded the development of taste buds. However, further investigations are required to examine such a characteristic model for the neurogenic theory of taste induction.

Abnormalities of somatic peptide-containing nerves supplying the pelvic floor of women with genitourinary prolapse and stress urinary incontinence

Objectives To test the hypothesis that genital prolapse may be related to peripheral nerve abnormalities, we examined the changes occurring to peptide-containing nerve processes supplying the periurethral muscles in women with stress urinary incontinence associated with prolapse. Methods Thirty patients with genital prolapse and 10 age-matched control subjects entered the study. All patients were evaluated by urodynamic investigations. Ten of 30 patients had pure stress urinary incontinence; none of the control subjects was incontinent. During surgery, four biopsy samples were obtained from each woman from the periurethral and perirectal muscles. The muscle sections were processed for immunohistochemistry using specific antibodies to glial (S-100 protein) and general neuronal markers (neuron-specific enolase) and neuropeptides, including neuropeptide Y, vasoactive intestinal polypeptide, and substance P. The evaluation of immunolabeled nerves was based on a semiquantitative analysis that allowed for a four-point ordinate scale score. Results S-100 and neuron-specific enolase immunoreactive nerve fibers, running either singly or in small bundles, along with a dense network of neural processes containing neuropeptide Y, vasoactive intestinal polypeptide, and substance P, were found throughout the connective tissue and striated muscle of the control specimens. In contrast, in the muscle specimens from those with genitourinary prolapse, both the density and the intensity of neuropeptide Y, vasoactive intestinal polypeptide, and substance P immunoreactive nerves were markedly reduced compared with the control specimens. Conclusions The evidence of a reduced peptide-containing nerve supply to the perineal muscles provides a morphologic basis suggesting that neural abnormalities contribute to the pathogenesis of genital prolapse and urinary incontinence.

Basiepidermal nervous system in Nemertoderma westbladi (Nemertodermatida): GYIRFamide immunoreactivity

The Nemertodermatida are a small group of microscopic marine worms. Recent molecular studies have demonstrated that they are likely to be the earliest extant bilaterian animals. What was the nervous system (NS) of a bilaterian ancestor like? In order to answer that question, the NS of Nemertoderma westbladi was investigated by means of indirect immunofluorescence technique and confocal scanning laser microscopy. The antibodies to a flatworm neuropeptide GYIRFamide were used in combination with anti-serotonin antibodies and phalloidin-TRITC staining. The immunostaining revealed an entirely basiepidermal NS. A ring lying outside the body wall musculature at the level of the statocyst forms the only centralisation, the “brain”. No stomatogastric NS has been observed. The GYIRFamide immunoreactive part of the “brain” is formed of loosely packed nerve fibres with multiple small neurones and a few large ones. The peptidergic and aminergic patterns of the NS do not correspond to each other: the former is more developed on the ventral side, the latter is more pronounced on the dorsal side. A pair of GYIRFamide immunoreactive nerve cords innervates the ventral side of the animal, the mouth and the male genital opening. The nemertodermatids studied to-date display no common NS pattern. Possible synapomorphies of the Acoelomorpha are discussed. The study demonstrates that the nemertodermatid NS possesses a number of plesiomorphic features and appears more primitive than the NS in other worms, except the Xenoturbellida. The bilaterian ancestor supposedly possessed only a basiepidermal nerve net and had no centralised brain-like structures and no stomatogastric NS.

Demonstration of an orexinergic central innervation of the pineal gland of the pig.

Orexins/hypocretins, two isoforms of the same prepropeptide, are widely distributed throughout the brain and are involved in several physiological and neuroendocrine regulatory patterns, mostly related to feeding, sleep, arousal, and cyclic sleep-wake behaviors. Orexin-A and orexin-B bind with different affinities to two G-protein-coupled transmembrane receptors, orexin-1 and orexin-2 receptors (OR-R1 and OR-R2, respectively). Because of the similarities between the human and the swine brain, we have studied the pig to investigate the orexinergic system in the diencephalon, with special emphasis on the neuroanatomical projections to the epithalamic region. By using antibodies against orexin-A and orexin-B, immunoreactive large multipolar perikarya were detected in the hypothalamic periventricular and perifornical areas at the light and electron microscopic levels. In the region of the paraventricular nucleus, the orexinergic neurons extended all the way to the lateral hypothalamic area. Immunoreactive nerve fibers, often endowed with large varicosities, were found throughout the hypothalamus and the epithalamus. Some periventricular immunoreactive nerve fibers entered the epithalamic region and continued into the pineal stalk and parenchyma to disperse among the pinealocytes. Immunoelectron microscopy confirmed the presence of orexinergic nerve fibers in the pig pineal gland. After extraction of total mRNA from the hypothalamus and pineal gland, we performed RT-PCR and nested PCR using primers specific for porcine orexin receptors. PCR products were sequenced, verifying the presence of both OR-R1 and OR-R2 in the tissues investigated. These findings, supported by previous studies on rodents, suggest a hypothalamic regulation of the pineal gland via central orexinergic nervous inputs.

Motor, sensory and autonomic nerve terminals containing NAP-22 immunoreactivity in the rat muscle

Neuron-enriched acidic protein having a molecular mass of 22 kDa, NAP-22, is a Ca 2+-dependent calmodulin-binding protein and is phosphorylated with protein kinase C (PKC). This protein is localized to the biological membrane via myristoylation and found in the membrane fraction of the brain and in the synaptic vesicle fraction. Recent studies showed that NAP-22 is localized in the membrane raft domain in a cholesterol-dependent manner and suggest a role for NAP-22 in maturation and/or maintenance of nerve terminals by controlling cholesterol-dependent membrane dynamics. The present study revealed the immunohistochemical distribution of NAP-22 in the peripheral nerves in rat muscles. In all examined muscles, nerve terminals in the motor endplates showed NAP-22 immunoreactivity associated with the membranes of synaptic vesicles and nerve terminals. In the muscle spindles, annulospiral endings, which made spirals around the intrafusal muscles, showed intense NAP-22 immunoreactivity. Autonomic nerve fibers around the intramuscular blood vessels also showed the immunoreactivity for NAP-22. NAP-22 immunoreactivity in these peripheral nerves was observed from birth to adulthood (100 days after birth). Though growth-associated protein-43 (GAP-43) immunoreactivity in these nerves was observed from birth, this immunoreactivity decreased from 20 days after birth. These findings suggest that NAP-22 is distributed and regulates functions in the motor, sensory and autonomic nerve terminals in the peripheral nervous system.

Distribution of calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) immunoreactive nerve fibers in the seminal vesicle and prostate of the male sheep

Double immunohistochemistry was used to determine the occurrence and distribution pattern of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) in seminal vesicles and prostate of the male sheep. Numerous CGRP- and SP-immunoreactive (IR) nerve fibres were found in the mucosal layer and smooth musculature of the seminal vesicles and prostate. In both glands nerve terminals immunoreactive to CGRP were more numerous than SP-IR ones. The majority of CGRP-IR nerve fibers showed colocalization of this peptide and SP. In both layers of the seminal vesicle and prostate, rare nerve terminals immunoreactive to GAL were also found. Immunoreactivity to SP was also found in all GAL-IR nerve fibers. The presence of numerous CGRP- and SP-IR nerve fibers in the seminal vesicle and prostate of the male sheep suggests that these neuropeptides may be involved in the sensory transmission and/or control of smooth muscle contractility. On the other hand, a relatively low number of GAL-IR nerve fibers of the seminal vesicle and prostate suggest that this peptide may act as an anti-nociceptive agent. It cannot be excluded that, in the seminal vesicle, GAL may also be involved in the control of the smooth muscle fiber activity. The possible role of CGRP, SP and GAL in the regulation of functions of the accessory sexual glands needs to be determined in further physiological studies.

Galanin- but not galanin-like peptide-containing axon terminals innervate hypophysiotropic TRH-synthesizing neurons in the hypothalamic paraventricular nucleus

Galanin and galanin-like peptide (GALP) are both orexigenic peptides involved in the regulation of food intake and energy metabolism. To determine whether these peptides may directly influence the hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons, double-labeling immunocytochemistry was performed at light and electron microscopic levels using antisera against proTRH, galanin and GALP. Galanin-IR axons densely innervated all of the major parvocellular subdivisions of the PVN where proTRH neurons were identified. The periventricular and anterior parvocellular subdivisions exhibited a prominent network of galaninergic nerve fibers, while the density of fibers was less intense in the medial parvocellular subdivision. Galanin-immunoreactive (IR) axon varicosities were juxtaposed to the majority of TRH-synthesizing neurons in the anterior, medial and periventricular subdivisions of the PVN. Ultrastucturally, galanin-IR nerve terminals established symmetric type synapses with the perikarya of proTRH-IR neurons, suggesting an inhibitory nature of these contacts. In contrast, GALP immunoreactive fibers and nerve terminals concentrated primarily in the anterior parvocellular subdivision of the PVN and were found in association with only few proTRH-IR neurons in the periventricular and medial parvocellular subdivisions. In conclusion, the dense innervation of TRH neurons in all subdivisions of the PVN by galanin-IR axons indicates that galanin may be involved in the central regulation of the hypothalamic–pituitary–thyroid axis. In contrast, the relative paucity of GALP-containing axons in juxtapsoition to TRH neurons in the medial and periventricular parvocellular subdivisions of the PVN, the origin of hypophysiotropic TRH neurons, makes it unlikely that GALP similarly exerts direct regulatory effects on hypophysiotropic TRH neurons.

Attenuation of knee joint inflammation by peripherally administered endomorphin-1.

Endomorphin-1 is a selective endogenous ligand for the micro-opioid receptor, and this study investigated the effect of endomorphin-1 on rat knee joint inflammation by examining the ability of the neuropeptide to modulate synovial protein extravasation. Acute joint inflammation was induced by intraarticular injection of 2% kaolin followed by 2% carrageenan and the animals allowed to recover for 3 h. Immunohistochemical examination of these inflamed joints revealed endomorphin-1-like immunoreactive nerves in deep synovium with a proportion of the nerve fibers occurring in close proximity to synovial blood vessels. Perfusion of inflamed knees with exogenous endomorphin-1 across the dose range 10-9-10-6 M produced a significant reduction in synovial vascular permeability with the 10-7M dose producing the greatest fall in protein exudation (approx 55%). These effects were blocked by the specific micro-opioid receptor antagonist CTOP. Destruction of knee joint unmyelinated afferent nerve fibers by capsaicin treatment significantly attenuated the anti-inflammatory effects of endomorphin-1, suggesting that the peptide is acting via a neurogenic mechanism. The findings of this study indicate that endomorphin-1 acts peripherally in knee joints to reduce synovial protein extravasation. These anti-inflammatory effects are mediated by micro-opioid receptors located on capsaicin-sensitive afferent nerves.

Development and distribution of mast cells and neuropeptides in human fetus duodenum.

To study the developmental regularities and heterogeneity of mast cells (MC) in human fetus duodenum and the distribution and developmental regularities of substance P(SP), calcitonin gene-related peptide (CGRP)-immunoreactive (IR) peptidergic nerves in fetus duodenum, as well as the relationship between MC, SP and CGRP- IR peptidergic nerves. Duodena from 21 cases of human fetus and one term infant were stained by hematoxylin-eosin (HE), toluidine blue (TB) and immunohistochemical avidin-biotinylated peroxidase complex (ABC) method. Lobe-shape intestinal villi in duodenum were already developed at the twelfth week. At the 21st wk, muscular mucosa appeared gradually, and four layers were observed in the wall of duodenum. TB staining showed that the granules in the immature MC were pale violet, while the mature MC were strong violet in color by TB staining. Connective tissue MC (CTMC) appeared occasionally in submucosa and muscular layer of duodenum at the 16th wk. While the mucosa MC (MMC) appeared at the 18th wk. At the 22nd wk, both CTMC and MMC were activated, and distributed in the surrounding blood vessels and ganglions. The verge of some MC were unclear, and showed degranular phenomena. At the 14th wk, SP and CGRP-IR nerve fibers and cells appeared in the myenteric and submucous plexuses in small intestine, and the responses were turn strongly. Neurons were light to deep brown, and nerve fibers were present as varicose and liner profiles. On the corresponding site of serial sections, SP and CGRP immunohistochemical reactions were coexisted in one nerve fiber or cell. Some of MC showed SP and CGRP-IR positive staining. There are two heterogeneous kinds of MC in duodenum, MMC and CTMC. MC might play an important role in regulating blood circulation and sensation.

Change of substance P containing neural pathway in rat model of irritable bowel syndrome

AIM: To investigate the expression of substance P (SP) and c-fos in the enteric nervous system (ENS) and central nervous system (CNS) of a rat model of constipation-pre- dominant irritable bowel syndrome (C-IBS), and to explore the possible role of SP-immunoreactive nerve fibers in IBS. METHODS: The rat model was established by intragastrical injection with ice-cold water (0-4 ). The expressions of SP and c-fos in the ENS and CNS of model group (n = 10) and control group (n = 10) were detected by immunohis- tochemistry, and analyzed semi-quantitatively by comput- erized color image analyzer. The statistical differences of the opacity density (OD) and immunoreactive areas (IA) between the two groups were examined by t-test. RESULTS: The expressions of SP in ileocecus, colonic myenteric plexus, spinal dorsal horn, hypothalamus, and the fore part of gyrus cinguli were significantly higher in model group than those in the corresponding regions of control group (OD: 176.6 vs 155.5, 172.3 vs 152.0, 182.1 vs 160.2, 128.3 vs 117.9, 127.9 vs 114.5, P <0.05; IA: 169318 vs 137655, 144728 vs 114403, 145117 vs 117612 except in ileocecus and colonic myenteric plexus, P <0.05). The expression of c-fos in the above tissues was significantly higher in model group than that in the corresponding regions in control group (OD: 120.9 vs 109.0, 101.3 vs 92.2, 125.4 vs 88.7, 115.5 vs 88.6, 120.6 vs 105.1, P<0.05; IA: 191 712 vs 154 363, 73 597 vs 41 179, 46 258 vs 33 238, 302 369 vs 204 563, 708 506 vs 409 856, P <0.05). There was a close correlation between the expression of SP and c-fos in the same region (r = 0.594-0.721, P <0.05). CONCLUSION: The abnormal SP immunoreactive nerve fibers may be involved in the pathogenesis of IBS. SP containing neural pathway may play an important role in the regulation of the gastrointestinal function.

Substance P immunoreactive nerve fibres in the domestic chick ankle joint before and after acute urate arthritis

Substance P immunoreactive (SP-IR) nerve fibres were identified using fluorescence immunohistochemistry and the length of the labelled individual nerve fibres was measured using confocal microscopy of whole mounts of the lateral wall of the ankle joint in 2-week-old domestic chicks. In the normal ankle there was an extensive network of SP-IR fibres in the synovial and subsynovial tissue of the joint capsule. Fours hours after injection of sodium urate into the joint space the joint was inflamed and there was a significant reduction in the labelled nerve fibres. These results demonstrate experimentally that painful gouty arthritis produces a neurogenic depletion of substance P from peripheral nerve fibres in the synovial and subsynovial tissue.

Evidence for vesicular glutamate transporter synapses onto gonadotropin‐releasing hormone and other neurons in the rat medial preoptic area

The medial preoptic area is a key structure in the control of reproduction. Several data suggest that excitatory amino acids are involved in the regulation of this function and the major site of this action is the medial preoptic region. Data concerning the neuromorphology of the glutamatergic innervation of the medial preoptic area are fragmentary. The present investigations were focused on: (i) the morphology of the vesicular glutamate transporter 1 (VGluT1)- and vesicular glutamate transporter 2 (VGluT2)-immunoreactive nerve terminals, which are considered to be specific to presumed glutamatergic neuronal elements, in the medial preoptic area of rat; and (ii) the relationship between these glutamate transporter-positive endings and the gonadotropin-releasing hormone (GnRH) neurons in the region. Single- and double-label immunocytochemistry was used at the light and electron microscopic level. There was a weak to moderate density of VGluT1- and a moderate to intense density of VGluT2-immunoreactive elements in the medial preoptic area. Electron microscopy revealed that both VGluT1- and VGluT2-immunoreactive boutons made asymmetric type synaptic contacts with unlabelled neurons. VGluT2-labelled, but not VGluT1-labelled, axon terminals established asymmetric synaptic contacts on GnRH-immunostained neurons, mainly on their dendrites. The present findings are the first electron microscopic examinations on the glutamatergic innervation of the rat medial preoptic area. They provide direct neuromorphological evidence for the existence of direct glutamatergic innervation of GnRH and other neurons in the rat medial preoptic area.

Genioglossal hypoglossal motoneurons contact substance P-like immunoreactive nerve terminals in the cat: a dual labeling electron microscopic study

This study investigated the synaptic interactions between hypoglossal motoneurons that project to the genioglossus muscle and substance P (SP) containing immunoreactive nerve terminals. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the right half of the genioglossus muscle in four anesthetized cats. Two days later, the animals were perfused with acrolein fixative. Tetramethylbenzidine (TMB) was the chromogen used to detect retrogradely labeled cells containing CTB-HRP. The tissues were then processed for immunocytochemistry using an antiserum raised against SP with diaminobenzidine (DAB) as the chromogen. At the light microscopic level, labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed at the level of the area postrema. At the electron microscopic level, SP-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled dendrites and perikarya. Nineteen percent of the terminals that contacted retrogradely labeled cells contained SP. These are the first ultrastructural studies demonstrating synaptic interactions between protruder hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, may be modulated by SP. Thus, SP may play a role in the control of protrusive movements of the tongue acting via neurokinin receptors.

Similar effects of cocaine and immobilization stress on the levels of heat-shock proteins and stress-activated protein kinases in the rat hippocampus, and on swimming behaviors: the contribution of dopamine and benzodiazepine receptors.

Cocaine (COC) has been reported to cause effects similar to physiological stressors in the brain neuroendocrinal system, including heat-shock protein (HSP) expression, although these effects have not been elucidated in detail. In the present study, we examined the effects of repeated (4 days) treatments with cocaine hydrochloride (35 mg/kg, i.p.) and 10 min immobilization stress (IM) on the distribution of HSP (HSP27, HSP60, HSP70, HSC70) and stress-activated protein kinase (SAPK) (SAPKalpha, SAPKbeta, SAPKgamma) immunoreactive nerve cells (positive cells) in the rat hippocampus. The swimming behaviors of the rats in the forced swimming test were also examined. In both COC and IM groups, an early enhancement (5 h time point) of hippocampal HSP (HSP27, HSP60, HSP70, HSC70) and SAPK (SAPKbeta, SAPKgamma) positive cells was observed, whereas a recovery (SAPKs) or attenuation (HSP60 and HSC70) was observed at the 24 h time point. In both groups, a depression of the swimming behaviors (attenuation in the activity counts and time until immobility) below the control level was observed at the 5 h point, but a recovery was observed at the 24 h time point. At the 48 h time point, all parameters returned to the control level. These alterations in the levels of HSPs and SAPKs, and the swimming behaviors were similar to those observed in the stress (IM) group, and were characteristic in that all of these alterations were attenuated by the benzodiazepine inverse agonist, Ro 15-4513 (5 mg/kg, i.p.), and the dopamine D1 receptor antagonist, SCH23390 (0.5 mg/kg, i.p.), which was not observed in the groups treated with another stressor-like drug (bicuculline).

Immunoelectron microscopic studies on protein gene product 9.5 and calcitonin gene-related peptide in vallate taste cells and related nerves in the guinea pig.

On the basis of our previous report that protein gene product 9.5 (PGP 9.5)-immunoreactive nerve fibers and taste cells and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers are found in guinea pig vallate papillae [Huang and Lu (1996b) Arch. Histol. Cytol. 59:433-441]. We speculated that PGP 9.5 might be a marker for taste receptor cells and that CGRP might play an important role in taste transmission. We, therefore, performed an immunohistochemical and ultrastructural analysis of taste cells and related nerves in guinea pig vallate papillae. In the connective tissue of the vallate papilla, the ultrastructural data revealed that the PGP 9.5-immunoreactive nerve fibers were both myelinated and unmyelinated. The CGRP-immunoreactive nerve fibers were unmyelinated and surrounded by the cytoplasm of Schwann cells as were the non-immunoreactive fibers. In the vallate taste buds, only type III cells, which make synaptic contacts with intragemmal nerves, were PGP 9.5-immunoreactive, while the nerve terminals making synaptic contact with the underlying type III cells were CGRP-immunoreactive. From these observations, we conclude that: (1) PGP 9.5 might be a useful specific marker for type III cells in guinea pig vallate taste buds; and (2) CGRP-containing nerve fibers might be primarily involved in the neural transmission of taste stimuli.