Neuropeptide Tyrosine Research Articles

Deletion of tyrosine hydroxylase gene reveals functional interdependence of adrenocortical and chromaffin cell system in vivo.

Catecholamines are produced in the medulla of the adrenal gland and may participate in the intraglandular regulation of its cortex. We analyzed the adrenal structure and function of albino tyrosine hydroxylase-null (TH-null) mice that are deficient in adrenal catecholamine production. Adrenal catecholamines were markedly reduced, and catecholamine histofluorescence was abrogated in 15-day-old TH-null mice. Chromaffin cell structure was strikingly altered at the ultrastructural level with a depletion of chromaffin vesicles and an increase in rough endoplasmic reticulum compared with wild-type mice. Remaining chromaffin vesicles lined up proximally to the cell membrane in preparation for exocytosis providing a "string-of-pearls" appearance. There was a 5-fold increase in the expression of proenkephalin mRNA (502.8 +/- 142% vs. 100 +/- 17.5%, P = 0.016) and a 2-fold increase in the expression of neuropeptide Y (213.4 +/- 41.2% vs. 100 +/- 59.9%, P = 0.014) in the TH-null animals as determined by quantitative TaqMan (Perkin-Elmer) PCR. Accordingly, immunofluorescence for met-enkephalin and neuropeptide tyrosine in these animals was strongly enhanced. The expression of phenylethanolamine N-methyl transferase and chromogranin B mRNA was similar in TH-null and wild-type mice. In TH-null mice, adrenocortical cells were characterized by an increase in liposomes and by tubular mitochondria with reduced internal membranes, suggesting a hypofunctional state of these steroid-producing cells. In accordance with these findings, plasma corticosterone levels were decreased. Plasma ACTH levels were not significantly different in TH-null mice. In conclusion, both the adrenomedullary and adrenocortical systems demonstrate structural and functional changes in catecholamine-deficient TH-null mice, underscoring the great importance of the functional interdependence of these systems in vivo.

Long-term repetitive transcranial magnetic stimulation increases the expression of brain-derived neurotrophic factor and cholecystokinin mRNA, but not neuropeptide tyrosine mRNA in specific areas of rat brain.

Repetitive transcranial magnetic stimulation (rTMS) is increasingly used as a therapeutic tool in various neurological and psychiatric disorders, and we recently found that it has a neuroprotective effect both in vitro and in vivo. However, the neurochemical mechanisms underlying the therapeutic effects are still unknown. We investigated the effects of long-term rTMS on the expression of brain-derived neurotrophic factor (BDNF), cholecystokinin (CCK), and neuropeptide tyrosine (NPY) mRNA in rat brain. In situ hybridization revealed a significant increase in BDNF mRNA in the hippocampal areas CA3 and CA3c, the granule cell layer, as well as in the parietal and the piriform cortex after rTMS. BDNF-like immunoreactivity was markedly increased in the same areas. A significant increase in CCK mRNA was observed in all brain regions examined. NPY mRNA expression, in contrast, was not altered. The present results suggest that BDNF may contribute to the neuroprotective effects of rTMS. Furthermore, the rTMS-induced changes in BDNF and CCK expression are similar to those reported after antidepressant drug treatment and electroconvulsive seizures, suggesting that a common molecular mechanism may underlie different antidepressant treatment strategies.

Neurochemical and cellular markers in human cervix of late pregnant, postpartal and non-pregnant women

The aim was to evaluate the peptidergic innervation and the dendritic cell content in the cervix uteri. Cervical biopsies were obtained from late pregnant (n=5), postpartal (n=5) and non-pregnant (n=5) women. The samples were prepared for immunohistochemistry using antibodies to protein S-100 (S-100), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), human peptide histidine isoleucine amide (PHM 27), neuropeptide tyrosine (NPY), and human histocompatibility complex class II subregion DR (HLA-DR). Nerve fibers positive for protein S-100, and dendritic cells positive for S-100 and HLA-DR were abundant in the cervix, especially at late pregnancy. CGRP, VIP, PHM-27 and NPY positive nerve fibers were present in non-pregnant, short nerve fibers and scattered immunoreactivity at term, and further scattered immunoreactivity after parturition. NPY positive nerve fibers were decreased at term, and after parturition a scattered immunoreactivity was observed. The abundant protein S-100 positive nerve fibers implies an impact of myelinated nerves in the cervix uteri during pregnancy. The abundant dendritic cells, positive for HLA-DR and S-100, especially at term, indicates a general activation of the immune system until late pregnancy and parturition. The changed occurrence and distribution of immunoreactivity for CGRP, VIP and PHM-27 suggest a release of these neuropeptides until term. The changes in NPY immunoreactivity indicate a release of NPY around parturition.

Glibenclamide but not other sulphonylureas stimulates release of neuropeptide Y from perifused rat islets and hamster insulinoma cells.

We have studied the effects of first and second generation sulphonylureas on the release of insulin and neuropeptide tyrosine (NPY) from hamster insulinoma tumour (HIT T15) cells and isolated rat islets. In the presence of 5.5 mmol/l glucose all sulphonylureas stimulated insulin release from the HIT cells (P<0.01 ANOVA, n> or =4) but only glibenclamide (GLIB, 10 micromol/l) stimulated the release of NPY (mean+/-s.e.m. control 11.1+/-1.3 vs GLIB 28.4+/-4.1 fmol/h per 10(6) cells, P<0001, n=16). In isolated perifused rat islets both glibenclamide (10 micromol/l) (control 3.5+/-0.3 vs GLIB 6. 3+/-0.2 fmol/min per islet, P<0.01, n=6) and tolbutamide (50 micromol/l) (control 4.7+/-0.1 vs TOLB 6.7+/-0.3 fmol/min per islet, P<0.01, n=6) enhanced glucose (8 mmol/l)-stimulated insulin release. However, only glibenclamide stimulated the release of NPY from the islets (control 3.4+/-0.8 vs GLIB 24.5+/-5 attomol/min per islet, P<0.01, n=6). Similar results were obtained in islets isolated from dexamethasonetreated rats. Glibenclamide treatment of HIT cells showed a prompt insulin release (10 min) while NPY secretion was slower (60 min), suggesting that internalization of the sulphonylurea is required to stimulate NPY release. Glibenclamide, the most common oral therapeutic agent in type 2 diabetes mellitus, is associated with release of the autocrine insulin secretion inhibitor, NPY.

Association of neuropeptide Y Y1 receptors with glutamate-positive and NPY-positive neurons in rat hippocampal cultures.

The hippocampus is particularly enriched with neuropeptide tyrosine (NPY) and NPY receptors including the Y1, Y2 and Y5 subtypes. We have previously reported on the enrichment of cultured rat hippocampal neurons in specific [125I][Leu31, Pro34]PYY/BIBP3226-sensitive (Y1) binding sites and Y1 receptor mRNAs [St-Pierre et al. (1998) Br. J. Pharmacol., 123, p183]. We have now identified which cell types express the Y1 receptor. The majority of Y1 receptors, visualized using either the radiolabeled probe [125I][Leu31,Pro34]PYY or two antibodies directed against distinct domains of the Y1 receptor, was expressed in neurons as revealed by neuron-specific enolase (NSE) immunostaining. One antibody was directed against the second extracelllular loop of the Y1 receptor (amino acids 185-203) whereas the second was directed against the intracellular C-terminal loop (amino acids 355-382). The labelling was evident over both perikarya and processes. Neurons labelled by the various Y1 receptor probes were mostly glutamate-positive as revealed by double immunostaining. Most interestingly, a number of NPY-positive cultured hippocampal neurons were also enriched with the Y1 receptor, suggesting that this subtype may act as an autoreceptor to regulate NPY release in the hippocampus. These results thus provide an anatomical basis for the modulation of glutamate and NPY release by the Y1 receptor in the hippocampus.

Distribution of GAD-immunoreactive neurons in the diencephalon of the African lungfishProtopterus annectens: Colocalization of GAD and NPY in the preoptic area

The distribution of GABAergic neurons was investigated in the diencephalon of the African lungfish, Protopterus annectens, by using specific antibodies directed against glutamic acid decarboxylase (GAD). A dense population of immunoreactive perikarya was observed in the periventricular preoptic nucleus, whereas the caudal hypothalamus and the dorsal thalamus contained only scattered positive cell bodies. Clusters of GAD-positive cells were found in the intermediate lobe of the pituitary. The diencephalon was richly innervated by GAD-immunoreactive fibers that were particularly abundant in the hypothalamus. In the periventricular nucleus, GAD-positive fibers exhibited a radial orientation, and a few neurons extended processes toward the third ventricle. More caudally, a dense bundle of GAD-immunoreactive fibers coursing along the ventral wall of the hypothalamus terminated into the median eminence and the neural lobe of the pituitary. Double-labeling immunocytochemistry revealed that GAD and neuropeptide tyrosine (NPY)-like immunoreactivity was colocalized in a subpopulation of perikarya in the periventricular preoptic nucleus. The proportion of neurons that coexpressed GAD and NPY was higher in the caudal region of the preoptic nucleus. The distribution of GAD-immunoreactive elements in the diencephalon and pituitary of the African lungfish indicates that GABA may act as a hypophysiotropic neurohormone in Dipnoans. The coexistence of GAD and NPY in a subset of neurons of the periventricular preoptic nucleus suggests that GABA and NPY may interact at the synaptic level.

Identification of the Pancreatic Endocrine Cells of Pseudemys scripta elegans by Immunogold Labeling

The endocrine pancreatic cells of Pseudemys scripta elegans were investigated immunocytochemically by light and electron microscopy. Insulin-, somatostatin (SST)-1, SST-28 (1-12)-, salmon (s)SST-25-, glucagon-, pancreatic polypeptide (PP)-, peptide tyrosine tyrosine (PYY)-, and neuropeptide tyrosine (NPY)-like immunoreactivities were observed. Insulin cells were immunogold labeled with bonito insulin antiserum and secretory granules were characterized by a wide halo and a dense core of varying shape. Consecutive PAP-immunostained sections showed that SST-28 (1–12), SST-14, and sSST-25 immunoreactivities occurred in the same cells. However, preabsorption tests demonstrated that anti-sSST-25 serum detected the invariant SST-14 molecule. The SST-28 (1–12)/SST-14-immunogold-labeled cells mainly had round or ovoid medium electron-dense granules. Glucagon-IR cells were characterized by round secretory granules with an electron-dense core, with or without a narrow clear halo. There were PP, PYY, and NPY (NPY-like) immunoreactivities in a population of glucagon-IR cells in the pancreatic duodenal region (glucagon/NPY cells). Most of the secretory granules of these glucagon/NPY-like cells had an electron-dense content and were round, although there were also pyriform or ovoid secretory granules which were smaller than those of glucagon-IR cells. Preabsorption tests proved that the NPY-like peptides detected in the endocrine pancreas of P. scripta elegans were more similar to NPY or PYY than to PP.

Cloning neuropeptide tyrosine cDNA.

AbstractThe nervous system is composed of a diverse population of cells selectively expressing different genes which ultimately determine the type of neurotransmitters and neuropeptides produced and released. Neuropeptide tyrosine (NPY) is one of the most abundant and widely expressed peptides in the mammalian nervous system (1). It was originally isolated by virtue of a chemical assay that detects the presence of a carboxyl-terminal amide in proteins (2); a hallmark of potential hormonal function. Its widespread expression in the central and peripheral nervous systems, in addition to the carboxyl-terminal amide, suggested that NPY could be a critical neurotransmitter in a variety of neuronal processes. We were therefore interested in obtaining the cDNA for human NPY in order to study NPY gene expression and screen for the NPY gene (3,4). The only available information was the amino acid sequence of porcine NPY and the amino acid sequences of two related peptides, pancreatic polypeptide and peptide YY (5,6). These experiments were performed in the early 1980s before the advent of genome sequencing and polymerase chain reaction (PCR) technology. Today, experiments to obtain cDNAs for proteins for which only amino acid sequence is available are designed very differently and include computer searches of nucleotide databases and/or various PCR strategies. The experiments described in this chapter represent the classical way by which cDNAs were cloned from corresponding protein sequence.KeywordsPancreatic PolypeptideStrand SynthesisAvian Myeloblastosis Virus Reverse TranscriptaseCellulose ChromatographyGuanidinium IsothiocyanateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The nervous system of the chicken proventriculus: an immunocytochemical and ultrastructural study.

The proventriculus constitutes the glandular region of the chicken stomach. This organ is innervated by two parasympathetic networks, the myenteric and submucous plexus, and here we present a systematic study of this system by immunohistochemistry and electron microscopy. All the neurons and fibres were positive for the neural markers, protein gene product 9.5 and the amidating enzymes. Immunoreactivities for the constitutive neuronal isoform of the enzyme nitric oxide synthase and the vasoactive intestinal peptide were present in neuronal bodies suggesting an intrinsic origin for the similarly immunoreactive fibres found in the proventriculus. On the other hand, immunoreactivity to gastric inhibitory peptide was only found in varicose fibres making contact with the blood vessels and the glandular epithelium, but never in the neuronal somas, suggesting that this substance may be provided by an extrinsic nervous system whose neuronal bodies are located elsewhere. Electron microscopy revealed frequent neuromuscular and neuroepithelial connections in the muscle layers, the wall of the blood vessels and the epithelium. In addition, synapsis-like structures were identified in the proximity of cells belonging to the diffuse endocrine system, providing a new example of neuroendocrine contacts. No positivity was found for antibodies against other neural substances including somatostatin, peptide histidine-isoleucine, peptide tyrosine-tyrosine, neuropeptide tyrosine, bombesin, met-enkephalin, serotonin, substance P, galanin, calcitonin gene-related peptide and S-100 protein.

Peptidergic innervation of the human clitoris

In the present study, the distributions of neuropeptides in the normal human clitoris and in a clitoris from an adrenogenital syndrome (AGS) was demonstrated by immunohistochemistry (IHC). Immunohistochemical screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), neuropeptide tyrosine (neuropeptide Y), C-flanking peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP) and substance P immunoreactivities. Special attention was given to the VIP-related peptide helospectin, that has been detected in neuronal elements in the clitoris. No visible differences between the localization and distribution of peptidergic nerve fibers of normal and hypertrophic clitoris from AGS have been observed. Co-localization studies showed the co-existence of VIP, PHM and partly helospectin and neuropeptide Y with CPON within nerve fibers in the cavernous tissue and substance P and CGRP co-expression in nerve fibers especially underneath and within the glans clitoris.

An immunohistochemical marker for Wallerian degeneration of fibers in the central and peripheral nervous system

This work was prompted by the accidental observation that a newly developed, affinity purified polyclonal antibody against the C-terminus of the neuropeptide tyrosine (NPY) Y1-receptor protein decorates degenerating fibers in the central nervous system (CNS). This staining did not appear in control animals in which the antibody marked perikarya and dendrites at previously described locations [X. Zhang, L. Bao, Z.-Q. Xu, J. Kopp, U. Arvidsson, R. Elde, T. Hökfelt, Localization of neuropeptide Y Y1-receptors in the rat nervous system with special reference to somatic receptors on small dorsal root ganglion neurons, Proc. Natl. Acad. Sci. USA 91 (1994) 11738–11742]. Three models of experimental lesions were studied: sciatic nerve transection, spinal cord transection and parietal cortex thermocoagulation. In each model, animals were divided in groups ( n=2) and processed for indirect immunofluorescence at different time intervals up to 28 days post-lesion (PL) (see below). All three experimental lesions produced a very intense immunolabeling of fibers in the projection pathways of the lesioned structures, strongly reminding of Wallerian degeneration (WD). In the sciatic nerve, the staining first appeared on day 1 PL, was strongly increased on day 3 PL, then declined after 7 days and had almost completely disappeared after 14 days. In the CNS, the staining appeared later and was first observed on day 3 PL and remained for a longer period, thus showing different time courses in the brain and spinal cord as compared to the sciatic nerve. The labeling was completely abolished, both in the CNS and in the sciatic nerve, by pre-incubation of the Y1-R antibody with the immunogenic peptide at a dilution of 10 −6 M. The appearance of the staining and its time course strongly suggest that the process was related to degenerating axons. Although the protein actually detected remains to be determined, it is suggested that the staining ability of this antibody could be used as a positive marker of axonal degeneration following experimental or naturally occurring lesions of the nervous system.

Localization of dopamine beta-hydroxylase (DBH) and neuropeptide tyrosine (NPY) mRNA in the grey monkey locus coeruleus and adrenal medulla.

Localization of dopamine beta-hydroxylase (DBH) and neuropeptide tyrosine (NPY) mRNA in the grey monkey locus coeruleus and adrenal medulla.

Autoradiographic distribution of neuropeptide tyrosine binding sites in the brain of the African lungfish, Protopterus annectens

The distribution of neuropeptide tyrosine (NPY) binding sites in the brain of the African lungfish, Protopterus annectens, was studied by autoradiography using radioiodinated NPY as a tracer. The highest concentrations of binding sites were found in the dorsal and intermediate parts of the medial pallium, the dorsal pallium, and in the medial and lateral subpallium. These observations, together with the finding of a moderate density of binding sites in the olfactory bulbs, suggest that NPY may be involved in the processing of olfactory information and/or neuromodulation of limbic activities. High densities of binding sites were also found in several rhombencephalic nuclei, including the nucleus fascicoli solitarii, the nucleus motorius nervi vagi, the spinal motor column and all components of the reticular formation, indicating that NPY may play a role in the regulation of neurovegetative functions. Concurrently, the presence of high concentrations of binding sites in the hypophysis suggests that, in the lungfish, NPY may exert a direct control of pituitary hormone secretion.

Effect of peripheral axotomy on dorsal root ganglion neuron phenotype and autotomy behaviour in neuropeptide Y-deficient mice

The lumbar 5 (L5) dorsal root ganglia (DRGs) were studied in neuropeptide tyrosine (NPY)-deficient (−/−) and wild type (+/+) mice after unilateral sciatic nerve transection using in situ hybridization and immunohistochemistry. NPY, galanin and two NPY receptors (Y-Rs) were analyzed as well as self-mutilation behaviour (autotomy) and nociceptive thresholds. No difference between wild type and NPY-deficient mice was seen in the tail-flick or hot plate test. However, −/− mice showed a much stronger autotomy behaviour than wild type mice. NPY was not found in L5 DRGs in −/− mice, not even after axotomy. Galanin was upregulated to the same extent after axotomy in NPY-deficient and wild type mice. Y1- and Y2-R mRNAs were found mainly in small DRG neuron profiles. Both receptor mRNAs were downregulated after axotomy, to about the same extent in NPY-deficient as in wild type mice. In control and contralateral ganglia the mRNA levels of both receptors were lower in NPY-deficient mice than in wild type mice. The contralateral Y2-R mRNA levels did not reach control values in the NPY-deficient mice, as they did in the wild type mice. In both strains the Y1-R protein was decorating the somatic plasmalemma. The present results suggest that lack of NPY may cause exaggerated autotomy, a self-mutilation behaviour possibly related to pain sensation, in agreement with the described analgesic effect of NPY. Although significant differences in levels of Y1- and especially Y2-R mRNAs were observed between wild type and NPY-deficient mice, they were only moderate. These findings suggest that expression, regulation, localization and possible function of Y1- and Y2-Rs are not dependent on presence of the endogenous ligand. Also, deletion of NPY does not seem to influence the expression of the partly coexisting peptide galanin.

Sympathetic axons surround neuropeptide-negative axotomized sensory neurons

Nerve injury can lead to sympathetically dependent neuropathic pain. A possible site of sympathetic-sensory interaction is the dorsal root ganglion (DRG), where sympathetic axons form pericellular 'baskets' around a subpopulation of DRG neurons. Since these structures possibly represent functional units of sympathetic pain, we attempted to characterize the neuropeptidergic phenotype of basketed DRG neurons. We performed double-labeling immunohistochemistry for tyrosine hydroxylase and neuropeptides on DRG sections, 2 weeks following L5 spinal nerve ligation (a well-characterized animal model of sympathetic pain). We found that basketed DRG neurons typically do not contain substance P, calcitonin gene-related peptide, galanin, neuropeptide tyrosine, or vasoactive intestinal polypeptide, and we conclude that if sympathetic baskets contribute to neuropathic pain, the involvement of these neuropeptides is unimportant.

Aminopeptidase P--a cell-surface antigen of endothelial and lymphoid cells: catalytic and immuno-histotopical evidences.

The physiological function of the GPI-anchored ectoenzyme aminopeptidase P (APP) is still elusive. Most researchers suppose that this enzyme inactivates biologically active peptides like bradykinin, neuropeptide tyrosine (NPY) and others (Vanhoof et al., 1995). We demonstrate by immunohistology with a specific antibody raised in rabbits and measurement of enzymatic activity in suspensions and of confluent monolayers on microscopic coverslips ('monolayer kinetics') that APP is a cell surface enzyme (ectoenzyme) of endothelial and lymphoid cells.

Galanin/GMAP- and NPY-like immunoreactivities in locus coeruleus and noradrenergic nerve terminals in the hippocampal formation and cortex with notes on the galanin-R1 and -R2 receptors.

By using immunofluorescence methodology, extensive galanin (GAL) and GAL message-associated peptide (GMAP)-positive terminal networks were observed in the hippocampal formation. The majority of the GAL/GMAP fibers were dopamine beta-hydroxylase- (DBH) positive, that is, they were noradrenergic. This finding was established with GAL/GMAP-DBH double-staining and with 6-hydroxy-dopamine treatment, which totally abolished all fibers in which GAL/GMAP and DBH coexisted. Also, reserpine treatment caused a marked depletion of GAL. No evidence for GAL/GMAP coexistence with 5-hydroxytryptamine was obtained. In the ventral hippocampus, GAL/GMAP-, DBH-negative fibers were seen in the stratum oriens, the anterior stratum radiatum, along the granule cell layer and in the strata oriens and alveus. In the locus coeruleus (LC), around 80% of the GMAP-positive neurons contained neuropeptide tyrosine (NPY), and about 40% of the NPY-positive neurons expressed GMAP. GAL-R1 receptor mRNA was expressed in Barrington's nucleus (close to the LC), but was not detected in the hippocampal formation/dorsal cortical areas. GAL-R2 receptor mRNA was found in the granule cell layer in the dentate gyrus. The present results show that most, but not all, immunohistochemically detectable GAL/GMAP in the hippocampal formation/dorsal cortex is present in noradrenergic nerve terminals originating in the LC, which has a robust GAL/GMAP synthesis. The functional role of GAL may be related to noradrenaline, possibly by a presynaptic action. However, the presence of GAL in other systems and of GAL-R2 receptor mRNA in granule cells also indicates other targets.

Effects of increasing gestation, cortisol and maternal undernutrition on hypothalamic neuropeptide Y expression in the sheep fetus.

We have characterized the localization and the ontogenetic changes in Neuropeptide tyrosine (NPY) before birth and investigated the regulation of NPY expression by cortisol and undernutrition in the fetal sheep hypothalamus during late gestation. Using immunohistochemistry, we have identified NPY-containing neurons in the infundibular nucleus and the internal layer of the median eminence in fetal hypothalami collected between 110 and 147 days gestation. NPY projections were also present in the paraventricular nucleus (PVN) of fetal hypothalami at all ages between 110 days gestation and term. There was a significant increase in the amount of immunoreactive NPY/g hypothalamus between 87 and 113 days and 131-140 days gestation and a further significant increase after 141 days gestation. The total hypothalamic content of immunoreactive NPY increased significantly between 87 and 113 days and 141-145 days gestation. The levels of NPY mRNA: 18S rRNA in the mediobasal region of the fetal hypothalamus were significantly higher at 145-146 days gestation than at any earlier gestational age between 116 and 141 days gestation. Cortisol (2.5-3.0 mg/24 h) was infused intrafetally between 109 and 116 days gestation. The ratio of NPY mRNA: 18s rRNA in the mediobasal region of the fetal hypothalamus was significantly higher in the cortisol-infused group when compared with the saline-infused control group at 116 days gestation. Maternal, and hence fetal undernutrition, was induced between 110 and 146 days gestation. At 145-146 days gestation the ratio of NPY mRNA: 18S rRNA in the mediobasal region of the fetal hypothalamus was significantly higher in the undernutrition group when compared with control animals. We have therefore demonstrated that NPY is present in the hypothalamus of the sheep fetus before birth and that hypothalamic NPY content and NPY mRNA increase before delivery. We have also found that glucocorticoids and undernutrition stimulate increases in NPY mRNA levels in the hypothalamus before birth.

Ontogeny of innervation of rat and ovine fetal adrenals

The formation of adrenocortical zonation occurs in rats during late gestation. Since adult cortical function is modulated by neural mediators, it is possible that the development of differentiated function is dependent on cortical innervation. The goal of this study was to compare the pattern and timing of rodent and ovine adrenal innervation during late organogenesis by staining with antibodies directed against the neuropeptides vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY) and the catecholamine biosynthetic enzyme, tyrosine hydrorylase (TOH). Rat adrenals were collected from fetal days 17–21 (term=21 days) and ovine adrenals from fetal days 101–136 (term=145 days). Adrenals were fixed, cryosectioned at 100μ and immunostained using Cy3-conjugated secondary antibodies. In both species, staining of VIP, CGRP, NPY and TOH fibers was observed in the capsule and subcapsular layers of the cortex during gestation. In late gestation, VIP- and NPY-positive ganglions cells were observed near the medulla extending processes toward the outer cortex; in ovine adrenals, fibers from ganglion cells appeared to surround nests of outer cortical (presumably, zona glomerulosa) cells. These data show that phenotypically distinct neural elements appear at different stages of adrenocortical development. The presence of neural elements in contact with adrenal cortical cells supports the possibility for neural control of adrenocortical development.

Localization of biogenic amines and neuropeptides in adrenal medullary cells of birds.

The present article reviews the immunohistochemical findings on the localization of biogenic amines and neuropeptides in adrenal medullary cells of birds. In the chicken, about 70% of medullary cells are adrenaline-containing cells and the rest of cells seem to be noradrenaline-containing cells. The ratio of adrenaline-cells to noradrenaline-cells extremely varies among avian species. Besides adrenaline and noradrenaline, medullary cells of birds contain many kinds of biogenic amines and neuropeptides: serotonin, galanin, cholecystokinin, somatostatin, enkephalin, neuropeptide tyrosine and atrial natriuretic peptide. The existence of these bioactive substances in medullary cells also exhibits interspecies heterogeneity. In the chicken, serotonin and galanin are contained in both adrenaline- and noradrenaline-cells of the adrenal gland. Cholecystokinin- and somatostatin-immunoreactivity is restricted to adrenaline-containing cells. Enkephalin-immunoreactivity is seen in both adrenaline- and noradrenaline-cells, but in about half of medullary cells. Neuropeptide tyrosine-immunoreactivity is found in the adrenal gland of the chick embryo and newly hatched chick, but not in the adult chicken. Serotonin and these neuropeptides may be selectively coreleased with adrenaline and/or noradrenaline from adrenal medullary cells of the chicken.