Expression and distribution of GABA and GABAB‐receptor in the rat adrenal gland

The inhibitory effects of gamma‐aminobutyric acid (GABA) in the central and peripheral nervous systems and the endocrine system are mediated by two different GABA receptors: GABA_A‐receptor (GABA_A‐R) and GABA_B‐receptor (GABA_B‐R). GABA_A‐R, but not GABA_B‐R, has been observed in the rat adrenal gland, where GABA is known to be released. This study sought to determine whether both GABA and GABA_B‐R are present in the endocrine and neuronal elements of the rat adrenal gland, and to investigate whether GABA_B‐R may play a role in mediating the effects of GABA in secretory activity of these cells. GABA‐immunoreactive nerve fibers were observed in the superficial cortex. Some GABA‐immunoreactive nerve fibers were found to be associated with blood vessels. Double‐immunostaining revealed GABA‐immunoreactive nerve fibers in the cortex were choline acetyltransferase (ChAT)‐immunonegative. Some GABA‐immunoreactive nerve fibers ran through the cortex toward the medulla. In the medulla, GABA‐immunoreactivity was seen in some large ganglion cells, but not in the chromaffin cells. Double‐immunostaining also showed GABA‐immunoreactive ganglion cells were nitric oxide synthase (NOS)‐immunopositive. However, neither immunohistochemistry combined with fluorescent microscopy nor double‐immunostaining revealed GABA‐immunoreactivity in the noradrenaline cells with blue‐white fluorescence or in the adrenaline cells with phenylethanolamine N‐methyltransferase (PNMT)‐immunoreactivity. Furthermore, GABA‐immunoreactive nerve fibers were observed in close contact with ganglion cells, but not chromaffin cells. Double‐immunostaining also showed that the GABA‐immunoreactive nerve fibers were in close contact with NOS‐ or neuropeptide tyrosine (NPY)‐immunoreactive ganglion cells. A few of the GABA‐immunoreactive nerve fibers were ChAT‐immunopositive, while most of the GABA‐immunoreactive nerve fibers were ChAT‐immunonegative. Numerous ChAT‐immunoreactive nerve fibers were observed in close contact with the ganglion cells and chromaffin cells in the medulla. The GABA_B‐R‐immunoreactivity was found only in ganglion cells in the medulla and not at all in the cortex. Immunohistochemistry combined with fluorescent microscopy and double‐immunostaining showed no GABA_B‐R‐immunoreactivity in noradrenaline cells with blue‐white fluorescence or in adrenaline cells with PNMT‐immunoreactivity. These immunoreactive ganglion cells were NOS‐ or NPY‐immunopositive on double‐immunostaining. These findings suggest that GABA from the intra‐adrenal nerve fibers may have an inhibitory effect on the secretory activity of ganglion cells and cortical cells, and on the motility of blood vessels in the rat adrenal gland, mediated by GABA‐Rs.     

Immunohistochemical features of substance P-immunoreactive chromaffin cells and nerve fibers in the rat adrenal gland

The distribution of substance P (SP) immunoreactivity and the colocalization of SP with other bioactive substances in chromaffin cells and nerve fibers were investigated in the rat adrenal gland at the light microscopic level. In the capsule and cortex, SP immunoreactivity was seen in some nerve fibers around blood vessels and in thick nerve bundles passing through the cortex directly into the medulla. In the medulla, the SP immunoreactivity was observed in a small number of chromaffin cells; these SP-immunoreactive chromaffin cells were either phenylethanolamine N-methyltransferase (PNMT) immunoreactive or immunonegative, indicating that they were either adrenaline cells or noradrenaline (NA) cells. SP-immunoreactive varicose nerve fibers were also found in the medulla and were in contact with a cluster of the NA cells showing catecholamine fluorescence, which suggests that SP from medullary nerve fibers may regulate the secretory activity of the NA cells. Because no SP-immunoreactive ganglion cell was present in the rat adrenal gland, the intra-adrenal nerve fibers were considered to be extrinsic in origin. The double-immunostaining method further revealed that the SP-immunoreactive chromaffin cells also exhibit immunoreactivities for calcitonin gene-related peptide (CGRP), and neuropeptide tyrosine (NPY), suggesting that these peptides can also be released from the chromaffin cells by certain stimuli. The intra-adrenal nerve fibers in the medulla were composed of SP-single immunoreactive, and SP/CGRP-, SP/choline acetyltransferase (ChAT)-, SP/nitric oxide synthase (NOS)-, SP/pituitary adenylate cyclase activating polypeptide (PACAP)-, ChAT/NOS-, and ChAT/PACAP-immunoreactive nerve fibers, which may affect the secretory activity of the NA cells. In the adrenal capsule, the nerve fibers were present around blood vessels and showed immunoreactivities for SP/ CGRP, SP/NPY, SP/NOS, and SP/vasoactive intestinal polypeptide, suggesting that the origin of nerve fibers in the capsule may differ from those in the medulla.     

Acetylcholinesterase Activity,Choline Acetyltransferase and Vesicular Acetylcholine Transporter Immunoreactivities in the Rat Adrenal Gland During Postnatal Development

From postnatl‐day‐0 to postnatal‐day‐2, a few acetylcholinesterase (AChE)‐active and choline acetytransferase (ChAT)‐immunoreactive nerve fibers and relatively numerous vesicular acetylcholine transporter (VAChT)‐immunoreactive puncta were observed in the rat adrenal medulla. Despite relatively numerous clear vesicles in the nerve fibers, the synthesis and hydrolysis of acetylcholine may not be fully activated until postnatal‐day‐2. The number of AChE‐active and ChAT‐immunoreactive nerve fibers dramatically increased and that of VAChT‐immunoreactive puncta gradually increased from postnatal‐day‐3 to postnatal‐week‐1. The synthesis and hydrolysis of acetylcholine may be dramatically activated in the nerve fibers of the medulla until postnatal‐week‐1. From postnatal‐week‐2 to postnatal‐week‐3, the number of AChE‐active and the ChAT‐immunoreactive nerve fibers gradually increased and reached the adult levels. The VAChT‐immunoreactive puncta per unit area was maximum number at postnatal‐week‐2. The synthesis and hydrolysis of acetylcholine in the nerve fibers of the medulla may be completed between postnatal‐week‐2 to postnatal‐week‐3. The diameter of the VAChT‐immunoreactive puncta gradually increased from postnatal‐day‐0 with aging. However, the number of the VAChT‐immunoreactive puncta gradually decreased from postnatal‐week‐2 onwards. In electron‐microscopy, the VAChT‐immunoreactive deposits were seen in clusters of clear vesicles, and the diameter of the nerve fibers and the number of clear vesicles at postnatal‐week‐8 increased compared with those at postnatal‐week‐2. The AChE‐active, ChAT‐immunoreactive, and VAChT‐immunoreactive nerve fibers observed around noradrenaline (NA) cells were denser than those around adrenaline (A) cells in the medulla at postnatal‐week‐8. These suggest that the preferential innervation of NA and A cells may cause the differential secretion NA and A. Anat Rec, 292:371–380, 2009. © 2009 Wiley‐Liss, Inc.     

Immunoelectron microscopic study of tyrosine hydroxylase immunoreactive nerve fibers and ganglion cells in the rat adrenal gland.

The present immunocytochemical study used an antiserum to tyrosine hydroxylase (TH), the first enzyme in the biosynthetic pathway of catecholamines, and revealed TH immunoreactivity in the ganglion cells and in the varicose nerve fibers of the cortex and medulla in the rat adrenal gland. TH immunoreactive nerve fibers in the cortex and medulla contained large and small granular vesicles, and also small clear vesicles. The immunoreactive nerve fibers were in close apposition to cortical cells in the cortex and in apposition to smooth muscle cells of blood vessels in both the cortex and medulla. Furthermore, TH immunoreactive nerve fibers were sometimes in close apposition to pericytes of blood vessels in the cortex and chromaffin cells in the medulla. The present results suggest that the catecholaminergic nerve fibers in the rat adrenal gland may be both intrinsic and extrinsic in origin.     

Immunoelectron microscopic study of tyrosine hydroxylase immunoreactive nerve fibers and ganglion cells in the rat adrenal gland

The present immunocytochemical study used an antiserum to tyrosine hydroxylase (TH), the first enzyme in the biosynthetic pathway of catecholamines, and revealed TH immunoreactivity in the ganglion cells and in the varicose nerve fibers of the cortex and medulla in the rat adrenal gland. TH immunoreactive nerve fibers in the cortex and medulla contained large and small granular vesicles, and also small clear vesicles. The immunoreactive nerve fibers were in close apposition to cortical cells in the cortex and in apposition to smooth muscle cells of blood vessels in both the cortex and medulla. Furthermore, TH immunoreactive nerve fibers were sometimes in close apposition to pericytes of blood vessels in the cortex and chromaffin cells in the medulla. The present results suggest that the catecholaminergic nerve fibers in the rat adrenal gland may be both intrinsic and extrinsic in origin.     

Neuropeptide tyrosine (NPY)-like immunoreactivity in adrenal chromaffin cells and intraadrenal nerve fibers of rats

The present peroxidase-antiperoxidase immunohistochemical study demonstrated that approximately 50% of the total chromaffin cells of the rat adrenal medulla exhibited NPY-like immunoreactivity. The immunoreactive material was localized in the core of the chromaffin granules as well as diffusely in the cytoplasm. By combination of immunohistochemistry with noradrenaline-fluorescence microscopy, all NPY-immunoreactive chromaffin cells are nonfluorescent, indicating that all NPY-chromaffin cells co-store adrenaline. A comparison of two consecutive sections, each of which was processed for the immunostaining with anti-NPY and anti-Met-Enk-Arg-Gly-Leu antisera, respectively, indicated that NPY and preproenkephalin A and its derivatives coexist in approximately one-fifth of the total NPY-immunoreactive cells. In addition to the NPY-immunoreactive cells, a plexus of NPY-immunoreactive nerve fibers with varicosities was found in the subcapsular regions of the adrenal gland. The nerve fibers were often associated with small blood vessels and extended into the zona glomerulosa. Single NPY-immunoreactive fibers were sparsely distributed in the deeper regions of the cortex and in the medulla. Ganglion cells in the adrenal gland were not seen exhibiting intensely positive NPY-like immunoreactivity. The NPY-immunoreactive nerve fibers contained abundant small clear vesicles mixed with a few small and large granular vesicles. The immunoreactive material appeared on the granular cores as well as in the axoplasm. The NPY fibers were closely apposed to smooth muscle cells and pericytes of small blood vessels in the cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-localization of vasoactive intestinal polypeptide and neuropeptide Y immunoreactivities in the nerve fibers of the rat adrenal gland

The co-localization of Vasoactive Intestinal Polypeptide (VIP) with Neuropeptide Y (NPY) or its C-flanking peptide (C-PON) was investigated with immunocytochemistry methods in the adrenal gland of the rat. Most of the VIP immunoreactive (+) nerve fibers found in the capsule/glomerular zone also exhibited NPY or C-PON immunoreactivity (IR). We found that at least two populations of VIP varicose nerve fibers can be observed, the most prevalent exhibited both VIP/NPY or VIP/C-PON IR and the other which was rather scarce lacked NPY or C-PON IR. In the superficial cortex VIP/NPY or VIP/C-PON IR nerve fibers were often associated with capsular or subcapsular vascularization and extended into the zona glomerulosa. In the deeper layers of the adrenal cortex radial fibers were closely associated with the inner vascularization of the zona fasciculata and reticularis. In the adrenal medulla NPY or C-PON immunoreactivity was associated with ganglion neurons as well as chromaffin cells; these last cells were always VIP (-). VIP and NPY/C-PON IR could be co-localized in catecholaminergic nerve terminals of the adrenal cortex but not in the adrenal medulla.     

Differential distribution of neuropeptides and serotonin in pig adrenal glands

A differential distribution of vasoactive neuropeptides and serotonin in chromaffin cells and nerve fibers within the adrenal glands of the pig (Sus scrofa) was found using immunohistochemical methods. Met- and leu-enkephalins, present at high levels in the medulla (measured by radioimmunoassay), occurred in adrenaline storing cells, some of which contained calcitonin gene-related peptide. Islets of chromaffin cells beneath the capsule also contained enkephalins and calcitonin gene-related peptide. Nerve fibers with enkephalin-like immunoreactivity were sparse, but many varicose fibers in the inner cortex and medulla showed calcitonin gene-related peptide immunofluorescence in a pattern similar to vasoactive intestinal polypeptide. Neuropeptide Y was mainly associated with perivascular fibers and neither neuropeptide Y nor vasoactive intestinal polypeptide immunoreactive chromaffin cells were detected. In contrast to the neuropeptides, most serotonin-like immunoreactivity coincided with noradrenaline histofluorescence. It is concluded that the distribution of nerve fibers with calcitonin gene-related peptide and vasoactive intestinal polypeptide would allow interactions between chromaffin and inner cortical cells. Stimuli activating noradrenaline chromaffin cells could release serotonin while stimulation of adrenaline storage cells would release enkephalin and, to a lesser extent, calcitonin gene-related peptide. Met-enkephalin, which occurs 3 4:1 over leu-enkephalin, is the most likely of the co-released peptides to reach distant receptors via the venous outflow.     

大鼠延髓头端腹内侧区内G蛋白偶联雌激素受体与 多种神经元的免疫荧光共定位关系

目的：探讨大鼠延髓头端腹内侧区（RVM）内G蛋白偶联型雌激素受体（GPER）与多种神经元的免疫荧光 共定位关系。方法：取6 周雄性SD大鼠脑组织,采用免疫荧光染色技术,对大鼠的RVM区域GPER和5- 羟色胺 （5-HT）能神经元标志物色氨酸羟化酶（TPH）、儿茶酚胺能神经元标志物酪氨酸羟化酶（TH）、神经元型一氧化 氮合酶（nNOS）、胆碱能神经元标志物胆碱乙酰转移酶（ChAT）以及脑啡肽（ENK）进行双重免疫组织化学显色。 结果：RVM内GPER免疫反应（GPER-ir）阳性神经元均不表达TPH,其周围有TPH 免疫反应（TPH-ir）阳性神 经元及纤维分布。RVM核心区域中缝大核内未检测到TH 或ChAT的免疫活性。网状巨细胞旁核内有TH 和ChAT 免疫反应阳性胞体或纤维分布,但它们极少与GPER-ir 共定位。nNOS的免疫活性（nNOS-ir）在RVM中表达丰富, 而少部分GPER-ir 弱阳性神经元可检测到nNOS-ir。RVM区域内有大量ENK免疫反应阳性纤维分布,且末梢紧 邻GPER-ir 阳性神经元胞体。结论：GPER特异性表达在RVM的非5-HT 能神经元,可能是雌激素调节痛觉和阿 片镇痛效应的结构和分子基础之一。     

GABAB and group I metabotropic glutamate receptors in the striatopallidal complex in primates

Glutamate and GABA neurotransmission is mediated through various types of ionotropic and metabotropic receptors. In this review, we summarise some of our recent findings on the subcellular and subsynaptic localisation of GABA_B and group I metabotropic glutamate receptors in the striatopallidal complex of monkeys. Polyclonal antibodies that specifically recognise GABA_BR1, mGluR1a and mGluR5 receptor subtypes were used for immunoperoxidase and pre‐embedding immunogold techniques at the light and electron microscope levels. Both subtypes of group I mGluRs were expressed postsynaptically in striatal projection neurons and interneurons where they aggregate perisynaptically at asymmetric glutamatergic synapses and symmetric dopaminergic synaptic junctions. Moreover, they are also strongly expressed in the main body of symmetric synapses established by putative intrastriatal GABAergic terminals. In the globus pallidus, both receptor subtypes are found postsynaptically in the core of striatopallidal GABAergic synapses and perisynaptically at subthalamopallidal glutamatergic synapses. Finally, extrasynaptic labelling was commonly seen in the globus pallidus and the striatum. Moderate to intense GABA_BR1 immunoreactivity was observed in the striatopallidal complex. At the electron microscope level, GABA_BR1 immunostaining was commonly found in neuronal cell bodies and dendrites. Many striatal dendritic spines also displayed GABA_BR1 immunoreactivity. Moreover, GABA_BR1‐immunoreactive axons and axon terminals were frequently encountered. In the striatum, GABA_BR1‐immunoreactive boutons resembled terminals of cortical origin, while in the globus pallidus, subthalamic‐like terminals were labelled. Pre‐embedding immunogold data showed that postsynaptic GABA_BR1 receptors are concentrated at extrasynaptic sites on dendrites, spines and somata in the striatopallidal complex, perisynaptically at asymmetric synapses and in the main body of symmetric striatopallidal synapses in the GPe and GPi. Consistent with the immunoperoxidase data, immunoparticles were found in the presynaptic grid of asymmetric synapses established by cortical‐ and subthalamic‐like glutamatergic terminals. These findings indicate that both GABA and glutamate metabotropic receptors are located to subserve various modulatory functions of the synaptic transmission in the primate striatopallidal complex. Furthermore, their pattern of localisation raises issues about their roles and mechanisms of activation in normal and pathological conditions. Because of their ‘modulatory’ functions, these receptors are ideal targets for chronic drug therapies in neurodegenerative diseases such as Parkinson's disease.     

Axonal GABA receptors are selectively present on normal and regenerated sensory fibers in rat peripheral nerve

Summary A sucrose gap chamber was used to study the effect of gamma-aminobutyric acid (GABA) on normal and regenerating rat peripheral nerve fibers. Sciatic nerves and dorsal roots were depolarized by GABA and the GABA_A receptor subtype agonist muscimol, but not by the GABA_B receptor subtype agonist baclofen. Ventral root fibers were not affected by these agents, suggesting a selective presence of axonal GABA receptors on sensory fibers of mammalian peripheral nerves. Regenerating dorsal and ventral root fibers were studied 13 to 20 days following nerve crush. The regenerated dorsal root fibers were depolarized by GABA or muscimol, but the regenerated ventral root fibers were not. These results indicate that GABA receptors are selectively present on normal mammalian sensory axons, and are reestablished on regenerated sensory axons.     

Enkephalin-like immunoreactivity in gland cells and nerve terminals of the adrenal medulla.

Met-enkephalin-like immunoreactivity has been observed in adrenal medullary gland cells of the rat, guinea-pig and cat. There were marked quantitative differences in untreated animals. Most medullary gland cells were positive in the cat, a large proportion in the guinea-pig but only a few in the rat. After sectioning of the splanchnic nerve, however, a large proportion of the gland cells were met-enkephalin positive also in the rat. Occasionally the met-enkephalin-like immunoreactivity had a granular appearance suggesting that the storage sites could be vesicular.In the guinea-pig a moderate number of met-enkephalin immunoreactive nerve fibers were observed in the adrenal medulla. They disappeared to a large extent after sectioning of the splanchnic nerve. A few fibers were also seen in the rat adrenal medulla, and in the cat some fibers could be seen in areas with few immunoreactive cells.The present findings suggest that in the adrenal medulla an enkephalin-like peptide(s) is present both in gland cells and in nerve terminals arising mainly from fibers in the splanchnic nerve. Thus, the possibility exists that in the adrenal medulla opioid peptides may be released from the gland cells into the blood as hormones as well as from nerve terminals to act as a modulator or transmitter. It should, however, be emphasized that the well-documented metabolic instability of met-enkephalin may be somewhat difficult to reconcile with a hormonal role of this peptide.     

Rodent and primate adrenal medullary cells in vitro: phenotypic plasticity in response to coculture with C6 glioma cells or NGF

Summary In order to maintain a chronic supply of growth factor for medulla cells in vitro, chromaffin cells from rat, African green monkeys and man were co-cultured with C₆ glioma cells, which secrete growth factors that sustain sympathetic neurons in vitro. The response of chromaffin cells to coculture was compared to treatment of medullary cells with nerve growth factor (NGF) alone. Dispersed chromaffin cell preparations were obtained by a trypsin-collagenase procedure, and subjected to differential plating on collagen-coated surfaces. With both human and monkey tissue, non-chromaffin cells did attach to the culture plates and an enriched chromaffin cell population could be replated. Rat adrenal medulla cells survived very poorly in vitro and were not enriched in this procedure. Cultured human and monkey chromaffin cells survived as epithelial cells (50%) and showed neuritic outgrowth on 55 to 66% of the cells after eight days when treated with nerve growth factor (NGF). These cells showed strong catecholamine histofluorescence, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) immunoreactivity. In contrast, only ten percent of adult rat chromaffin cells survived in culture, although NGF treatment rescued an additional 20% of the cells and induced neuritic outgrowth after one week in vitro. C₆ glioma cells were treated with mitomycin C bromodeoxyuridine to inhibit mitosis and were plated with the various medulla cells in a one to one ratio. Both human and monkey chromaffin cells expressed extensive and enhanced neuritic arborization within eight days of co-culture, (64–82% respectively) and exhibited intimate contact with the glioma cells as seen at the ultrastructural level. Importantly, survival of adult rat adrenal medulla cells was enhanced to 50% or more with 40% of the cells extending neurites when co-cultured with glioma cells for seven days. Chromaffin cells from all three species reacted for TH, DBH and PNMT in co-culture and were histofluorescent. The majority of these cells were also immunoreactive for serotonin and enkephalin, while only 37% of chromaffin cells indicated the presence of NPY. These data indicate that adrenal medulla can be maintained in vitro as the neuronal phenotype when co-cultured with growth factor producing cells and that this strategy may be useful for in vivo transplantation studies.     

Nerve growth factor receptor expression in peripheral and central neuroectodermal tumors, other pediatric brain tumors, and during development of the adrenal gland.

Nerve growth factor (NGF) is important to the survival, development, and differentiation of neurons. Its action is mediated by a specific cell surface transmembrane glycoprotein, nerve growth factor receptor (NGFR). In this study, NGFR expression by human fetal and adult adrenal medullary tissue, peripheral nervous system (PNS) neuroectodermal tumors (neuroblastoma, ganglioneuroblastoma, ganglioneuroma), pediatric primitive neuroectodermal tumors (PNETs) of the central nervous system (CNS), and CNS gliomas was examined by an immunohistochemical technique. Sixty-nine tumors in total were probed in this manner. Nerve growth factor receptor immunoreactivity was confined to nerve fibers and clusters of primitive-appearing cells in the fetal adrenal, and to nerve fibers and ganglion cells of the adult adrenal medulla; adrenal chromaffin cells were negative. In PNS neuroectodermal tumors, there was NGFR expression in tumor cells of 6 of 11 neuroblastomas and 6 of 6 ganglioneuroblastomas or ganglioneuromas. Thirteen of thirty-five CNS PNETs showed NGFR positivity. In most CNS PNETs, NGFR was restricted to scattered single or small groups of cells, but two tumors with astroglial differentiation showed much more extensive immunoreactivity. Most astrocytomas (11 of 14) and all ependymomas (3 of 3) were intensely NGFR positive.     

Immunohistochemical localization of neurotensin in hamster adrenal medulla

Neurotensin-like immunoreactivity was localized in nerve fibers and terminals of hamster adrenal medulla at light and electron microscopy using the peroxidase-antiperoxidase method. Numerous varicose neurotensin-immunoreactive nerves and terminals were found among nonlabeled cell groups situated peripherally in the adrenal medulla. Combined formaldehyde-glutaraldehyde (Faglu) fluorescence and immunohistochemistry of the same vibratome section showed that only norepinephrine cells were innervated by neurotensin-immunoreactive nerves. All norepinephrine cells seemed to be innervated by neurotensin-immunoreactive nerves. Neurotensin-immunoreactive nerves disappeared after extrinsic denervation of the adrenal gland. By electron microscopy numerous neurotensin-immunoreactive terminals were seen to make synaptic contacts with norepinephrine cells and with autonomic ganglion cells present in small numbers among norepinephrine cells. In the terminals neurotensin-like immunoreactivity was localized mainly in large dense-cored vesicles, but some precipitates were also associated with small vesicles, diffusely scattered in the axoplasm. The present findings suggest that in the hamster adrenal medulla part of the nerve terminals arising from splanchnic nerves contain neurotensin-like peptide. The functional significance of these nerves in the hamster adrenal medulla remains to be elucidated.     

Phospholipase C isozymes are differentially distributed in the rat adrenal medulla

Immunohistochemistry has been used to examine the distribution of selected phospholipase C (PLC) isozymes within the adrenal medulla of the rat. PLCbeta isozymes were expressed at moderate levels in the chromaffin cells but more strongly in association with ganglion cell clusters. PLCbeta2 and PLCbeta3 staining of clusters did not overlap suggesting selective PLC isozyme expression in two distinct ganglionic types. The distribution of PLCbeta4 immunoreactivity was very similar to PLCbeta3 with the strongest staining observed in the same cell clusters. Antibodies to PLCbeta1 labelled multiple bands on Western blots and were not therefore used for immunohistochemistry. The chromaffin cells were also immunoreactive for PLCgamma1, although the strongest staining with this antibody was seen in cells surrounding large sinus vessels. PLCdelta1 and PLCdelta2 had quite distinct distributions, with the former selectively localized to an endothelial cell population surrounding the chromaffin cells. This observation was supported by experiments on isolated bovine adrenal medullary cells where PLCdelta1 expression was lost when the cell preparation was enriched for chromaffin cells. Antibodies to PLCdelta2 labelled a network of nerve fibres throughout the medulla and clusters of ganglion cells located primarily at the medullary-cortical boundary. PLCdelta2 immunoreactivity was also present in nerve fibres within the adrenal capsule where it appeared to be co-localized with PLCbeta4 staining.     

GABA regulates corticotropin releasing hormone levels in the paraventricular nucleus of the hypothalamus in newborn mice

The paraventricular nucleus of the hypothalamus (PVN) is a major regulator of stress responses via release of corticotropin releasing hormone (CRH) to the pituitary gland. Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is characteristic of individuals with major depressive disorder (MDD). Postmortem data from individuals diagnosed with MDD show increased levels of CRH mRNA and CRH immunoreactive neurons in the PVN. In the current study, an immunohistochemical (IHC) analysis revealed increased levels of CRH in the PVN of newborn mice lacking functional GABA_B receptors. There was no difference in the total number of CRH immunoreactive cells. By contrast, there was a significant increase in the amount of CRH immunoreactivity per cell. Interestingly, this increase in CRH levels in the GABA_B receptor R1 subunit knockout was limited to the rostral PVN. While GABAergic regulation of the HPA axis has been previously reported in adult animals, this study provides evidence of region-specific GABA modulation of immunoreactive CRH in newborns.     

Neuronal nitric oxide synthase in the olfactory system,forebrain, pituitary and retina of the adult teleost Clarias batrachus

Immunocytochemical application of antibodies against nNOS to the brain sections of Clarias batrachus revealed intense immunoreactivity in several olfactory receptor neurons (ORNs), in their axons over the olfactory nerve, and terminals in the olfactory glomeruli. Several basal cells in the olfactory epithelium showed NOS immunoreactivity. Application of post-embedding immunoelectron microscopy showed nNOS labeled gold particles in apical cilia, dendrites and soma of the ORNs and also in the axon terminals in the glomeruli of the olfactory bulb. nNOS containing fibers were also encountered in the medial olfactory tracts (MOTs). Bilateral ablation of the olfactory organ resulted in total loss of nNOS immunoreactivity in the fascicles of the olfactory nerve layer and also in the MOT. nNOS immunoreactivity was seen in several cells of the nucleus preopticus (NPO) and their axons that innervate the pituitary gland. Some cells in the floor of the tuberal area were stained positive with nNOS antibodies. nNOS immunolabeled cells were seen in all the three components of the pituitary gland with light as well as post-embedding immunoelectron microscopy. While several nNOS immunoreactive fibers were seen in rostral pars distalis, a much limited fiber population was seen in the proximal pars distalis. In addition, conspicuous immunoreactivity was noticed in some ganglion cells in the retina and in some fibers of the optic nerve traceable to the optic tectum. The NO containing system in this fish appears to be similar to that in other fishes.