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The theory holds that the anterior pituitary in mammals receives humoral regulation. Previous studies have reported that the pars distalis of the anterior pituitary of several mammalian species contains substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive nerve fibers, but the origins of these nerve fibers are unclear. Removal of the pituitary gland, also called hypophysectomy, involves methods that access the pituitary gland via the transauricular or parapharyngeal pathways. However, these methods are not applicable for viral tracer injection to investigate the innervation of the anterior pituitary. The transauricular technique leads to inaccuracies in locating the pituitary gland, while the parapharyngeal approach causes high mortality in animals. Here, we introduce a protocol that accesses the pituitary gland in the rat via the transsphenoidal pathway. This method imitates surgical manipulations such as endotracheal intubation and sphenoid bone drilling, which involve the use of custom-made devices. Using the transsphenoidal pathway greatly improves the survival rate of rats because no additional dissection of blood vessels and nerves is required. Moreover, the pituitary gland can be viewed clearly and directly during the operation, making it possible to accurately inject pseudorabies virus (PRV) 152-expressing enhanced green fluorescent protein (EGFP) into the anterior or posterior pituitary, respectively. After injecting PRV 152 into the anterior pituitary, we found no evidence of direct innervation of the anterior pituitary in the rat brain. However, PRV 152 injection into the posterior pituitary revealed retrograde transneuronal cell bodies in many brain areas, including the CA1 field of the hippocampus, the basolateral amygdaloid nucleus, posterior part (BLP), the arcuate hypothalamic nucleus (Arc), the dorsal portion of the dorsomedial hypothalamic nucleus (DMD), the suprachiasmatic nucleus (SCh), and the subfornical organ (SFO). In the present study, we provide a description of a possible model of hypophysectomy or pituitary injection, and identify brain regions involved in regulating the rat pituitary gland using transneuronal retrograde cell body labeling with PRV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2020 Li, Su, Zhao, Li, Xu, Gao, Sun, Yang, Jin and Ke.)

The influence of chronic stress, induced by food deprivation (FD) and/or high stocking density (HSD), was assessed on stress, vasotocinergic and isotocinergic pathways of the gilthead sea bream (Sparus aurata). Fish were randomly assigned to one of the following treatments: (1) fed at low stocking density (LSD-F; 5kg·m -3 ); (2) fed at high stocking density (HSD-F, 40kg·m -3 ); (3) food-deprived at LSD (LSD-FD); and (4) food-deprived at HSD (HSD-FD). After 21days, samples from plasma, liver, hypothalamus, pituitary and head-kidney were collected. Both stressors (FD and HSD) induced a chronic stress situation, as indicated by the elevated cortisol levels, the enhancement in corticotrophin releasing hormone (crh) expression and the down-regulation in corticotrophin releasing hormone binding protein (crhbp) expression. Changes in plasma and liver metabolites confirmed a metabolic adjustment to cope with energy demand imposed by stressors. Changes in avt and it gene expression, as well as in their specific receptors (avtrv1a, avtrv2 and itr) at central (hypothalamus and pituitary) and peripheral (liver and head-kidney) levels, showed that vasotocinergic and isotocinergic pathways are involved in physiological changes induced by FD or HSD, suggesting that different stressors are handled through different stress pathways in S. aurata., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Male testosterone levels decline by 1% per year from the age of 40 years. Whilst a primary testicular deficit occurs, hypothalamic or pituitary dysregulation may also coexist. This study aimed to compare the hypothalamic response to kisspeptin-54 and the pituitary response to gonadotropin-releasing hormone (GnRH) of older men with those of young men., Methods: Following 1 h of baseline sampling, healthy older men (n = 5, mean age 59.3 ± 2.9 years) received a 3-h intravenous infusion of either vehicle, kisspeptin-54 0.1, 0.3, or 1.0 nmol/kg/h or GnRH 0.1 nmol/kg/h, on five different study days. Serum gonadotropins and total testosterone were measured every 10 min and compared to those of young men (n = 5/group) (mean age 28.9 ± 2.0 years) with a similar body mass index (24 kg/m2) who underwent the same protocol., Results: Kisspeptin-54 and GnRH significantly stimulated serum gonadotropin release in older men compared to vehicle (p < 0.001 for all groups). Gonadotropin response to kisspeptin-54 was at least preserved in older men when compared to young men. At the highest dose of kisspeptin-54 (1.0 nmol/kg/h), a significantly greater luteinising hormone (LH) (p = 0.003) response was observed in older men. The follicle-stimulating hormone (FSH) response to GnRH was increased in older men (p = 0.002), but the LH response was similar (p = 0.38). Serum testosterone rises following all doses of kisspeptin-54 (p ≤ 0.009) were reduced in older men., Conclusions: Our data suggest that healthy older men without late-onset hypo-gonadism (LOH) have preserved hypothalamic response to kisspeptin-54 and pituitary response to GnRH, but impaired testicular response. Further work is required to investigate the use of kisspeptin-54 to identify hypothalamic deficits in men with LOH., (The Author(s). Published by S. Karger AG, Basel.)

The dopamine D 2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D 2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D 2 receptor. D 2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as "intermediate zone". This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D 2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D 2 receptors. D 2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland.

Cytosolic Ca(2+) buffers bind to a large fraction of Ca(2+) as it enters a cell, shaping Ca(2+) signals both spatially and temporally. In this way, cytosolic Ca(2+) buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca(2+) entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca(2+) buffers in controlling pituitary Ca(2+) signaling is poorly understood. Here, cytosolic Ca(2+) buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca(2+) indicator fluo-8 revealed stepwise increases in free Ca(2+) after a series of brief depolarizing pulses in rapid succession. These Ca(2+) increments grew larger as free Ca(2+) rose to saturate the cytosolic buffers and reduce the availability of Ca(2+) binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a Kd of 1.5-4.7 µM, and approximately half contained an additional higher-affinity buffer with a Kd of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca(2+) buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca(2+) signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones., (© 2016 McMahon et al.)

Orexin receptors type 1 (OX1R) and type 2 (OX2R) are G protein-coupled receptors whose structure is highly conserved in mammals. OX1R is selective for orexin A, and OX2R binds orexin A and orexin B with similar affinity. Orexin receptor expression was observed in human, rat, porcine, sheep as well as Xenopus laevis pituitaries, both in the adenohypophysis and in the neurohypophysis. The expression level is regulated by gonadal steroid hormones and GnRH. The majority of orexins reaching the pituitary originate from the lateral hypothalamus, but due to the presence of the receptors and the local production of orexins in the pituitary, orexins could deliver an auto/paracrine effect within the gland. Cumulative data indicate that orexins are involved in the regulation of LH, GH, PRL, ACTH, and TSH secretion by pituitary cells, pointing to orexins' effect on the functioning of the endocrine axes. Those hormones may also serve as a signal linking metabolic status with endocrine control of sleep, arousal, and reproduction processes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The pituitary gland is an endocrine organ that is developmentally derived from a fold in the oral ectoderm and a juxtaposed fold in the neural ectoderm. Here, we show that the absence of Vax1, a homeodomain transcription factor known for its role in eye and optic chiasm development, causes the rostral oral ectoderm to form an ectopic fold that eventually develops into a separate second pituitary with all the pituitary cell types and neuronal fibers characteristic of the normal pituitary. The induction of the second pituitary is associated with a localized ectopic expression of Fgf10, a gene encoding a growth factor known to recruit oral ectodermal cells into the pituitary. Interestingly, there are rare cases of pituitary duplications in humans that are also associated with optic nerve dysplasia, suggesting that VAX1 might be involved in the pathogenesis of this disorder.

This review aims at synthesizing the most relevant information regarding the neuroendocrine circuits controlling reproduction, mainly gonadotropin release, in teleost fish. In teleosts, the pituitary receives a more or less direct innervation by neurons sending projections to the vicinity of the pituitary gonadotrophs. Among the neurotransmitters and neuropeptides released by these nerve endings are gonadotrophin-releasing hormones (GnRH) and dopamine, acting as stimulatory and inhibitory factors (in many but not all fish) on the liberation of LH and to a lesser extent that of FSH. The activity of the corresponding neurons depends on a complex interplay between external and internal factors that will ultimately influence the triggering of puberty and sexual maturation. Among these factors are sex steroids and other peripheral hormones and growth factors, but little is known regarding their targets. However, very recently a new actor has entered the field of reproductive physiology. KiSS1, first known as a tumor suppressor called metastin, and its receptor GPR54, are now central to the regulation of GnRH, and consequently LH and FSH secretion in mammals. The KiSS system is notably viewed as instrumental in integrating both environmental cues and metabolic signals and passing this information onto the reproductive axis. In fish, there are two KiSS genes, KiSS1 and KiSS2, expressed in neurons of the preoptic area and mediobasal hypothalamus. Pionneer studies indicate that KiSS and GPR54 expression seem to be activated at puberty. Although precise information as to the physiological effects of KiSS1 in fish, notably on GnRH neurons and gonadotropin release, is still limited, KiSS neurons may emerge as the "gatekeeper" of puberty and reproduction in fish as in mammals., (Copyright 2009 Elsevier Inc. All rights reserved.)

Developing neurons are guided to their appropriate targets by specific guidance substances that have neurotrophic actions. The aim of the present study was to elucidate the mechanism by which hypothalamic neurons reach the pars intermedia (PI) by correlating the development of dopaminergic (DA) neurons arising in the periventricular nucleus (PeV) of fetal rats with the expression of brain-derived neurotrophic factor (BDNF) in the rat pituitary. The differentiation of DA neurons was observed by immunohistochemistry using an antibody against tyrosine hydroxylase (TH), whereas the ontogenesis of BDNF mRNA in the PI was examined by in situ hybridization and RT-PCR. Immunoreactive TH-neurons were first observed in the PeV at embryonic day (E) 16.5, following which time their axons elongated toward the pituitary. TH-positive reactions were observed in the connective tissue between the PI and the pars nervosa at E20.5. Innervation of the PI by TH-positive neurons was determined at postnatal day (P) 1.5; however, BDNF mRNA was first detected in the PI cells at E17.5, with an increase in its expression clearly visible at E21.5 and continuing high expression levels in the PI thereafter. These results suggest that BDNF is a specific guidance cue for DA neurons elongating from the PeV to the PI.

Interoceptive feedback signals from the body are transmitted to hypothalamic neurons that control pituitary hormone release. This review article describes the organization of central neural pathways that convey ascending visceral sensory signals to endocrine neurons in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus in rats. A special emphasis is placed on viscerosensory inputs to corticotropin releasing factor (CRF)-containing PVN neurons that drive the hypothalamic-pituitary-adrenal axis, and on inputs to magnocellular PVN and SON neurons that release vasopressin (AVP) or oxytocin (OT) from the posterior pituitary. The postnatal development of these ascending pathways also is considered.

Genistein is a phytoestrogen, particularly abundant in soybeans that can bind estrogen receptors and sex hormone binding proteins, exerting both estrogenic and antiestrogenic activity. In this study we used the Japanese quail embryo as a test end-point to investigate the effects of early embryonic exposure to genistein on male copulatory behavior and on vasotocin parvocellular system. Both differentiate by the organizational effects of estradiol during development and may therefore represent an optimal model to study the effects of xenoestrogens. We injected two doses of genistein (100 and 1000 microg) into the yolk of 3-day-old Japanese quail eggs. Other eggs were treated with either 25 microg of estradiol benzoate or sesame oil as positive and negative controls. At the age of 6 weeks, behavioral tests revealed a significant decrease of all aspects of copulatory behavior (in comparison to the control group) in estradiol-treated birds. In contrast, genistein-treated animals demonstrated various degrees of decrease in the mean frequencies of some aspects of the sexual behavior. The computerized analysis of vasotocin innervation in medial preoptic, stria terminalis and lateral septum nuclei revealed a statistically significant decreased immunoreactivity in treated animals compared to control ones. These results demonstrate that genistein, similarly to estradiol, has an organizational effect on quail parvocellular vasotocin system and on copulatory behavior. In conclusion, present results confirm, in this avian model, that embryonic exposure to phytoestrogens may have life-long effects on sexual differentiation of brain structures and behaviors.

Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated.

We elucidated the structure of the principle factors regulating the initiation of the acute stress response in common carp: corticotrophin-releasing hormone (CRH), CRH-receptor 1 (CRH-R1) and CRH-binding protein (CRH-BP). Phylogenetic analyses reveal that these proteins are evolutionarily well conserved in vertebrates. CRH and CRH-BP expression are not co-localised in the same hypothalamic perikarya. On the contrary, CRH-BP expression is limited to the perimeter of the nucleus preopticus (NPO), but is abundant in other regions, including an area directly rostral from, and in close proximity to, the NPO. Despite the lack of co-expression, the nerve fibres projecting onto both the rostral pars distalis (rPD) as well as the large fibre bundles projecting onto the pars intermedia (PI) contain CRH as well as CRH-BP, suggesting that both ACTH release from the rPD as well as the release of PI melanotrope content is regulated via CRH and CRH-BP. Finally, we show via real-time quantitative PCR that expression of hypothalamic CRH and CRH-BP following a 24 h restraint significantly increases, whereas PD CRH-R1 expression decreases; this reflects desensitisation of the PD for hypothalamic CRH output. We conclude that these factors are actively involved in the regulation of acute stress responses in the teleost fish.

An attempt has been made to immunocytochemically visualize the effect of neuropeptide Y (NPY) on the GnRH fibers and luteinizing hormone (LH) secreting cells in the pituitary of the catfish, Clarias batrachus. Two hours following the intracranial administration of NPY at the dose of 20 ng/g body wt, a significant increase in the area occupied by the GnRH-like immunoreactive fibers, and population and size of the LH cells was observed. The treatment also resulted in considerable augmentation of immunoreactivity in the LH cells. Double immunolabeling revealed NPY fibers directly terminating on the LH cells. The results suggest that NPY may (a) stimulate some GnRH containing hypophysiotropic neuronal group in the brain, (b) promote anterograde transport of GnRH to the pituitary gland, and (c) up-regulate the LH cells.

To elucidate which factors regulate growth hormone (GH) secretion in rainbow trout, dopaminergic innervation of the rainbow trout pituitary along with the action of dopamine in vitro, were studied. Brains with attached pituitaries were double-labeled for putative dopaminergic neuronal fibers and somatotropes, using fluorescence immunohistochemistry. A direct dopaminergic innervation to the proximal pars distalis (PPD) with dopaminergic fibers terminating adjacent to somatotropes was demonstrated. Growth hormone secretion from whole pituitaries was measured in perifusate using a homologous GH-RIA. Dopamine (DA; 10(-7)-2x10(-6) g ml(-1)) increased basal GH secretion, with the GH secretion normalizing again after the DA exposure was halted. When pituitaries were pre-treated with somatostatin-14 (SRIF-14; 10(-12)-10(-9) g ml(-1)), before being exposed to different doses of DA, there was an inhibition of GH secretion which was not reversed after treatment of SRIF-14 was halted, unless DA was added. It is concluded that dopamine can function as a GH secretagogue in the rainbow trout pituitary gland.

The development of the catfish gonadotropin-releasing hormone (cfGnRH) fiber network in the pituitary of male African catfish (Clarias gariepinus) was investigated in relation to puberty. Double immunolabeling studied by confocal laser scanning microscopy revealed a concomitant development of gonadotropes and of pituitary cfGnRH innervation during the first wave of spermatogenesis. Catfish GnRH-immunoreactive fibers in the proximal pars distalis (PPD) of the pituitary were initially observed at the age of 10 weeks (onset of spermatogonial proliferation) and gradually reached the adult pattern at the age of 20 weeks (spermatozoa present in the testis). The content of cfGnRH-associated peptide (cfGAP, part of the prohormone) in the pituitary similarly increased during puberty. At the electron microscopical level, fibers containing cfGAP-ir granules came into close proximity of the gonadotropes at 18 weeks of age. In vitro studies indicated a progressively increasing basal and cfGnRH-stimulated luteinizing hormone (LH) secretion during pubertal development. The LH secretion patterns were similar in response to exogenous cfGnRH (0.1 microM) or to endogenous cfGnRH, the release of which was induced by forskolin (1 microM). Castration experiments demonstrated that the innervation of the pituitary with cfGnRH fibers continued after surgery, accompanied by an increase in the cfGAP levels. However, gonadotrope development was retarded, suggesting a differential regulation of the two maturational processes. Since testosterone stimulates both processes, other testicular factors may also be involved. Puberty-associated changes in LH release patterns appear to reflect changes in the GnRH sensitivity and in the pool of releasable LH, while availability of cfGnRH does not appear to be a limiting factor., (Copyright 2000 S. Karger AG, Basel)

The release of a bioassayable form of growth hormone (BGH), distinct from growth hormone as measured by immunoassay (IGH), from the rat pituitary into the blood is differentially regulated by afferent input from fast and slow skeletal muscles. Specifically, activation of low-threshold fast muscle afferents for 15 min increased plasma BGH by 217 and 295% and decreased pituitary BGH by 68 and 45% in male and female rats, respectively. In contrast, activation of slow muscle afferents inhibited BGH release, decreasing plasma BGH by approximately 60% and increasing pituitary BGH by 30-50% in male rats. Female rats from which food had been withheld for approximately 12 h had elevated basal plasma BGH levels, which then were decreased by 81% after slow muscle nerve stimulation. Plasma IGH concentrations were unchanged after any nerve stimulation condition. These results demonstrate that regulation of BGH release can be differentially mediated through low-threshold afferent inputs from fast or slow skeletal muscle. Furthermore, the results indicate that BGH responses are independent of gender or feeding status.

L1 is a murine multidomain glycoprotein implicated in cell aggregation, fasciculation. neurite outgrowth and synaptogenesis. Laminin, a trimeric polypeptide, is implicated in neuronal survival, growth cone guidance, neurite outgrowth and cell differentiation. Laminin can also interact with the cell adhesion molecule L1. Their expressions were investigated from embryonic day 15 (E15) to adult in the rat hypophysis, and in adult neurohemal zones. Detected in the neural lobe from E17, the L1 immunoreactivity increased during prenatal development and persisted in adulthood mainly related to the neuropeptidergic fibers. Pituicytes were only labelled on the plasmalemma apposed to axons. In the intermediate lobe, L1 appeared at birth on folliculo-stellate cells extensions, constituting a network which densified during postnatal development. L1 is also expressed in all neurohemal areas on neuronal profiles. Laminin was clearly detectable in the hypophysis at E15 before the first blood vessels penetrate the Rathke pouch. At E20, all the basal membranes of the blood vessels were stained. In the intermediate lobe, a spotted laminin immunoreactivity was detected at E21. At this stage, we observed the staining of intercellular spaces and the intracellular labelling of melanotrophs, concerning reticulum or vesicles. The staining of melanotrophs seemed to maintain during adulthood. In contrast with blood vessels of the adult cerebral tissue, adult capillaries of the neural lobe and the others neuro-hemal zones were intensely labelled with the anti-laminin antibody. These results suggest that neurite outgrowth and neurite guidance could be promoted by L1 and laminin in the neurointermediate lobe. The "intercellular tunnels" could also be an important guidance cue for migrating cells in the intermediate lobe. These data also demonstrate that melanotrophic cells. secreting the laminin, have a role in the ontogenesis of the gland. Finally, we suggest that L1 and laminin can collaborate to reinforce "neurons-capillaries" interactions in neurohemal zones.

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.

The pituitary intermediate lobe was studied by immunocytochemistry on semithin sections and by electron microscopy in gerbils (Gerbillus pyramidum) caught in summer and winter in the natural biotope or experimentally submitted to chronic hydration or dehydration. In the gerbil, the intermediate lobe was formed by a predominating population of tightly packed melanotrophic cells the immunocytochemical and morphological features of which were comparable to those described in other mammals. A few typical corticotrophic cells were scattered in the contact zone with the neural lobe. Folliculostellate cells labelled with antibodies against glial fibrillary acid protein and vimentin were interspersed between the glandular cells; they formed small follicles in the vicinity of which the apical cytoplasm contained conspicuous dense granules. Both glandular cell types were innervated by axons most probably colocalizing dopamine and gamma-aminobutyric acid. In the pituitaries from the gerbils caught in winter, as from those having free access to hydrated food, the melanotrophic cells exhibited morphological characteristics of high functional activity. In the gerbils caught in summer or receiving exclusively dry food, the secretory activity of the cells was obviously depressed. The corticotrophic cells were unaffected. These observations raise the question of the role of the intermediate lobe in the adaptation to desert life.

Recent molecular studies have noted the affinity among cephalochordates and vertebrates. In particular, a cluster of vertebrate-like homeobox genes regulates the development of the lancelet Branchiostoma lanceolatum. A previous study has outlined the expression pattern of the pituitary-specific transcription factor Pit-1 in adult lancelets. Pit-1 belongs to the POU family of transcription factors, which, like homeotic proteins, are members of the helix-turn-helix superfamily of proteins. POU is an acronym for Pit-1, Oct-1 and Oct-2, and Unc-86. In the present work, we investigated the head region of premetamorphic larvae of B. lanceolatum, by means of scanning electron microscopy, wholemount and tissue sections immunocytochemistry, and Western blotting assay, to verify the presence and distribution of Pit-1. Immunoreactive Pit-1 protein was detected in the rostral nerves and in a cluster of photoreceptor cells of the frontal eye. At the same time, an electrophoretic band of 33 kDa was shown from extracts of premetamorphic larvae and recognized by a monoclonal antibody to rat Pit-1. On the basis of the immunocytochemical and electrophoretic results, we can assume that Pit-1 may play a neuromodulatory role in the larval central nervous system. Moreover, the spatial and temporal distribution of Pit-1 protein in larva and adult lancelets agrees only in part with that described in embryonic and adult mice, suggesting different molecular controls of regional identity in the nervous system of cephalochordates and vertebrates.

Synaptophysin (SN) is a synaptic-vesicle-associated membrane protein whose presence is indicative of intact, functional synapses. This study examines the presence of SN in pituitary gland innervation after neurotoxin-induced denervation followed by reinnervation. Immunostaining of rat pituitary neurointermediate lobe tissue for SN reveals a pattern of dot-like densities in the intermediate lobe and intensely stained dispersed regions in the neural lobe of normal animals. In rats treated with 6-hydroxydopamine (6-OHDA), a catecholamine neurotoxin, by peripheral injection, there is a significant depletion of the SN immunostaining in the intermediate lobe, as well as a significant reduction of SN immunoreactivity in the neural lobe, in animals studied 1 wk after drug treatment, with computer analysis of the tissue sections. At 3 wk after 6-OHDA, there is a partial recovery of immunoreactivity for SN in the neural lobe in many tissue sections, and the intermediate lobe also contains only relatively sparse staining for the synaptic protein. Computer analysis revealed that at 3 wk after 6-OHDA, both lobes still had reduced SN immunoreactivity, but the difference in levels measured did not achieve statistical significance. These results contrast with the prior finding of significant recovery of immunoreactivity for GAP-43, a growth and regeneration-associated protein, in intermediate lobe innervation of rats treated with the same drug regimen. We suggest that 6-OHDA treatment damages synaptic vesicle integrity in both the intermediate and neural lobes of the pituitary, and that recovery is in progress, but not complete at 3 wk after the drug is administered.

In most teleosts, there are three groups of gonadotropin-releasing hormone (GnRH) neurons. In this study we addressed the question of GnRH neuronal innervation of the pituitary in the dwarf gourami and the tilapia using immunocytochemistry combined with biocytin tract tracing. Biocytin was applied to the pituitary attached to the brain in vitro. Similar results were obtained in both species. GnRH neurons retrogradely labeled with biocytin were observed only in the preoptic area. These results indicate that preoptic GnRH neurons innervate the pituitary. Negative labeling of biocytin in the terminal-nerve and midbrain GnRH neurons suggests that these two GnRH neuronal populations do not project to the pituitary. Biocytin-positive but GnRH-negative neurons were also observed in the preoptic area and the ventromedial parts of the hypothalamus, suggesting neuropeptidergic and aminergic innervation of the pituitary besides GnRH.

Dopamine and GABA were detected in intermediate lobe axons around birth, and early axons were closely apposed to glial cells and processes, possibly using them for guidance. In the adult, axons containing colocalized dopamine and GABA were distributed in a distinct pattern within the lobe, with plexuses located dorsally and ventrally. Axons preferentially followed glial processes in interlobular septa, yet were also interspersed between melanotropes. Individual melanotropes were contacted by varying numbers of axon terminals, with some devoid of contacts. Boutons contained both small clear vesicles and large dense-cored vesicles; membrane specializations were not well-developed. From these findings we concluded that in addition to direct synaptic inhibition, dopamine and GABA could stimulate their receptors by mechanisms similar to "parasynaptic" [Schmitt (1984) Neuroscience, 13:991-1001] or "volume" [Agnati et al. (1995) Neuroscience, 69:711-726] transmission as proposed for the CNS. Humoral agents passing into the intermediate lobe from portal vessels, thus acting as classical hormones, further regulate the melanotropes. Moreover, approximately 50% of the axonal elements were closely apposed to glia, suggesting that glia could have regulatory roles. Previous studies from our laboratory [Chronwall et al. (1987) Endocrinology, 120:1201-1211; Chronwall et al. (1988) Endocrinology, 123:1992:1202] demonstrated heterogeneity in proopiomelanocortin (POMC) biosynthesis among individual melanotropes, prompting the hypothesis that the degree of innervation could govern the expression of certain molecules. We combined immunohistochemistry and in situ hybridization histochemistry to evaluate whether melanotrope molecular heterogenity is spatially correlated with axons and terminals. Tentatively, melanotropes expressing low levels of POMC and alpha1A subunit P/Q type Ca2+ channel mRNAs often were apposed to axons, whereas those with low levels of D2L receptor mRNA rarely were contacted by axons, suggesting that innervation could be one of the factors inducing and maintaining heterogeneity.

In most vertebrates, multiple gonadotropin-releasing hormone (GnRH) neuronal groups have been reported. In tilapia three GnRH neuronal groups (terminal nerve, preoptic, midbrain) have been reported. Which of the three GnRH cell groups regulate the pituitary is not well known. We performed brain lesions of each neuronal group and studied immunocytochemically the changes of GnRH fiber distribution in the pituitary. Lesions of the preoptic cell group resulted in almost complete absence of GnRH fibers in the neurohypophysis of the proximal pars distalis. After lesions of the terminal nerve GnRH cell group, no changes were observed in the distribution of GnRH fibers in the pituitary. Lesions of the midbrain cell group were unsuccessful because of high mortality. The present study indicates that the preoptic GnRH cell group is the main contributor of the pituitary innervation.

The biosynthetic activity of rat intermediate lobe melanotropes in vivo is inhibited by stimulation of dopamine D2 receptors. Individual melanotropes are innervated differentially by dopaminergic axons and vary in their levels of pro-opiomelanocortin (POMC) mRNA. We tested the hypothesis that placement of the lobe in primary culture, which removes the inhibitory innervation, would increase POMC mRNA levels and abolish the heterogeneity in POMC expression. POMC mRNA levels increased successively in untreated melanotropes when tested on culture Days 10, 16, and 20; however, some heterogeneity in POMC expression persisted. If treated with a D2 receptor agonist (1 microM bromocriptine) from culture Day 1, POMC mRNA levels were decreased significantly throughout the testing period when compared to untreated cells with the same time in culture. Although some melanotropes still expressed high POMC levels, preparations appeared more homogeneous by Day 20. Melanotrope responses were reversible, since POMC mRNA levels were down-regulated by application and up-regulated by withdrawal of a D2 receptor agonist. A short agonist treatment resulted in subpopulations that responded differently to the agonist, possibly representing a mechanism for fine-tuning peptide hormone release.

The glandular activity of the vertebrate pituitary intermediate lobe (IL) is regulated by direct cellular innervation, in contrast with the purely humoral regulation of adjacent pituitary anterior lobe (AL). Thus in the rat IL, melanotrophs receive a dopaminergic and GABAergic innervation from the basal hypothalamus, which tonically inhibit their glandular activity. We studied this model of neuron-target interactions in cocultures in defined medium of fetal hypothalamic neurons with neonate pituitary glandular cells. In the cocultures with IL cells, neuroglandular contacts occurred after 4 days in vitro (DIV) but required another 8 DIV to exhibit ultrastructural and immunocytochemical features of fully differentiated functional synapses; by contrast, neuroneuronal synapses developed much faster and could already be detected after 4 DIV. In the cocultures with AL cells, neuroglandular contacts never mature in differentiated synapses. Confocal microscope observation revealed that dopaminergic neurons, which represented less than 1% of total neurons in the cocultures, established 50% of the synapses detected on the melanotrophs. These cells are thus able, contrary to the AL cells, to promote the establishment of functional synapses and, to some extent, to select their specific innervation.

1. A novel slowly activating voltage-dependent K+ current was observed in isolated nerve terminals from rat neurohypophysis using the whole-cell configuration of the patch-clamp technique. 2. The activation kinetics of the slow current could be fitted assuming Hodgkin--Huxley-type kinetics, an exponential, n, of 1.3 and activation time constants decreasing from 4 s at -50 mV to 0.7s at +40 mV. 3. A positive shift of reversal potential was observed when [K+] was increased in the bath solution. The current is carried mainly but not exclusively by K+ ions. 4. When intracellular free [Mg2+] was low (approximately 60 microM), average current density was 74 pA pF-1 at membrane potentials around 0 mV. In 83% of nerve terminals current amplitude was > 10 pA pF-1. 5. The slow current was never observed when the pipette contained 4.6 mM free Mg2+. At a physiological level of free Mg2+ (0.5 mM) the average current density was 16 pA pF-1. 6. When nerve terminals were analysed after patch-clamp experiments for vasopressin content by immunodetection, no difference in current amplitude was found between the terminals containing vasopressin and all analysed terminals. 7. The voltage dependence of activation was fitted by a Boltzmann equation giving a half-activation potential of -37 mV and a slope factor of about 9 mV. 8. Tail current deactivation kinetics was biexponential with time constants of 0.12 and 1.5s. Kinetics was dependent on the duration of the activating pulse. 9. Noise analysis of the slow current indicated a single-channel current of 0.33 pA at +6 mV, corresponding to a single-channel conductance of 4.3 pS. 10. This is the first demonstration of a current similar to the slow K+ current, IKs, in a neurone, suggesting that a protein similar to the IKs-inducing channel protein IsK (minK) may be present in peptidergic nerve terminals. 11. The activation properties are consistent with a role of the slow current in inhibition of excitability, at least at the level of the nerve terminal.

The retinoblastoma gene (RB) is the prototypic tumor suppressor. Studies to date have demonstrated cancer suppression with tumor cells reconstituted with RB ex vivo and implanted into immunodeficient mice, as well as with germline transmission of a human RB transgene into tumor-prone Rb +/- mice. To mimic the therapy of cancer more closely, spontaneous pituitary melanotroph tumors arising in immunocompetent Rb +/- mice were treated with a recombinant adenovirus carrying RB cDNA. Intratumoral RB gene transfer decreased tumor cell proliferation, reestablished innervation by growth-regulatory dopaminergic neurons, inhibited the growth of tumors, and prolonged the life spans of treated animals.

Neuropeptide Y (NPY)-containing neural projections to the rat pituitary gland were studied by combining NPY immunohistochemistry with retrograde tracing with Fluorogold as well as central and peripheral denervations. Numerous pituitary-projecting, i.e. Fluorogold-labelled, neurons in the superior cervical ganglion, as well as in the hypothalamic magnocellular nuclei were NPY-immunoreactive (NPY-IR). In contrast, no other hypothalamic NPY-IR neurons, e.g. in the arcuate nucleus or the preoptic area, were observed to be projecting into the pituitary. Within the posterior lobe of the pituitary gland two morphologically distinct NPY-IR fiber populations were discovered, namely thinner parenchymal terminals, distinct from the neurosecretory terminals, and thicker, perivascular fibers. Neurosecretory nerve terminals, in contrast, were devoid of NPY-IR, being consistent with the previous reports on their sensitivity to osmotic stimulation. On the other hand, the anterior and intermediate lobes contained no NPY-IR fibers. Bilateral extirpation of the superior cervical ganglion resulted in disappearance of the perivascular NPY-IR fibers leaving the parenchymal NPY-IR fibers unaffected, while transection of the pituitary stalk abolished all of the parenchymal NPY-IR neurons, leaving the perivascular fibers unaffected. These findings together with the observed colocalization of tyrosine hydroxylase and NPY in the posterior lobe perivascular fibers indicated that they are sympathetic nerve endings. The thin parenchymal terminals, instead, are suggested to stem from central sources other than hypothalamus. Our findings indicate that the pituitary gland receives NPY-containing innervation from at least three distinct sources, and NPY may thus affect pituitary functions in various ways, such as blood flow and vasopressin release.

To better understand the cell lineage-specific character of retinoblastoma (Rb) gene inactivation during tumor formation, the earliest stages of spontaneous melanotroph carcinogenesis in Rb+/- heterozygous mice have been subjected to sequential analyses. The first atypical cells are detected in the pituitary intermediate lobe during a period corresponding to the cessation of melanotroph proliferation between 35 and 60 days after birth. Atypical cells contain no wild-type copy of the Rb gene and synchronously form early atypical proliferates (EAP) in the subsequent 30-60 day period. In contrast to surrounding mature melanotrophs with the wild-type Rb gene, Rb-negative cells in EAP continue to proliferate well past postnatal day 60, and fail to be innervated by growth inhibitory dopaminergic nerve terminals. Atypical melanotrophs remain competent for dopamine D2 receptor stimulation and undergo S-phase apoptosis in close proximity to nerve terminals. These results indicate a key role for the Rb protein in the onset of neuron-neuroendocrine cell interactions. This role may explain cell-type-specific neuroendocrine carcinogenesis associated with inactivation of the ubiquitously expressed Rb gene.

Acetylcholine (ACh) plays an important role in pituitary gland function. Little is known, however, about the source and trajectory of pituitary ACh, the location of pituitary cholinergic receptors, and the pathways along which the release of pituitary ACh is controlled. Therefore choline acetyltransferase (CHAT) immunoreactive profiles have been investigated in the rat median eminence and pituitary. Furthermore, both muscarinic- (mAChRp-L) and nicotinic receptor proteinlike (nAChRp-L) immunoreactivity have been examined in the rat, rabbit, and cat pituitary. The results have demonstrated that the rat pituitary ChAT network is composed of neurons in the hypothalamic arcuate nucleus and a great number of terminals in the median eminence. In the pituitary, ChAT immunolabeled profiles were virtually absent. This suggests that much of the ACh acting on pituitary cells is released as a humoral factor from the median eminence. All the examined animals expressed mAChRp-L immunostained endocrine cells in the intermediate lobe. Apart from this, marked species differences in AChRp-L immunolabeled profiles have been found. In addition, strong mAChRp-L immunoreactive rod to cone-shaped bodies were detected associated with blood vessels of the anterior and intermediate lobes in the rat and rabbit, but not in the cat. The immunolabeling was present in particles on the body plasma membrane. These characteristics suggest that the function of these structures might be to sense pituitary blood ACh levels. Consequently the name blood acetylcholine reading bodies (BARBs) was adopted to indicate these structures. It is proposed that the BARBs may play a role in the feedback control of ACh release from the median eminence.

Early chemical desympathisation with 6-hydroxydopamine decreased the contents of the follicle-stimulating hormone in the blood serum of 1-month old rats. The steroidogenesis disturbances were more obvious in sexually immature rats in the general desympathisation. In sexually mature rats, these disturbances disappeared. Sympathetic innervation seems to play a major part in development of the reproductive system in rats.

Neural cell adhesion molecules (NCAMs) can undergo post-translational modifications, such as the addition of polysialic acid chains, thus generating PSA-NCAMs, which are expressed mainly during development. Since polysialylation considerably modifies NCAM adhesivity, expression of NCAMs and PSA-NCAMs has been investigated in the developing hypophysis by immunohistochemistry. At embryonic day 13 (E13), an antibody against NCAM outlined all cellular profiles in the entire Rathke's pouch; this labelling persisted until adulthood. NCAM expression increased in all lobes during development and concerned all pituitary cell types. In contrast, at E13, PSA-NCAMs were only detected in the neural lobe, solely constituted of pituicytes at this stage, and the tuberal lobe, the only lobe expressing hormonal mRNA at the same stage. PSA-NCAMs expression increased in the neural lobe at E17 with the arrival of the neurosecretory fibres and persisted into adulthood. In the anterior lobe, PSA-NCAMs appeared at E15 where their distribution was similar to that of the differentiating corticotrophic cells; at subsequent stages, their expression extended to the whole anterior lobe. Only two cell types, corticotrophic and somatotrophic cells, remained labelled in the adult gland. In the intermediate lobe, melanotrophic cells never expressed PSA-NCAMs but these were expressed on folliculo-stellate cells at birth, preceding the onset of innervation. These results suggest that NCAMs and PSA-NCAMs play a role in pituitary histogenesis, cell differentiation and neurointermediate lobe innervation.

The neuroanatomical organization of neurons projecting to the pituitary and the origin of pituitary dopamine and nonapeptides were investigated in the brain of the Atlantic salmon (Salmo salar). Carbocyanine tract tracing in combination with tyrosine hydroxylase, arginine vasotocin and isotocin immunocytochemistry for double labelling revealed a previously unknown organization of hypophysiotrophic cell groups and their extrahypothalamic projections, and provide the first direct identification in a teleost fish of the origin of the dopaminergic and nonapeptidergic innervation of the pituitary. The present data include identification of (1) hypophysiotrophic neurons in the ventral telencephalon and in the periventricular preoptic nucleus, (2) large (magnocellular) vasotocinergic hypophysiotrophic neurons in the most rostral extension of the preoptic area, (3) a distinct neuronal group located in a supraoptic/suprachiasmatic position in the anterior periventricular nucleus, that seems to be the major source of dopaminergic innervation of the pituitary, (4) the nonapeptidergic hypophysiotrophic neurons in the preoptic nucleus, (5) hypophysiotrophic neurons in the ventral and posterior hypothalamus of which some are of liquor-contacting type, (6) projections from hypophysiotrophic and non-hypophysiotrophic neurons in the preoptic nucleus to extrahypothalamic areas such as thalamic and periventricular pretectal nuclei, and (7) subdivisions within the preoptic nucleus that exhibit different combinations of hypophysiotrophic and extrahypothalamic efferent connections. Together with previous studies of retinohypothalamic projections and neurochemical organization of hypothalamic/preoptic areas, the present data suggest that the preoptic nucleus and the anterior periventricular nucleus in teleosts possess functional subdivisions with features that resemble those of the paraventricular, supraoptic and suprachiasmatic nuclei of other vertebrates. In the Atlantic salmon, specific dopaminergic and nonapeptidergic neuronal subdivisions are proposed to play a role for photoperiod control of endocrine activity.

The nerve terminals in the intermediate and posterior lobes of the rat pituitary gland are reported to show colocalization of serotonin and tyrosine hydroxylase. This study examined the extent of this colocalization in the pituitary gland and in the nuclei considered to project to the pituitary. In the intermediate lobe, two types of nerve fibers were encountered, one containing serotonin (5-HT-IR) and tyrosine hydroxylase (TH-IR) immunoreactivities and the other showing 5-HT-IR only. Instead, there was no colocalization in the posterior lobe. In the hypothalamus, colchicine treatment with L-tryptophan and pargyline injections resulted in 5-HT-IR in some neurons in the dorsomedial, periventricular and arcuate nuclei, some of which in the arcuate and periventricular nuclei were also TH-IR. In the raphe nuclei no colocalization of 5-HT-IR and TH-IR was observed. Catecholamine neurotoxin, 6-hydroxydopamine, abolished the 5-HT-IR and dramatically reduced the TH-IR in the intermediate lobe nerve fibers. Both effects were prevented by cocaine, a monoamine uptake inhibitor, but not by fluoxetine, a specific serotonin uptake inhibitor. Serotonin neurotoxin p-chloroamphetamine (PCA) had no effect on intermediate lobe fibers, although it caused complete disappearance of 5-HT-IR from the posterior lobe nerve fibers. This effect was prevented by fluoxetine. Our results indicate, that colocalization of serotonin and TH observed in the intermediate lobe occurs both in the nerve terminals within the lobe and in some nuclei that innervate it. Furthermore, drug treatments suggest that serotonin in the intermediate lobe is localized in catecholaminergic fibers, which do not posses a specific serotonin uptake mechanism.

Following administration of met-enkephalin into the third ventricle of rats of both sexes, an ultrastructural-morphometric study was carried out of the neural lobe of the hypophysis, comparing the results with those obtained in untreated animals, controls (injected intraventricularly with distilled water) and animals previously receiving naloxone intraperitoneally. In the untreated and control animals, both males and females, there was a high percentage (about 70%) of neurosecretory axons considered to have a normal morphology; after met-enkephalin administration, this percentage decreased and was accompanied by a rise (slightly more pronounced in the females) in the percentage of degranulated axons and a slight rise in axons with a morphology indicative of regenerative and degenerative phases. Previous administration of the opiate antagonist naloxone led to a rise in the number of axons in the degenerative phase with respect to the other groups of animals; this occurred in both sexes.

Afferent projections to the pituitary pars intermedia were studied using the DiI tract-tracing technique in two amphibian species, the urodelan Triturus carnifex, and the anuran Rana esculenta. After DiI crystal application into the pituitary intermediate lobe, in both species cells were retrogradely labeled in the preoptic nucleus, in the supra- and retro-chiasmatic hypothalamus and in the brainstem (especially in the area indicated as locus coeruleus). The findings are discussed in relation to data on the neurochemical nature of the innervation of the pars intermedia in amphibians.

Innervation within the aging male rat pituitary neurointermediate lobe was examined using immunocytochemical methods with antisera to rate-limiting enzymes for serotonin (tryptophan hydroxylase, TpOH) or catecholamine (tyrosine hydroxylase TH) synthesis. Immunostaining for both enzymes was observed in both neural and intermediate lobes in younger rats (age 6 weeks or 7 months), but TpOH was absent from the intermediate lobe in older groups (10-12, 15-16, and 18-19 months). Colocalization of TpOH and TH was demonstrated in some nerve fibers, using immunofluorescence. The oldest age group showed a reduced staining pattern for TH. 5-hydroxytryptophan, the immediate precursor for serotonin (5-HT), was taken up and converted to immunoreactive 5-HT in all age groups. Overall, the pattern of staining for regulatory neurotransmitters or enzymes which ultimately affect pituitary intermediate lobe secretory function is significantly modulated during the aging process.

The hypothalamic hypophysiotrophic neurones are densely innervated by adrenergic and noradrenergic nerve terminals. Activation of alpha 1-adrenoceptors located in the brain stimulates the secretion of ACTH, prolactin and TSH. The effects of the alpha 1-adrenoceptors seem to be exerted on hypothalamic neurones that secrete vasopressin, CRH-41 and TRH. These mechanisms are important in the physiological control of the secretion of ACTH and TSH in humans. alpha 2-Adrenoceptors are not involved in the control of secretion of these hormones under basal conditions in humans. However, alpha 2-adrenoceptors exert an inhibitory effect that acts as a negative feedback mechanism, limiting excessive secretion of these hormones. There is no convincing evidence for the involvement of beta-adrenoceptors in the control of the secretion of these three hormones in humans. Studies on cultured anterior pituitary cells suggested that adrenaline and noradrenaline may influence the secretion of ACTH, prolactin and TSH directly at the level of the pituitary. However, these effects are not demonstrable in humans, and are likely to be due to alterations in the pituitary adrenoceptors during culture. In the case of growth hormone, activation of alpha 2-adrenoceptors located in the brain stimulates secretion of this hormone both by increasing the secretion of GHRH and by inhibiting the secretion of somatostatin. Activation of beta-adrenoceptors inhibits the secretion of growth hormone via an increase in the secretion of somatostatin. The effects of the central alpha 2- and beta-adrenoceptors are important in the physiological control of growth hormone secretion in humans. A considerable amount of evidence implicates brain alpha 1-adrenoceptors in the control of secretion of the gonadotrophins in experimental animals, but, despite intensive study, no convincing evidence has been found in humans of reproductive age.

The development of the intermediate lobe of the rabbit pituitary was investigated by light and electron microscopy and by using immunocytological techniques. The first immunoreactive melanotrophic cells were detected at the fetal day 17 in the dorsal zone of Rathke's pouch epithelium facing the neural lobe; this coincided ultrastructurally with the appearance in this area of a few cells exhibiting secretory vesicles and granular condensations in the Golgi saccules. The differentiation of the gland probably required an infundibular inductive effect. Secretory cells increased in number following a dorsoventral gradient during the next fetal and neonatal stages until postnatal day 20, the stage at which the intermediate lobe exhibited its definitive organization. The gland innervation occurred during the first days after birth. The advent of these oxytocin- and neurophysin-immunoreactive fibres coincided with an obvious stimulation of the synthetic activity of the melanotrophic cells. The possible neurotrophic effect of these cells on their innervating system remains to be established.

Patch-clamp recording techniques were applied to thin slices of the rat pituitary gland in order to study synaptic transmission between hypothalamic nerve terminals and neuroendocrine cells of the intermediate lobe. Inhibitory postsynaptic currents (IPSCs) could be evoked by electrical stimulation of afferent neuronal fibres in the surrounding tissue of the slice. The IPSCs could be evoked in an all-or-nothing mode depending on the stimulus intensity, suggesting that single afferent fibres were stimulated. They had a chloride-dependent reversal potential and were blocked by bicuculline (Kd = 0.1 microM), indicating that they were mediated by gamma-aminobutyric acid A (GABAA) receptors. In symmetrical chloride solutions the current/voltage relation of the IPSC peak amplitudes was linear. The IPSCs were characterized by a fast (1-2 ms) rise time and a biexponential decay, with time constants of 21 +/- 4 ms and 58 +/- 14 ms at a holding potential of -60 mV (n = 6 cells). Both decay time constants increased with depolarization in an exponential manner. Spontaneously occurring IPSCs had a time course that was similar to that of evoked IPSCs. These miniature IPSCs, recorded in 1 microM tetrodotoxin, displayed an amplitude distribution that was well fitted by single Gaussian functions, with a mean value of its maxima of 18.1 +/- 2.3 pA (n = 4 cells). Amplitude histograms of evoked IPSCs were characterized by multiple peaks with a modal amplitude of about 18 pA (n = 6 cells). These findings indicate the quantal nature of GABAergic synaptic transmission in this system, with a quantal conductance step of about 280 pS. Single-channel currents underlying the IPSCs were studied by bath application of GABA to outside-out patches excised from intermediate lobe cells. Such GABA-induced currents revealed two conductance levels of 14 pS and 26 pS. In conclusion, GABAergic synaptic transmission in neuroendocrine cells of the pituitary has properties that are quite similar to those observed in neurones of the central nervous system.

The distribution of FMRFamide-like immunoreactivity was determined in the brain and the pituitary of the goldfish, Carassius auratus. Immunoreactive perikarya were observed in the olfactory nerve, nucleus entopeduncularis, nucleus anterioris periventricularis, nucleus posterioris periventricularis, lateral part of the nucleus lateralis tuberis pars posterioris, midbrain tegmentum, and medulla oblongata. Immunoreactive fibers were widely distributed in the brain, in particular in the ventral telencephalon and the hypothalamus. A few immunoreactive nerve fibers were observed in the pituitary. The findings are discussed in relation to male sexual behavior and the involvement of FMRFamide-like peptide in pituitary functions in the goldfish.

Certain thick FMRFamide-like immunoreactive fibers arising from the ganglion cells of nervus terminalis in the olfactory bulb of Clarias batrachus can be traced centripetally through the medial olfactory tract, telencephalon, lateral preoptic area, tuberal area, and hypothalamohypophysial tract to the pituitary. Following 6 days of bilateral olfactory tract transection, the immunoreactivity in the thick fibers, caudal to the lesion site, was partially eliminated, whereas after 10 and 14 days, it was totally abolished in the processes en route to the pituitary. The results indicate a direct innervation of the pituitary gland by the FMRFamide-like peptide containing fibers of the nervus terminalis.

The activities of periventricular-hypophysial dopaminergic (DA) neurons were compared in male and female rats by measuring dopamine synthesis (accumulation of 3,4-dihydroxyphenylalanine [DOPA] after inhibition of L-aromatic amino acid decarboxylase) and metabolism (concentrations of 3,4-dihydroxyphenylacetic acid [DOPAC]) in terminals of these neurons in the intermediate lobe of the pituitary. For comparison, the synthesis and metabolism of dopamine in the neural lobe of the pituitary and median eminence were also determined. The concentrations of DOPAC and accumulation of DOPA were higher in females than in males in both the intermediate lobe and median eminence, revealing a sexual difference in the basal activity of periventricular-hypophysial and tuberoinfundibular DA neurons. In contrast, there were no differences between male and female rats in activity of DA neurons terminating in the neural lobe. One week following gonadectomy, DOPA accumulation in the median eminence was decreased in females and increased in males, but remained unchanged in the intermediate lobe. These results indicate that sexual differences in the activity of periventricular-hypophysial DA neurons terminating in the intermediate lobe are not dependent upon the presence of circulating gonadal steroids, and in this respect, these neurons differ from tuberoinfundibular DA neurons.

The aim of the present experiments was to determine whether some rationale for the presence of the several inhibitory neurotransmitters in the innervation to the toad melanotroph might be found in differences in their individual effects or in possible cooperative interactions affecting secretion. Measurements of peptide release from isolated, perifused neurointermediate lobes of the toad Xenopus laevis showed that each of the three identified inhibitory transmitters, dopamine, GABA and NPY, was able to inhibit secretion profoundly and no less effectively than omission of Ca. Moreover, the inhibitory effects were rather similar in onset, duration and recovery. Furthermore, there was no evidence of any cooperative interactions when the transmitters were given in various combinations. And finally, the inhibitory response to each of the transmitters was abolished by pretreatment with pertussis toxin. While not excluding differential postsynaptic effects on other parameters of melanotroph function, the similarities observed have encouraged alternative speculations on the significance of the apparent redundancy of inhibitory transmitters.

The distribution of the octapeptide Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL), a proenkephalin A-derived opioid peptide, in the rat and mouse pituitary gland was studied using the indirect immunofluorescence technique and immunoelectron microscopy. The anterior lobe contained a few MEAGL-immunoreactive cells but no nerve fibers. A previously unknown enkephalin-immunoreactive nerve fiber system was revealed in the intermediate lobe. These fibers originated in a dense MEAGL-immunoreactive plexus located along the border between the intermediate and posterior lobes and were distributed throughout the lobe. In the posterior lobe, MEAGL immunoreactivity was found in a very dense network of varicose fibers that was evenly distributed over the entire lobe. These results provide a morphological correlate for previous chemical studies and together with them suggest that MEAGL-immunoreactive innervation regulates endocrine functions of the intermediate and posterior lobes directly at the pituitary level.

The intermediate lobe of the pituitary is composed of a homogeneous population of endocrine cells, the melanotrophs, which secrete several bioactive peptides including alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. In contrast to most endocrine glands which are richly vascularized, the intermediate lobe of the pituitary contains very few blood vessels; in some species, the pars intermedia is virtually totally avascular. In contrast, pituitary melanotrophs are richly supplied by nerve fibers originating from the hypothalamus. The pars intermedia thus appears as a pure model of neuroendocrine communication, i.e. it is an archetype of the mode of transducing interface between the central nervous system and endocrine effectors. In mammalian species, different types of nerve terminals containing dopamine, norepinephrine, gamma-aminobutyric acid (GABA) and serotonin have been identified. In lower vertebrates, particularly in fish and amphibians, the pars intermedia is also innervated by peptidergic fibers which are though to take part in regulation of the secretory activity of the melanotroph. In these animals, the pars intermedia is regarded as a major center of neuroendocrine integration and an exceptional model to investigate the process of communication between the brain and the endocrine glands. The purpose of the present review is to summarize our current knowledge on the synthesis, processing and release of peptide hormones from pars intermedia cells and to survey the multiple regulatory mechanisms which are involved in the control of the activity of pituitary melanotrophs. Proopiomelanocortin, a multifunctional precursor. Pituitary melanotrophs synthetise a major precursor protein called proopiomelanocortin (POMC) which generates through proteolytic cleavage several biologically active peptides including adrenocorticotropic hormone (ACTH), endorphins and MSHs. In lower vertebrates, alpha-MSH is generally considered as the major hormone secreted by melanotrophs, in that it is involved in the process of skin colour adaptation. The post-translational processing of POMC, which yields to the mature hormones released by melanotrophs, includes a number of steps: glycosylation, phosphorylation, tissue-specific proteolytic cleavage, amidation and acetylation. Some of these posttranslational modifications can be regulated by neuroendocrine factors. For instance, in frogs, it has been shown that dopamine inhibits acetylation of alpha-MSH and thus reduces the secretion of the biologically active form of the peptide. The intermediate lobe of the pituitary: a model of neuroendocrine integration. In most vertebrate species, the intermediate lobe of the pituitary is innervated by catecholamine-containing fibers. In particular, the presence of dopaminergic nerve fibers has been observed in the pars intermedia of mammals and poikilotherms.(ABSTRACT TRUNCATED AT 400 WORDS)