Virus Injection to the Pituitary via Transsphenoidal Approach and the Innervation of Anterior and Posterior Pituitary of Rat.

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.

Hypothalamic Response to Kisspeptin-54 and Pituitary Response to Gonadotropin-Releasing Hormone Are Preserved in Healthy Older Men.

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.

Unraveling vasotocinergic, isotocinergic and stress pathways after food deprivation and high stocking density in the gilthead sea bream.

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.

Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland.

The dopamine D2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D2 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 D2 receptor. D2 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 D2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D2 receptors. D2 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.

Multiple cytosolic calcium buffers in posterior pituitary nerve terminals.

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.

Expression of orexin receptors in the pituitary.

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.

Lack of the ventral anterior homeodomain transcription factor VAX1 leads to induction of a second pituitary.

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.

Neuroendocrinology of reproduction in teleost fish.

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.

Visceral sensory inputs to the endocrine hypothalamus.

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.

Effects of early embryonic exposure to genistein on male copulatory behavior and vasotocin system of Japanese quail.

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.

Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary of the rat.

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.

Possible involvement of brain-derived neurotrophic factor (BDNF) in the innervation of dopaminergic neurons from the rat periventricular nucleus to the pars intermedia.

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.

Structural characterisation of a cyprinid (Cyprinus carpio L.) CRH, CRH-BP and CRH-R1, and the role of these proteins in the acute stress response.

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.

GnRH-LH secreting cells axis in the pituitary of the teleost Clarias batrachus responds to neuropeptide Y treatment: an immunocytochemical study.

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.

Dopaminergic innervation of the rainbow trout pituitary and stimulatory effect of dopamine on growth hormone secretion in vitro.

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.

Gonadotropin-releasing hormone fibers innervate the pituitary of the male African catfish (Clarias gariepinus) during puberty.

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.

Afferent input from rat slow skeletal muscle inhibits bioassayable growth hormone release.

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.

The pituitary intermediate lobe of the hydrated and dehydrated gerbil. An immunohistochemical and electron-microscopic study.

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.

L1 and laminin: their expression during rat hypophysis ontogenesis and in adult neurohemal areas.

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.

Serotonergic innervation of the pituitary pars intermedia of xenopus laevis.

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.