Protocol for targeting the magnocellular neuroendocrine cell ensemble via retrograde tracing from the posterior pituitary.

The hypothalamic magnocellular neuroendocrine cells (MNCs) project to the posterior pituitary (PPi), regulating reproduction and fluid homeostasis. It has been challenging to selectively label and manipulate MNCs, as they are intermingled with parvocellular neuroendocrine cells projecting to the median eminence. Here, we provide a step-by-step protocol for specifically targeting the MNCs by infusing retrograde viral tracers into the PPi. When combined with optogenetics, chemogenetics, and transgenic animals, this approach allows cell-type-specific manipulation of MNCs in multiple sites for functional dissection. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021) and Tang et al. (2020).

Vasopressin at Central Levels and Consequences of Dehydration.

Disorders of water balance are a common feature of clinical practice. An understanding of the physiology and pathophysiology of central vasopressin release and perception of thirst is the key to diagnosis and management of these disorders. Mammals are osmoregulators; they have evolved mechanisms that maintain extracellular fluid osmolality near a stable value, and, in animal studies, osmoregulatory neurons express a truncated delta-N variant of the transient receptor potential vannilloid (TRPV1) channel involved in hypertonicity and thermal perception while systemic hypotonicity might be perceived by TRPV4 channels. Recent cellular and optogenetic animal experiments demonstrate that, in addition to the multifactorial process of excretion, circumventricular organ sensors reacting to osmotic pressure and angiotensin II, subserve genesis of thirst, volume regulation and behavioral effects of thirst avoidance.

Architecture of the hypothalamo-posthypophyseal complex is controlled by monoamines.

The hypothalamo-neurohypophyseal system displays significant plasticity when subjected to physiological stimuli, such as dehydration, parturition, or lactation. This plasticity arises at the neurochemical and electrophysiological levels but also at a structural level. Several studies have demonstrated the role of monoaminergic afferents in controlling neurochemical and electrophysiological plasticity of the supraoptic nucleus (SON) and of the neurohypophysis (NH), but little is known about how the changes in structural plasticity are triggered. We used Tg8 mice, disrupted for the monoamine oxidase A gene, to study monamine involvement in the architecture of the SON and of the NH. SON astrocytes in Tg8 mice displayed an active status, characterized by an increase in S100ß expression and a significant decrease in vimentin expression, with no modification in glial fibrillary acidic protein (GFAP) levels. Astrocytes showed a decrease in glutamate dehydrogenase (GDH) levels, whereas glutamine synthetase (GS) levels remained constant, suggesting a reduction in astrocyte glutamate catabolism. Tenascin C and polysialic acid-neural cell adhesion molecule (PSA-NCAM) expressions were also elevated in the SON of Tg8 mice, suggesting an increased capacity for structural remodelling in the SON. In the NH, similar date were obtained with a stability in GFAP expression and an increase in PSA-NCAM immunostaining. These results establish monoamine (serotonin and noradrenaline) involvement in SON and NH structural arrangement. Monoamines therefore appear to be crucial for the coordination of the neurochemical and structural aspects of neuroendocrine plasticity, allowing the hypothalamo-neurohypopyseal system to respond appropriately when stimulated.

Oxytocin-induced elevation of ADP-ribosyl cyclase activity, cyclic ADP-ribose or Ca(2+) concentrations is involved in autoregulation of oxytocin secretion in the hypothalamus and posterior pituitary in male mice.

Locally released oxytocin (OT) activates OT receptors (2.1:OXY:1:OT:) in neighboring neurons in the hypothalamus and their terminals in the posterior pituitary, resulting in further OT release, best known in autoregulation occurring during labor or milk ejection in reproductive females. OT also plays a critical role in social behavior of non-reproductive females and even in males in mammals from rodents to humans. Social behavior is disrupted when elevation of free intracellular Ca(2+) concentration ([Ca(2+)](i)) and OT secretion are reduced in male and female CD38 knockout mice. Therefore, it is interesting to investigate whether ADP-ribosyl cyclase-dependent signaling is involved in OT-induced OT release for social recognition in males, independent from female reproduction, and to determine its molecular mechanism. Here, we report that ADP-ribosyl cyclase activity was increased by OT in crude membrane preparations of the hypothalamus and posterior pituitary in male mice, and that OT elicited an increase in [Ca(2+)](i) in the isolated terminals over a period of 5 min. The increases in cyclase and [Ca(2+)](i) were partially inhibited by nonspecific protein kinase inhibitors and a protein kinase C specific inhibitor, calphostin C. Subsequently, OT-induced OT release was also inhibited by calphostin C to levels inhibited by vasotocin, an OT receptor antagonist, and 8-bromo-cADP-ribose. These results demonstrate that OT receptors are functionally coupled to membrane-bound ADP-ribosyl cyclase and/or CD38 and suggest that cADPR-mediated intracellular calcium signaling is involved in autoregulation of OT release, which is sensitive to protein kinase C, in the hypothalamus and neurohypophysis in male mice.

Plasticity of nonapeptidergic neurosecretory cells in fish hypothalamus and neurohypophysis.

The structure and function of nonapeptidergic neurosecretory cells (NP-NSC) are considered in terms of comparative morphology. Among NSC of different ergicity for NP-NSC the most characteristic involve massive accumulation and storage of neurohormonal products. Only in NP-NSC are the secretory cycles of functioning clearly expressed. Their highest reactivity is established during experimental and physiological stresses. In contrast, liberinergic, statinergic, and monoaminergic NSC, unlike NP-NSC, are characterized even in the "norm" by a constantly high level of extrusion processes. As signs of maximum NP-NSC plasticity, we consider the largest size of elementary neurosecretory granules, the diversity of secretion forms, and the maximum development of Herring bodies-clear manifestations of secretory cycles of functioning. In particular, phases of massive storage of neurosecretory granules in the extrusion cycle of NP-NSC neurosecretory terminals express accumulation of neurosecretory products. It is concluded that a particularly high degree of plasticity of NP-NSC is provided by their capability for functional reversion. This reversion is manifested first in the form of the restoration of the initial moderate level of functioning and especially in the accumulation of neurosecretory products. The reversion is considered an important mechanism providing a high degree of NSC plasticity. This degree turns out to be sufficient for participation of NP-NSC in the integration of fish reproduction. It is shown that NP-NSC are organized by the principle of a triad of the balanced system. This system consists of two alternative states: accumulation and release of neurosecretory products and the center of control of dynamics of their interrelations, the self-regulating center. In the latter, the key role is probably played by the Golgi complex.

Coexistence of salusin and vasopressin in the rat hypothalamo-hypophyseal system.

Salusins are two newly discovered TOR-related peptides consisting of 28 and 20 amino acids and designated salusin-alpha and salusin-beta, respectively. Using immunohistochemistry techniques, salusin-like immunoreactivity was detected in the rat hypothalamo-neurohypophyseal tract and immunopositive cells were distributed in the suprachiasmatic, supraoptic and paraventricular nucleus. In the paraventricular nucleus, salusin-like immunoreactivity was observed both in parvocellular and magnocellular neurons. Many salusin-positive nerve fibers and their terminals were identified in the internal layer of the median eminence and posterior pituitary. Less intense salusin-positive staining of fibers and terminals was found in the suprachiasmatic nucleus and external layer of the median eminence. Dual immunostaining was performed to determine if salusin coexisted with vasopressin or oxytocin in the hypothalamus. Most of the salusin-like immunoreactivity was detected in vasopressin- but not in oxytocin-containing neurons in these nuclei. The functional significance of the coexistence of salusin with vasopressin is discussed, including the possibility that salusin participates in the regulation of blood pressure.

Localization of transforming growth factors, TGFbeta1 and TGFbeta3, in hypothalamic magnocellular neurones and the neurohypophysis.

The distribution of transforming growth factor beta (TGFbeta) in the rat and human hypothalamus and neurohypophysis was investigated by immunocytochemical techniques using rabbit polyclonal antisera against TGFbeta(1) and TGFbeta(3). Colocalization of TGFbeta(1) or TGFbeta(3) and arginine vasopressin (AVP) in the rat hypothalamus was studied by double immunolabelling in light microscopy, while their subcellular localization in the rat neurohypophysis was investigated by immunoelectron microscopy. TGFbeta(1) and TGFbeta(3) immunoreactivity was demonstrated in the cell bodies and processes of neurones in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). The TGFbeta-immunoreactive cells were more numerous in the SON compared to the PVN. TGFbeta/AVP double-labelled cells were seen in both nuclei, but some neurones in the SON were labelled for TGFbeta(1) or TGFbeta(3), although not for AVP. In the rat and human neurohypophysis, TGFbeta(3) immunolabelling was more diffuse and stronger than TGFbeta(1) immunolabelling. TGFbeta(1) expression was seen in axonal vesicles and in neurosecretory granules of the axonal endings, while TGFbeta(3) was observed in axonal fibres. Colocalization of TGFbeta(3) or TGFbeta(1) and AVP was observed in some neurosecretory granules, but many were either single-labelled for TGFbeta or AVP or unlabelled. Our results demonstrate, for the first time, the colocalization of TGFbeta and neurohypophysial hormones in magnocellular neurones. We suggest that TGFbeta secreted by the neurohypophysis regulates the proliferation and secretion of certain anterior pituitary cells.

Distribution of androgen-binding protein in the rat hypothalamo-neurohypophyseal system, co-localization with oxytocin.

Androgen-binding protein (ABP) is known to be expressed in the male and female rat hypothalamus. In the present study, we observed immunocytochemically ABP in neurons of the magnocellular hypothalamic nuclei, in the preoptic region and in the lateral hypothalamus. Dense fiber networks with varicosities, containing ABP immunofluorescence, were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed a partial coexistence of ABP-and oxytocin immunoreactivity in a portion of the magnocellular perikarya. ABP was isolated by affinity chromatography from hypothalamus homogenates. Western blots resulted in immunoreactive (IR) bands with an approximate molecular weight of 35 and 50 kDa. Mass spectrometry of these preparations confirmed the presence of ABP, which was almost identical to ABP isolated from rat testis. It is likely that ABP, expressed in magnocellular oxytocinergic neurons, is subject to axonal transport and release in the hypothalamo-neurohypophyseal system.

Alpha-melanocyte-stimulating hormone stimulates oxytocin release from the dendrites of hypothalamic neurons while inhibiting oxytocin release from their terminals in the neurohypophysis.

The peptides alpha-melanocyte stimulating hormone (alpha-MSH) and oxytocin, when administered centrally, produce similar behavioral effects. alpha-MSH induces Fos expression in supraoptic oxytocin neurons, and alpha-MSH melanocortin-4 receptors (MC4Rs) are highly expressed in the supraoptic nucleus, suggesting that alpha-MSH and oxytocin actions are not independent. Here we investigated the effects of alpha-MSH on the activity of supraoptic neurons. We confirmed that alpha-MSH induces Fos expression in the supraoptic nucleus when injected centrally and demonstrated that alpha-MSH also stimulates Fos expression in the nucleus when applied locally by retrodialysis. Thus alpha-MSH-induced Fos expression is not associated with electrophysiological excitation of supraoptic neurons because central injection of alpha-MSH or selective MC4 receptor agonists inhibited the electrical activity of oxytocin neurons in the supraoptic nucleus recorded in vivo. Consistent with these observations, oxytocin secretion into the bloodstream decreased after central injection of alpha-MSH. However, MC4R ligands induced substantial release of oxytocin from dendrites in isolated supraoptic nuclei. Because dendritic oxytocin release can be triggered by changes in [Ca2+]i, we measured [Ca2+]i responses in isolated supraoptic neurons and found that MC4R ligands induce a transient [Ca2+]i increase in oxytocin neurons. This response was still observed in low extracellular Ca2+ concentration and probably reflects mobilization of [Ca2+]i from intracellular stores rather than entry via voltage-gated channels. Taken together, these results show for the first time that a peptide, here alpha-MSH, can induce differential regulation of dendritic release and systemic secretion of oxytocin, accompanied by dissociation of Fos expression and electrical activity.

Neurons expressing neuropeptide Y mRNA in the infundibular hypothalamus of Japanese quail are activated by fasting and co-express agouti-related protein mRNA.

The neural mechanisms involved in the compensatory hyperphagia exhibited by many vertebrate species after a fast are not fully understood but, in mammals, appear to involve nutritionally-sensitive neurons that co-express neuropeptide Y (NPY) and agouti-related protein (AGRP) in the infundibular hypothalamus. We investigated whether these neurons have been evolutionarily conserved in a non-mammalian vertebrate, the Japanese quail. Birds exhibited compensatory hyperphagia 1 h after return of food following a 24-h fast. We addressed a potential regulatory role for NPY, first, by using in situ hybridisation (ISH) to map NPY gene expression in the hypothalamus. This revealed a strong signal in the infundibular nucleus (IN). Secondly, we quantified NPY gene expression in 24-h fasted birds compared to ad libitum fed controls using two independent methods. In whole hypothalamus, measured by ribonuclease protection assay, NPY mRNA increased 1.5-fold in fasted birds. A similar, 1.7-fold, increase was observed specifically in the IN when analysed by ISH. No differences in NPY expression between fed and fasted birds were observed in other brain regions. To determine whether NPY neurons in the avian IN co-express AGRP, we cloned a fragment of the quail AGRP gene and used it to localise AGRP mRNA by ISH. The gene was expressed exclusively in the hypothalamus, specifically in the IN, where its distribution matched that of NPY. Double-label ISH revealed that the majority of NPY neurons in the IN co-express AGRP mRNA. Collectively, these data indicate that this cell type has been neuroanatomically and functionally conserved during vertebrate evolution.

Corticotropin-releasing hormone-synthesizing neurons of the human hypothalamus receive neuropeptide Y-immunoreactive innervation from neurons residing primarily outside the infundibular nucleus.

Immunohistochemical single- and double-labeling studies were performed on the hypothalami of postmortem human brains to elucidate the distribution of corticotropin-releasing hormone (CRH)-immunoreactive (IR) neuronal elements and their interaction with the neuropeptide Y (NPY)-ergic neuronal system. The great majority of CRH-IR perikarya were found in the paraventricular nucleus (PVN), whereas a considerable number of CRH-IR neurons were also observed in the periventricular and infundibular nuclei. The dorsomedial nucleus and the perifornical region contained only scattered CRH-IR neurons. Dense CRH-IR fiber networks were found throughout the hypothalamus. However, the medial preoptic, the dorsolateral part of the supraoptic, the suprachiasmatic, the ventromedial, and the different mammillary nuclei showed a relative paucity of fibers. The terminal fields of NPY-IR axons overlapped the distribution of CRH-IR neurons in the hypothalamus. NPY-IR axon varicosities were juxtaposed to both dendrites and perikarya of the majority of CRH-IR neurons residing in the paraventricular, periventricular, and infundibular nuclei. These neurons were frequently contacted by multiple NPY axons that either formed baskets around their perikarya or completely ensheathed the emanating CRH dendrites. Because NPY and agouti-related protein (AGRP) are co-contained in neurons of the human infundibular nucleus, we used AGRP as a marker of NPY fibers originating exclusively from the infundibular nucleus. Only a small proportion of CRH neurons in the PVN was contacted by AGRP-IR axon varicosities, suggesting that NPY-IR innervation of CRH neurons in the PVN derive mainly from regions outside the infundibular nucleus. The present morphological findings support the view that NPY regulates the CRH system of the human hypothalamus and therefore at least some of the effects of NPY on metabolic, autonomic, and endocrine functions may be mediated through CRH.

Double immunocytochemistry of vasoactive intestinal peptide and cGnRH-I in male quail: photoperiodic effects.

Reproduction in Japanese quail is primarily regulated by photoperiod. Vasoactive intestinal peptide (VIP) has been suggested as a transducer of environmental information, especially photoperiodic cues, to the hypothalamo-pituitary-gonadal axis. To investigate the possible interaction of VIP and the reproductive (gonadotropin-releasing hormone, GnRH) system, double-immunocytochemical staining for VIP and cGnRH-I was conducted in sexually mature male quail held under a long-day photoperiod (16L:8D; LD) and in sexually quiescent males held under a short-day photoperiod (8L:16D; SD). VIP-immunoreactive (ir) cells were found primarily in three locations: lateral septal organ (LSO) in nucleus accumbens (Ac), ventral hypothalamus, and infundibular area. VIP-ir cells in LSO displayed characteristics typical of cerebrospinal fluid (CSF)-contacting cells, and co-existed with cGnRH-I-ir cells and beaded fibers. In contrast, VIP-ir cells in the infundibular area did not co-exist with cGnRH-I-ir structures. The number of visible VIP-ir cells in the infundibular area of SD males was significantly lower than that of LD males, while the number of visible VIP-ir cells in Ac/LSO was not altered by photoperiod. A cluster of cGnRH-I-ir cells in the caudalmost septal area was heavily innervated by VIP-ir fibers, which appeared to contact cGnRH-I-ir cells directly at this location. Both VIP- and cGnRH-I-ir fibers heavily innervated the external layer of the median eminence (ME). These data suggest that Ac/LSO, the caudalmost septal area, and ME are possible sites of interaction between the VIP and the GnRH systems.

A role of adhesion molecules in neuroglial plasticity.

Glial cells are exquisitely sensitive to changes in neuronal activity, and their capacity for structural plasticity including migration is critical for remodeling an repair of nervous tissue. Our in vitro studies suggest that isoforms of the neural cell adhesion molecule (NCAM) carrying an unconventional carbohydrate polymer, polysialic acid (PSA), are involved in these events. We have demonstrated that neurohypophyseal explants from newborn rats generate cellular outgrowth of immature astrocytes displaying the characteristics of oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells previously identified in the optic nerve. Treatment of O-2A cells with the enzyme Endo N, which specifically removes PSA from the cells surface, produced a complete blockade of the dispersion of the O-2A cell population from the explant. Identical effects of Endo N were observed in migration assays using cortical O-2A cells. Neurohypophyseal O-2A cells express functional NMDA class of glutamate receptors and the pharmacological blockade of these receptors inhibit PSA-NCAM biosynthesis and dramatically diminish O-2A cell migration from neurohypophyseal explants. This suggests a potential mechanism through which neuronal activity via glutamate release may regulate PSA-NCAM expression on immature glial cells, which in turn is critical for their migration.

Regulated expression of the cell adhesion glycoprotein F3 in adult hypothalamic magnocellular neurons.

F3, a glycoprotein of the immunoglobulin superfamily implicated in axonal growth, occurs in oxytocin (OT)-secreting and vasopressin (AVP)-secreting neurons of the adult hypothalamo-neurohypophysial system (HNS) whose axons undergo morphological changes in response to stimulation. Immunocytochemistry and immunoblot analysis showed that during basal conditions of HNS secretion, there are higher levels of this glycosylphosphatidyl inositol-anchored protein in the neurohypophysis, where their axons terminate, than in the hypothalamic nuclei containing their somata. Physiological stimulation (lactation, osmotic challenge) reversed this pattern and resulted in upregulation of F3 expression, paralleling that of OT and AVP under these conditions. In situ hybridization revealed that F3 expression in the hypothalamus is restricted to its magnocellular neurons and demonstrated a more than threefold increase in F3 mRNA levels in response to stimulation. Confocal and electron microscopy localized F3 in secretory granules in all neuronal compartments, a localization confirmed by detection of F3 immunoreactivity in granule-enriched fractions obtained by sucrose density gradient fractionation of rat neurohypophyses. F3 was not visible on any cell surface in the magnocellular nuclei. In contrast, in the neurohypophysis, it was present not only in secretory granules but also on the surface of axon terminals and glia and in extracellular spaces. Taken together, our observations reveal that the cell adhesion glycoprotein F3 is colocalized with neurohypophysial peptides in secretory granules. It follows, therefore, the regulated pathway of secretion in HNS neurons to be released by exocytosis at their axon terminals in the neurohypophysis, where it may intervene in activity-dependent structural axonal plasticity.

Combined use of immunocytochemistry and lectin histochemistry for the study of the hypothalamic neurosecretory system of the snake Natrix maura (L.).

Natrix maura snakes were processed for immunocytochemistry and lectin histochemistry at both light- and electron-microscopic levels. Antisera against bovine neurophysins, vasotocin and mesotocin were used as well as concanavalin A, wheat germ and Limax flavus agglutinin lectins. The hypothalamic supraoptic and paraventricular nuclei were studied. Vasotocin neurons should contain a glycopeptide and displayed large colloid droplets consisting of large cisternae filled with packed secretory material. Mesotocin was located in different neurons.

Pituitary of "cobalt" variant of the rainbow trout separated from the hypothalamus lacks most pars intermedial and neurohypophysial tissue.

Adenohypophysial cell types and neurohypophysial tissues were investigated in the cobalt variant of the rainbow trout, which possesses an irregularly-shaped pituitary. The pituitary remnant was completely detached from the hypothalamus in all but one fish, in which a remnant was associated with the hypothalamus. Prolactin (PRL) and growth hormone (GH) cells were predominant cell types in all pituitary remnants, forming PRL and GH areas, respectively. There were fewer somatolactin (SL) and melanophore-stimulating hormone cells than in normal fish. There were few corticotropin cells in cobalt and normal trout. Although aldehyde-fuchsin positive cells were also present, positively-staining fibers were not detected in any of remnants examined. Plasma SL levels were much lower in cobalt than in normal trout. Plasma levels of GH and T3 were significantly lower in cobalt than in normal fish. Plasma levels of PRL, T4 and cortisol, plasma osmolality, ions (Na+, K+, Ca2+, and Mg2+), glucose, triglyceride, free fatty acids, and amino nitrogen concentrations were similar in the two groups. Cobalt variants thus lack hypophysial pars intermedia and neurohypophysial tissues. Various abnormalities of the cobalt variant, such as the cobalt blue body color and the fat deposition in the abdominal cavity, may be related to the absence of the pars intermedia.

Vasoactive intestinal peptide (VIP) in magnocellular neurons of the hypothalamo-neurohypophysial system of the mink (Mustela vision) is co-localized with vasopressin or oxytocin.

The distribution of vasoactive intestinal peptide (VIP) was analysed in perikarya of the mink hypothalamus with immunohistochemistry and, surprisingly, a large population of magnocellular VIP-immunoreactive neurons was present in the paraventricular and supraoptic nuclei as well as in accessory hypothalamic nuclei. From perikarya in the paraventricular as well as supraoptic nuclei, a large number of VIP immunoreactive nerve fibers was observed to enter the hypothalamo-neurohypophysial tract. Within the median eminence, a high density of VIP-immunoreactive nerve fibers was present in the external and internal zones. Fibers in the external zone of the median eminence were endowed with varicosities and perivascular terminals, while fibers in the internal zone were smooth and without terminal specializations. From the internal zone of the median eminence, fibers coursed via the infundibular stalk to terminate in perivascularly situated terminals in the neurohypophysis. In addition, a substantial number of small VIP-immunoreactive perikarya was observed within the suprachiasmatic nucleus. These perikarya were immunoreactive to neither vasopressin nor neurophysin. To elucidate the co-existence of VIP-immunoreactivity with vasopressin, oxytocin or neurophysin, a sequential double immunoperoxidase procedure to localize antigens with diaminobenzidine and benzidine dihydrochloride as chromagens was performed. From these experiments it was evident that VIP in nearly all magnocellular hypothalamo-neurohypophysial neurons co-existed with neurophysin. Based on a semi-quantitative estimate, half the VIP-immunoreactive magnocellular perikarya co-stored vasopressin, while another half co-stored oxytoxin. The present study describes the presence of a large population of VIP-containing neurons in the hypothalamo-neurohypophysial system of the mink. These findings raise evidence that within the mink, VIP may be involved in neurohypophysial physiology.

Anatomical organization of the hypophysiotrophic systems in the electric fish, Apteronotus leptorhynchus.

The organization of afferents to the pituitary was investigated by applying DiI crystals to the pituitary or pituitary stalk of the gymnotiform electric fish, Apteronotus leptorhynchus. Most hypophysiotrophic cells were found in the hypothalamus and were distributed throughout its rostrocaudal extent: nucleus preopticus periventricularis, pars anterior and posterior; suprachiasmatic nucleus; anterior, dorsal, ventral, lateral, and caudal hypothalamic nuclei; and nucleus tuberis lateralis, pars anterior and posterior. In addition a small number of retrogradely labeled cells were found in the ventral telencephalon (area ventralis, pars ventralis) and, most surprisingly, in a thalamic nucleus (nucleus centralis posterioris). The nucleus preopticus periventricularis pars posterior and the anterior hypothalamic nucleus appear to correspond to the parvicellular and magnocellular divisions of the nucleus preopticus of other teleosts. Integration of these results with immunohistochemical localization of monoamines and neuropeptides in the apteronotid brain suggests many homologies between the hypophysiotrophic nuclei of teleosts and other vertebrates, including mammals. Apteronotus communicates electrically during agonistic and sexual interactions. There are numerous anatomical links between the hypophysiotrophic systems and the brain areas related to electrocommunication.

Immunocytochemical localization of VIP-immunoreactive neurons in the hypothalamus of immature gilts.

Immunocytochemical study using avidin-biotin complex method and peroxidase-antiperoxidase technique was carried out to determine the distribution and morphology of vasoactive intestinal polypeptide-immunoreactive (VIP-IR) neuronal structures in the hypothalamus of the immature female pig. VIP-IR neurons were found in the arcuate, paraventricular and supraoptic nuclei, as well as in the pituitary stalk. The median eminence disclosed an abundance of VIP-IR processes both in the internal and external layer. These results let us conclude that there exist convenient morphological conditions for the release of VIP into portal blood in the area of the porcine median eminence-pituitary stalk complex.

Cells of origin of histaminergic afferents to the cat median eminence.

The exact origin of histaminergic neuronal perikarya sending axons to the median eminence and posterior pituitary was investigated in the cat by using two colour double-immunostaining methods: unconjugated wheat germ agglutinin or cholera toxin as retrograde tracers combined with histamine (HA) immunohistochemistry. HA immunohistochemistry revealed the presence of HA-immunoreactive terminal-like fibers both in the external layer of the median eminence and neural lobe of the pituitary. The double-labeling studies further demonstrated the histaminergic innervation of the median eminence and neural lobe by a few HA-immunoreactive neuronal perikarya located in the posterior hypothalamus.