Photoperiodic co-regulation of kisseptin, neurokinin B and dynorphin in the hypothalamus of a seasonal rodent.

In many species, sexual activity varies on a seasonal basis. Kisspeptin (Kp), a hypothalamic neuropeptide acting as a strong activator of gonadotrophin-releasing hormone neurones, plays a critical role in this adaptive process. Recent studies report that two other neuropeptides, namely neurokinin B (NKB) and dynorphin (DYN), are co-expressed with Kp (and therefore termed KNDy neurones) in the arcuate nucleus and that these peptides are also considered to influence GnRH secretion. The present study aimed to establish whether hypothalamic NKB and DYN expression is photoperiod-dependent in a seasonal rodent, the Syrian hamster, which exhibits robust seasonal rhythms in reproductive activity. The majority of Kp neurones in the arcuate nucleus co-express NKB and DYN and the expression of all three peptides is decreased under a short (compared to long) photoperiod, leading to a 60% decrease in the number of KNDy neurones under photo-inhibitory conditions. In seasonal rodents, RFamide-related peptide (RFRP) neurones of the dorsomedial hypothalamus are also critical for seasonal reproduction. Interestingly, NKB and DYN are also expressed in the dorsomedial hypothalamus but do not co-localise with RFRP-immunoreactive neurones, and the expression of both NKB and DYN is higher under a short photoperiod, which is opposite to the short-day inhibition of RFRP expression. In conclusion, the present study shows that NKB and DYN display different photoperiodic variations in the Syrian hamster hypothalamus. In the arcuate nucleus, NKB and DYN, together with Kp, are down-regulated under a short photoperiod, whereas, in the dorsomedial hypothalamus, NKB and DYN are up-regulated under a short photoperiod.

Cannabinoid type-1 receptors in the paraventricular nucleus of the hypothalamus inhibit stimulated food intake.

Cannabinoid receptor type 1 (CB1)-dependent signaling in the brain is known to modulate food intake. Recent evidence has actually shown that CB1 can both inhibit and stimulate food intake in fasting/refeeding conditions, depending on the specific neuronal circuits involved. However, the exact brain sites where this bimodal control is exerted and the underlying neurobiological mechanisms are not fully understood yet. Using pharmacological and electrophysiological approaches, we show that local CB1 blockade in the paraventricular nucleus of the hypothalamus (PVN) increases fasting-induced hyperphagia in rats. Furthermore, local CB1 blockade in the PVN also increases the orexigenic effect of the gut hormone ghrelin in animals fed ad libitum. At the electrophysiological level, CB1 blockade in slices containing the PVN potentiates the decrease of the activity of PVN neurons induced by long-term application of ghrelin. Hence, the PVN is (one of) the site(s) where signals associated with the body's energy status determine the direction of the effects of endocannabinoid signaling on food intake.

Neurokinin B and the control of the gonadotropic axis in the rat: developmental changes, sexual dimorphism, and regulation by gonadal steroids.

Neurokinin B (NKB), encoded by Tac2 in rodents, and its receptor, NK3R, have recently emerged as important regulators of reproduction; NKB has been proposed to stimulate kisspeptin output onto GnRH neurons. Accordingly, NKB has been shown to induce gonadotropin release in several species; yet, null or even inhibitory effects of NKB have been also reported. The basis for these discrepant findings, as well as other key aspects of NKB function, remains unknown. We report here that in the rat, LH responses to the NK3R agonist, senktide, display a salient sexual dimorphism, with persistent stimulation in females, regardless of the stage of postnatal development, and lack of LH responses in males from puberty onward. Such dimorphism was independent of the predominant sex steroid after puberty, because testosterone administration to adult females failed to prevent LH responses to senktide, and LH responsiveness was not restored in adult males treated with estradiol or the nonaromatizable androgen, dihydrotestosterone. Yet, removal of sex steroids by gonadectomy switched senktide effects to inhibitory, both in adult male and female rats. Sexual dimorphism was also evident in the numbers of NKB-positive neurons in the arcuate nucleus (ARC), which were higher in adult female rats. This is likely the result of differences in sex steroid milieu during early periods of brain differentiation, because neonatal exposures to high doses of estrogen decreased ARC NKB neurons at later developmental stages. Likewise, neonatal estrogenization resulted in lower serum LH levels that were normalized by senktide administration. Finally, we document that the ability of estrogen to inhibit hypothalamic Tac2 expression seems region specific, because estrogen administration decreased Tac2 levels in the ARC but increased them in the lateral hypothalamus. Altogether, our data provide a deeper insight into relevant aspects of NKB function as major regulator of the gonadotropic axis in the rat, including maturational changes, sexual dimorphism, and differential regulation by sex steroids.

Co-localisation of kisspeptin with galanin or neurokinin B in afferents to mouse GnRH neurones.

The gonadotrophin-releasing hormone (GnRH) secreting neurones, which form the final common pathway for the central regulation of reproduction, are directly targeted by kisspeptin (KP) via the G protein-coupled receptor, GPR54. In these multiple labelling studies, we used ovariectomised mice treated with 17ß-oestradiol (OVX + E(2)) or vehicle (OVX + oil) to determine: (i) the ultrastructural characteristics of KP-immunoreactive (IR) afferents to GnRH neurones; (ii) their galanin or neurokinin B (NKB) content; and (iii) the co-expression of galanin or NKB with KP in the two major subpopulations of KP neurones located in the rostral periventricular area of the third ventricle (RP3V) and the arcuate nucleus (Arc). Electron microscopic investigation of the neuronal juxtapositions revealed axosomatic and axodendritic synapses; these showed symmetrical or asymmetrical characteristics, suggesting a phenotypic diversity of KP afferents. Heterogeneity of afferents was also demonstrated by differential co-expression of neuropeptides; in OVX + E(2) mice, KP afferents to GnRH neurones showed galanin-immunoreactivity with an incidence of 22.50 ± 2.41% and NKB-immunoreactivity with an incidence of 5.61 ± 2.57%. In OVX + oil animals, galanin-immunoreactivity in the KP afferents showed a major reduction, appearing in only 5.78 ± 1.57%. Analysis for co-localisation of galanin or NKB with KP was extended to the perikaryal level in animal models, which showed the highest KP incidence; these were OVX + E(2) females for the RP3V and OVX + oil females for the ARC. In the RP3V of colchicine-treated OVX + E(2) animals, 87.84 ± 2.65% of KP-IR neurones were galanin positive. In the Arc of the colchicine-treated OVX + oil animals, galanin immunoreactivity was detected in only 12.50 ± 1.92% of the KP expressing neurones. By contrast, the incidence of co-localisation with NKB in the Arc of those animals was 98.09 ± 1.30%. In situ hybridisation histochemistry of sections from OVX + E(2) animals identified galanin message in more than a third of the KP neurones in the RP3V (38.67 ± 11.57%) and in the Arc (42.50 ± 12.52%). These data suggest that GnRH neurones are innervated by chemically heterogeneous KP cell populations, with a small proportion deriving from the Arc group. The presence of galanin within KP axons innervating GnRH neurones and the oestrogen-dependent regulation of that presence add a new dimension to the roles played by galanin in the central regulation of reproduction.

The kisspeptin system of the human hypothalamus: sexual dimorphism and relationship with gonadotropin-releasing hormone and neurokinin B neurons.

Kisspeptin signaling via the kisspeptin receptor G-protein-coupled receptor-54 plays a fundamental role in the onset of puberty and the regulation of mammalian reproduction. In this immunocytochemical study we addressed the (i) topography, (ii) sexual dimorphism, (iii) relationship to gonadotropin-releasing hormone (GnRH) neurons and (iv) neurokinin B content of kisspeptin-immunoreactive hypothalamic neurons in human autopsy samples. In females, kisspeptin-immunoreactive axons formed a dense periventricular plexus and profusely innervated capillary vessels in the infundibular stalk. Most immunolabeled somata occurred in the infundibular nucleus. Many cells were also embedded in the periventricular fiber plexus. Rostrally, they formed a prominent periventricular cell mass (magnocellular paraventricular nucleus). Robust sex differences were noticed in that fibers and somata were significantly less numerous in male individuals. In dual-immunolabeled specimens, fine kisspeptin-immunoreactive axon varicosities formed axo-somatic, axo-dendritic and axo-axonal contacts with GnRH neurons. Dual-immunofluorescent studies established that 77% of kisspeptin-immunoreactive cells in the infundibular nucleus synthesize the tachykinin peptide neurokinin B, which is known to play crucial role in human fertility; 56 and 17% of kisspeptin fibers in the infundibular and periventricular nuclei, respectively, contained neurokinin B immunoreactivity. Site-specific co-localization patterns implied that kisspeptin neurons in the infundibular nucleus and elsewhere contributed differentially to these plexuses. This study describes the distribution and robust sexual dimorphism of kisspeptin-immunoreactive elements in human hypothalami, reveals neuronal contacts between kisspeptin-immunoreactive fibers and GnRH cells, and demonstrates co-synthesis of kisspeptins and neurokinin B in the infundibular nucleus. The neuroanatomical information will contribute to our understanding of central mechanisms whereby kisspeptins regulate human fertility.

An androgen-dependent sexual dimorphism visible at puberty in the rat hypothalamus.

Morphological studies in rodents have well documented the masculinization of the perinatal brain by estradiol derived from aromatized testosterone, and the resulting irreversible quantitative sex-differences generated in cell numbers or expression of chemical phenotypes. Here, using immunohistochemistry, we explored how this applies to the postnatal development and masculinization of the neurokinin B (NKB)-containing system of the arcuate nucleus/median eminence complex (ARC/ME). In adult rats, NKB-immunoreactive neurons exhibit an unusual, qualitative sexual dimorphism of their ventral axonal projections: to the neuropil in females, to capillary vessels in males. In adults, there was no sex-difference in the numbers of NKB-immunoreactive perikarya or capillary vessels in the ARC/ME, suggesting that this sexual dimorphism cannot be explained by the existence of supernumerary structures. At birth (day 0) the NKB system was immature in both sexes, and while its adult features emerged progressively until puberty in females, they did not develop before puberty (day 40) in males, revealing a sexual dimorphism only late postnatally. When males were orchidectomized at day 30, the masculine distribution of NKB-immunoreactive axons expected at day 40 was not seen, while it was apparent after chronic treatment with testosterone or dihydrotestosterone, suggesting a testicular masculinizing action via androgen receptors at puberty. Moreover in these prepubertal-orchidectomized males, the distribution of NKB-immunoreactive axons was surprisingly feminized by chronic estradiol alone, suggesting that NKB neurons are not irreversibly programmed before puberty. Last, in adult females, the distribution of NKB-immunoreactive axons was feminine 30 days after ovariectomy, and it was masculinized after concurrent chronic dihydrotestosterone, suggesting that NKB neurons remain responsive to androgens late in reproductive life. Thus, the sexual differentiation of the hypothalamus proceeds well beyond the perinatal period and includes the epigenetic action of non-aromatizable androgens upon subsets of neurons that have retained bipotent features.

Sexual dimorphism in the organization of the rat hypothalamic infundibular area.

The hypothalamic infundibular area is located outside the blood-brain barrier and includes, the ventromedial arcuate nucleus (vmARC) sensing circulating substances, and the median eminence (ME) where neurohormones are released into the hypothalamo-hypophysial vasculature. This integrated functional unit, pivotal in endocrine control, adjusts neuroendocrine output to feedback information. Despite a differing physiology in males and females, this functional unit has not appeared differently organized between sexes. Using immunocytochemistry, we describe here for the first time in adult rats, a conspicuous sex-difference in its axonal wiring by intrinsic glutamatergic neurons containing the neuropeptides neurokinin B (NKB) and dynorphin. In the male, NKB neurons send axons to capillary vessels of the vmARC and of the ME (only where gonadotropin-releasing hormone (GnRH) axons terminate). Electron microscopy revealed that NKB axons target the barrier of tanycytes around fenestrated capillary vessels (in addition to GnRH axons), suggesting a control of regional bidirectional permeability. In the female, NKB neurons send axons to the neuropile of the vmARC, suggesting a direct control of its sensor neurons. The other projections of NKB neurons, studied by surgical isolation of the ARC-ME complex and confocal microscopy, are not sexually dimorphic and target both integrative and neuroendocrine centers controlling reproduction and metabolism, suggesting a broad influence over endocrine function. These observations demonstrate that the mechanisms subserving hypothalamic permeability and sensitivity to feedback information are sexually dimorphic, making the infundibular area a privileged site of generation of the male-to-female differences in the adult pattern of pulsatile hormonal secretions.

Prostaglandins mediate lipopolysaccharide effects upon cholecystokinin and neurotensin phenotypes in neuroendocrine corticotropin-releasing hormone neurons: in situ hybridization evidence in the rat.

Intraperitoneal injection of the endotoxin lipopolysaccharide produces an inflammation accompanied by immune system activation and secretion of cytokines that stimulate the hypothalamo-pituitary-adrenal (HPA) axis to release the anti-inflammatory corticosterone. Upstream in HPA axis are neuroendocrine corticotropin-releasing hormone neurons in the paraventricular nucleus whose multipeptidergic phenotype changes during inflammation: coexisting corticotropin-releasing hormone and cholecystokinin mRNAs are up-regulated whereas neurotensin mRNA expression is induced de novo. These changes may be mediated by prostaglandins released from perivascular and microglial cells in response to circulating cytokines. We examined by quantitative in situ hybridization histochemistry whether blockade of prostaglandin synthesis by indomethacin alters phenotypic expression in paraventricular nucleus neurons after lipopolysaccharide. Because indomethacin also elevated circulating corticosterone, animals were adrenalectomized and corticosterone replaced. Results showed that i.p. indomethacin administration suppressed lipopolysaccharide effects in a phenotype non-specific manner: one injection was sufficient to prevent both the increase in corticotropin-releasing hormone and cholecystokinin mRNAs expression and the induction of neurotensin mRNA expression. Therefore, neuroendocrine corticotropin-releasing hormone neurons with different peptidergic phenotypes appear to respond as a whole in the acute phase response to systemic infection.

Localisation of GABA(A) receptor epsilon-subunit in cholinergic and aminergic neurones and evidence for co-distribution with the theta-subunit in rat brain.

In situ hybridisation and immunohistochemical methodologies suggest the existence of a large diversity of GABA(A) receptor subtypes in the brain. These are hetero-oligomeric proteins modulated by a number of clinically important drugs, depending on their subunit composition. We recently cloned and localised the rat GABA(A) receptor epsilon-subunit by in situ hybridisation and immunohistochemical procedures. Here, in a dual-labelling immunohistochemical study in the rat brain, we used our affinity-purified antiserum to epsilon with antisera to markers of cholinergic, catecholaminergic, and serotonergic neurones. As far as cholinergic systems were concerned, epsilon-immunoreactivity was expressed in all forebrain cell-groups, as well as in the caudal lateral pontine tegmentum and dorsal motor nucleus of the vagus nerve. As far as dopaminergic systems were concerned, epsilon-immunoreactivity was found to be expressed in a great number of hypothalamic cell-groups (A15, A14 and A12) and in the substantia nigra pars compacta. The noradrenergic, and to a lesser extent, adrenergic cell-groups were all epsilon-immunoreactive. Also, epsilon-immunoreactivity was detected in all serotonergic cell-groups. We also revealed by in situ hybridisation in a monkey brain that epsilon mRNA was expressed in the locus coeruleus, as previously observed in rats. Finally, by using in situ hybridisation in rat brains, we compared the distribution of the mRNA of epsilon with that of the recently cloned theta-subunit of the GABA(A) receptor. Both subunits showed strikingly overlapping expression patterns throughout the brain, especially in the septum, preoptic areas, various hypothalamic nuclei, amygdala, and thalamus, as well as the aforementioned monoaminergic cell-groups. No theta-mRNA signals were detected in cholinergic cell-groups. Taken together with previously published evidence of the presence of the alpha3-subunit in monoamine- or acetylcholine-containing systems, our data suggest the existence of novel GABA(A) receptors comprising alpha3/epsilon in cholinergic and alpha3/theta/epsilon in monoaminergic cell-groups.

CCK mRNA expression in neuroendocrine CRH neurons is increased in rats subjected to an immune challenge.

The expression of cholecystokinin (CCK) mRNA in neuroendocrine corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) of male rats was examined 8 h following an acute immune challenge by intraperitoneal lipopolysaccharide (LPS, 250 microg/kg). Both quantitative, macroautoradiographic, single-label radioactive in situ hybridization histochemistry (ISHH) and qualitative dual-label ISHH were performed. Compared to controls, LPS-injected rats displayed increased (185%) parvicellular CCK mRNA expression levels, occurring in a majority (70%) of CRH neurons as revealed by dual-label ISHH.

cDNA cloning and expression of a gamma-aminobutyric acid A receptor epsilon-subunit in rat brain.

A cDNA encoding a GABA(A) receptor subunit was isolated from rat brain. The predicted protein is 70% identical to the human epsilon-subunit. It was recently reported [Sinkkonen et al. (2000), J. Neurosci., 20, 3588-3595] that the rodent epsilon-subunit mRNA encoded an additional sequence ( approximately 400 residues). We provide evidence that human and rat epsilon-subunit are similar in size. The distribution of cells expressing the GABA(A) epsilon-subunit was examined in the rat brain. In situ hybridization histochemistry revealed that epsilon-subunit mRNA is expressed by neurons located in septal and preoptic areas, as well as in various hypothalamic nuclei, including paraventricular, arcuate, dorsomedial and medial tuberal nuclei. The mRNA was also detected in major neuronal groups with broad-range influence, such as the cholinergic (basal nucleus), dopaminergic (substantia nigra compacta), serotonergic (raphe nuclei), and noradrenergic (locus coeruleus) systems. Immunohistochemistry using an affinity-purified antiserum directed towards the N-terminal sequence unique to the rat epsilon-subunit revealed the presence of epsilon-subunit immunoreactivity over the somatodendritic domain of neurons with a distribution closely matching that of mRNA-expressing cells. Moreover, using in situ hybridization, alpha3, theta and epsilon GABA(A) subunit mRNAs were all detected with an overlapping distribution in neurons of the dorsal raphe and the locus coeruleus. Our results suggest that novel GABA(A) receptors may regulate, neuroendocrine and modulatory systems in the brain.

Identification of neurokinin B-expressing neurons as an highly estrogen-receptive, sexually dimorphic cell group in the ovine arcuate nucleus.

Studies were undertaken to examine the hypothesis that neurons expressing neurokinin B (NKB) may represent an estrogen-receptive input to GnRH neurons in the sheep. Cells immunoreactive for NKB were located almost exclusively within the arcuate nucleus of the ovine hypothalamus. Dual labeling experiments revealed that essentially all NKB neurons (97%) were immunoreactive for estrogen receptor alpha and that NKB-immunoreactive fibers were found in close proximity to approximately 40% of GnRH neurons located in the rostral preoptic area as well as intermingled with GnRH fibers in the median eminence. The analysis of male and female brains revealed a marked female-dominant sex difference in the numbers of NKB neurons, and sections obtained from in utero androgen-treated females indicated that this sex difference resulted from an organizational influence of testosterone during neural development. In adult ovariectomized ewes, in situ hybridization studies failed to detect any significant effect of 8- to 26-h exposure of estrogen on cellular NKB messenger RNA levels. Together, these studies identify the first sexually differentiated neuronal cell population in the ovine hypothalamus and, remarkably, show that essentially all of these female-dominant NKB neurons express estrogen receptors. Although these neurons may be involved in any number of steroid-dependent, sexually differentiated functions in the sheep, the neuroanatomical evidence for potential NKB inputs to GnRH neurons suggests a role for this novel population in the regulation of reproductive function.

Glucocorticosteroids up-regulate the expression of cholecystokinin mRNA in the rat paraventricular nucleus.

Adrenalectomy abolishes corticosteroid feedback onto the hypothalamic-pituitary-adrenal axis. This results in an increased biosynthetic and secretory activity of corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN), sustained in the absence of hormone replacement. In the PVN, cholecystokinin (CCK) is present both in parvicellular CRH-containing and in magnocellular oxytocin (OXY)-containing neurons. We presently studied the glucocorticoid feedback regulation of the expression of cholecystokinin (CCK) mRNA in rats after: (i) adrenalectomy, (ii) sham surgery or (iii) adrenalectomy with corticosterone replacement. Using 35S-labeled CRH and p-CCK cRNA probes and in situ hybridization, CRH and CCK mRNAs were radiolabeled. The total amount of hybridization labeling (integrated density), was quantified in adjacent series of cryosections regularly spaced throughout the PVN. The OXY mRNA detection served to identify PVN magnocellular areas. Adrenalectomy was shown to induce: (i) a 75% increase in CRH mRNA labeling in the PVN, (ii) a concomitant 43% decrease in CCK mRNA labeling but only in the anterior part of the PVN and occurring both in CCK/CRH area (two thirds of it) and CCK/OXY area (one third of it) and (iii) that they were fully reversed by corticosterone replacement. Thus, glucocorticoids that are well known to negatively feedback on CRH expression in parvicellular PVN neurons are also capable of positively regulating CCK expression in anterior PVN neurons, both in parvicellular and magnocellular areas.

Phenotypical segregation among female rat hypothalamic gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic coexpression of cholecystokinin and neurotensin.

The neuroendocrine control of the gonad is exerted primarily by the gonadotropin-releasing hormone neurons located in the septum and the hypothalamus. Despite their sexually dimorphic activity, tonic in males and phasic in females, these neurons have not appeared qualitatively different between sexes in intrinsic organization or chemical phenotype. Here, by using multiple-label immunocytochemistry, it is demonstrated that the phenotype of gonadotropin-releasing hormone neurons is sex specific. In females only, 54.5% of them co-expressed cholecystokinin immunoreactivity and 29.4% additionally expressed neurotensin immunoreactivity. These multipeptidergic neurons were observed in the hypothalamus but not in the septum. During postnatal development, cholecystokinin and neurotensin immunoreactivities were first detected in gonadotropin-releasing hormone-containing axons of the median eminence at vaginal opening, suggesting an involvement of the neuropeptides in peri-ovulatory events. This peptidergic phenotype was not apparent in females ovariectomized as adults but was reinstated by estradiol treatment. In adult males, the testicle does not control this phenotype because orchidectomized adults did not display it, whatever the post-operative delay (one to five weeks) or substitutive chronic steroid treatment (testosterone or estradiol). The testicle may, however, masculinize the phenotype neonatally because estradiol or testosterone treatment in adulthood induced an expression of cholecystokinin immunoreactivity in gonadotropin-releasing hormone-containing axons of the median eminence in both males and females that were gonadectomized at birth. This procedure, however, failed to significantly induce an expression of neurotensin immunoreactivity, suggesting a role of the postnatal ovary on this element of the chemistry of gonadotropin-releasing hormone neurons.Thus, the gonad permanently organizes the gonadotropin-releasing hormone neuronal population, resulting, at least in females, in a mosaic of phenotypically distinct, functional subunits.

Immune challenge-stimulated hypophysiotropic corticotropin-releasing hormone messenger RNA expression is associated with an induction of neurotensin messenger RNAs without alteration of vasopressin messenger RNAs.

The corticotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus are the final common pathway of the neuroendocrine adaptative response to a variety of stressors. To meet varied homeostatic needs, corticotropin-releasing hormone neurons exhibit a marked phenotypical plasticity, enabling them to rapidly modify their neuroendocrine output. In particular, they synthesize the neuropeptides vasopressin and neurotensin. Under many experimental circumstances, it is observed that corticotropin-releasing hormone and vasopressin are regulated in parallel, whereas the expression of neurotensin seems dissociated, in these neurons, evoking different transcriptional control over the co-existing neuropeptides depending on the adaptative response required. Using radioactive and dual-label in situ hybridization techniques, we have studied the respective expression of paraventricular corticotropin-releasing hormone, vasopressin and neurotensin messenger RNAs in the context of an immune challenge. A single intraperitoneal injection of the endotoxin lipopolysaccharide was administered to adult male rats that were killed 8 h later. Compared to control animals, lipopolysaccharide-injected rats showed elevated plasma corticosterone (614+/-65 vs 185+/-40 ng/ml in control) and increased expression of paraventricular corticotropin-releasing hormone messenger RNA (+200%); expression of neurotensin messenger RNA was induced in about one-third of corticotropin-releasing hormone neurons, whereas vasopressin messenger RNA expression remained unchanged. Therefore, in this experimental context and at the time-point examined, co-existing corticotropin-releasing hormone and vasopressin appeared differentially expressed, and an additional stimulus (inflammation) is demonstrated to result in neurotensin expression in neuroendocrine corticotropin-releasing hormone neurons. Neurotensin may be released in the pituitary portal blood to trigger pituitary response associated with mobilization of the immune system.

Ultrastructural colocalization of vesicular cholecystokinin and corticoliberin in the periportal nerve terminals of the rat median eminence.

Cholecystokinin (CCK) is present in axon terminals distributed around the fenestrated capillary loops of the hypothalamo-hypophysial portal system. In the hypothalamic paraventricular nucleus, CCK has been shown to coexist with corticoliberin (CRH). However, in the median eminence (ME) nothing is known about the chemical phenotype of the CCK immunoreactive terminals. This study, carried out in the male rat, was designed to examine the possibility of coexistence of CCK immunoreactivity (CCK-IR) and CRH-IR in fibres of the ME and to describe, at the electron microscopic level, the vesicular pattern of distribution of CCK-IR in the pericapillary endings of the ME. The use of the elution-restaining procedure showed notable similarities between stainings directed against CCK or CRH, respectively, suggesting a colocalization of both peptides in the same terminals. This result was confirmed using a simultaneous double-staining procedure. At the electron microscope level, double immunogold staining procedure enabled us to observe a consistent localization of CCK-IR and CRH-IR over dense-cored vesicles. Most of the terminals were seen to contain both immunoreactivities which, in addition, were often present together in the same vesicles. However, some rare endings remained exclusively stained either for CCK or for CRH. Our results provide evidence for a concomitant release of CCK and CRH into the portal blood.

Immunocytochemical detection of the neurokinin B receptor (NK3) on melanin-concentrating hormone (MCH) neurons in rat brain.

The presence of the neurokinin B receptor (NK3 receptor) in the rat lateral hypothalamus and the zona incerta was previously reported. The aim of the present study was to define its cellular localization in these areas. Investigations, coupling immunocytochemical and in situ hybridization techniques, focussed on two neuron populations: the melanin-concentrating hormone (MCH) neurons and a population of neurons recognized by an ovine prolactin antiserum (PRL-ir neurons). While PRL-ir neurons did not exhibit NK3 immunoreactivity, 57% +/- 6% of MCH neurons were strongly stained by the NK3 antiserum. These results suggest that neurokinin B is involved in the regulation of MCH neuron activity via the NK3 receptor; they provide new bases for further investigations on MCH role in the control of food and water intake.

Suckling-induced Fos-immunoreactivity in subgroups of hypothalamic POMC neurons of the lactating rat: investigation of a role for prolactin.

Attention has recently been focused on lactation-induced modifications of activity of neuronal populations in the arcuate nucleus (ARC) of the mediobasal hypothalamus. The ARC hosts the tubero-infundibular dopaminergic (TIDA system) responsible for the neuroendocrine control of prolactin (PRL), and other non-neuroendocrine neuronal populations, such as neuropeptide Y (NPY)- and proopiomelanocortin (POMC)-containing systems that are important modulators of hypothalamic gonadoliberin (GnRH) secretion. Our longstanding interest in the functional anatomy of the ARC led us to investigate whether the suckling stimulus would trigger an expression of Fos-ir in specific arcuate neuronal populations and to possibly characterize responsive neurons by using double-labeling immunohistochemistry. Freely nursing lactating females expressed strong Fos-ir in neurons of the ARC compared to diestrous females. Fos-ir was encountered in neurons not belonging to the TIDA system and that was for a large proportion identical to the POMCergic neurons. We showed that, in lactating females submitted to suppression of the suckling stimulus by removal of the pups, the pattern of expression of Fos-ir is similar to that seen in diestrous females and that, a pattern of expression of Fos-ir indistinguishable from that observed during free lactation is reinstated a short time after the return of the pups and restoration of the suckling stimulus, suggesting that this expression of Fos-ir strictly depends upon the presence of the newborns and the suckling stimulus. By lowering circulating levels of the PRL with bromocryptine-or PRL antiserum-treatment, we noticed a decrease in the number of (beta-endorphin + Fos)-ir neurons compared to non-injected freely nursing lactating females. By maintaining high levels of circulating PRL with haloperidol-treatment, we observed a number of colocalizations close to that observed in freely nursing lactating females. Our results suggest that during lactation a rostral subgroup of the arcuate POMCergic neuronal population is activated at least partially in response to the suckling-induced secretion of PRL and that this activation participates in maintaining the endocrine and/or metabolic demands of the lactational status.

Neuropeptide Y suppresses prolactin secretion from rat anterior pituitary cells: evidence for interactions with dopamine through inhibitory coupling to calcium entry.

Expression of neuropeptide Y (NPY) in the medial basal hypothalamus is increased during lactation, and at least part of this increase is due to the appearance of the peptide in hypothalamic tuberoinfundibular dopamine neurons, a cell population that does not exhibit NPY expression in other physiological conditions. The present studies tested the hypothesis that NPY affects PRL secretion by modulating the action of dopamine (DA) at the lactotroph. In static incubations of cultured anterior pituitary (AP) cells, the addition of either NPY or DA in concentrations of 0.1-500 nM resulted in dose-dependent inhibition of PRL secretion, and the combination of DA and NPY in submaximal concentrations produced an additive inhibition of PRL release. NPY also inhibited PRL secretion induced by TRH in perifused AP cells, and the effects were again additive with DA. The interactions of NPY and DA on TRH-induced elevations in cytosolic Ca2+ ([Ca2+]i) were examined by loading cultured AP cells of lactating rats with the fluorescent calcium probe fura-2 TRH produced a dose-dependent stimulation of [Ca2+]i, which was characterized by a rapid transient spike and a more prolonged plateau. Both phases were attenuated by either DA or NPY at 100 nM and were nearly abolished by the combination of DA and NPY, whereas neither DA nor NPY altered resting [Ca2+]i. DA and NPY also inhibited the increases in PRL secretion and [Ca2+]i induced by elevated extracellular K+ in an additive manner. Stimulation of AP cells with TRH in the absence of extracellular Ca2+ resulted in an attenuated spike of PRL release and [Ca2+]i and no plateau phase. Under these conditions, DA still inhibited the residual [Ca2+]i and PRL responses, but the inhibitory effects of NPY on PRL secretion and [Ca2+]i, and the potentiation by NPY of DA inhibition, were abolished. These results suggest that one physiological function of the NPY expressed in tuberoinfundibular dopamine neurons in lactation is to amplify the inhibitory action of DA on PRL secretion through negative coupling to the Ca2+ messenger system, particularly the entry of extracellular Ca2+.

Presence of nuclear androgen receptor-like immunoreactivity in neurokinin B-containing neurons of the hypothalamic arcuate nucleus of the adult male rat.

Dual label immunofluorescence was used in the brain of normal, non colchicine-treated, adult male rats in order to characterize the neurons possessing the nuclear androgen receptor in the hypothalamic arcuate nucleus. The affinity purified PG21 rabbit serum to the rat androgen receptor (AR) was used in conjunction with a polyclonal guinea pig antiserum to peptide 2, a sequence within the precursor to neurokinin B (NKB). All NKB-containing neurons were also immunoreactive for the AR, and made up a large proportion (about 60%) of the arcuate cells with detectable and specific AR immunoreactivity.