A method for drug infusion into the lateral median eminence and arcuate nucleus of sheep

The role of catecholamines in the control of the GnRH pulse generator is unclear as studies have relied on the use of peripheral or intracerebroventricular injections, which lack specificity in relation to the anatomical site of action. Direct brain site infusions have been used, however, these are limited by the ability to accurately target small brain regions. One such area of interest in the control of GnRH is the median eminence and arcuate nucleus within the medial basal hypothalamus. Here we describe a method of stereotaxically targeting this area in a large animal (sheep) and an infusion system to deliver drugs into unrestrained conscious animals. To test our technique we infused the dopamine agonist, quinpirole or vehicle into the medial basal hypothalamus of ovariectomised ewes. Quinpirole significantly suppressed LH pulsatility only in animals with injectors located close to the lateral median eminence. This in vivo result supports the hypothesis that dopamine inhibits GnRH secretion by presynaptic inhibition in the lateral median eminence. Also infusion of quinpirole into the medial basal hypothalamus suppressed prolactin secretion providing in vivo evidence that is consistent with the hypothesis that there are stimulatory autoreceptors on tubero-infundibular dopamine neurons. (C) 1997 Elsevier Science B.V.

Semaphorin7A regulates neuroglial plasticity in the adult hypothalamic median eminence.

Reproductive competence in mammals depends on the projection of gonadotropin-releasing hormone (GnRH) neurons to the hypothalamic median eminence (ME) and the timely release of GnRH into the hypothalamic-pituitary-gonadal axis. In adult rodents, GnRH neurons and the specialized glial cells named tanycytes periodically undergo cytoskeletal plasticity. However, the mechanisms that regulate this plasticity are still largely unknown. We demonstrate that Semaphorin7A, expressed by tanycytes, plays a dual role, inducing the retraction of GnRH terminals and promoting their ensheathment by tanycytic end feet via the receptors PlexinC1 and Itgb1, respectively. Moreover, Semaphorin7A expression is regulated during the oestrous cycle by the fluctuating levels of gonadal steroids. Genetic invalidation of Semaphorin7A receptors in mice induces neuronal and glial rearrangements in the ME and abolishes normal oestrous cyclicity and fertility. These results show a role for Semaphorin7A signalling in mediating periodic neuroglial remodelling in the adult ME during the ovarian cycle.

EFFECT OF TESTOSTERONE ON THE PRESSOR-RESPONSE TO COMMON CAROTID OCCLUSION IN GONADECTOMIZED CONSCIOUS MALE-RATS WITH LESION OF THE MEDIAN-EMINENCE

The influence of testosterone on the development of the pressor response to common carotid occlusion was investigated in control and median eminence-lesioned male rats. In control rats (N = 9), gonadectomy performed 21 days before the experiments reduced by 22% (from 51 +/- 2 to 40 +/- 2 mmHg) and treatment with testosterone (300-mu-g for 4 days before the measurements) increased the initial peak pressor response (from 51 +/- 2 to 57 +/- 2 mmHg) which depends on carotid innervation. The maintained response which is of central origin (probably ischemic) was less affected. In nongonadectomized rats (N = 6), lesions of the median eminence (6 days) decreased the initial peak by 19% (from 52 +/- 2 to 42 +/- 3 mmHg) and the maintained response by 56% (from 32 +/- 2 to 14 +/- 1 mmHg). Sham-operated rats served as controls. In gonadectomized animals (N = 6) the lesion reduced only the maintained response (from 23 +/- 2 to 11 +/- 1 mmHg). Testosterone supplementation restored the maintained response but did not alter the initial peak. These results indicate that the pressor response to common carotid occlusion in male rats is modulated by testosterone and that the depression in the maintained response caused by median eminence lesion can be reversed by steroid supplementation.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on hormone secretion from sheep pituitary cells in vitro

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 mu mol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l-1 mu mol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 mu mol/l; P<0.05), PACAP also stimulated FSH secretion in cells from some animals although this effect was small, The GH response to PACAP was inhibited by PACAP(6-38), a putative PACAP antagonist; but not by (N-Ac-Tyr(1), D-Arg(2))-GHRH(1-29)-NH2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1-24 h, VIP (1 mu mol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l-1 mu mol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l-1 mu mol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 mu mol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.

Activity of Hypothalamic Dopaminergic Neurones During the Day of Oestrus: Involvement in Prolactin Secretion

A secretory surge of prolactin occurs on the afternoon of oestrus in cycling rats. Pituitary prolactin is inhibited by dopamine. We evaluated the activity of the neuroendocrine dopaminergic neurones during oestrus and dioestrus, as determined by dopaminergic activity in the median eminence and neurointermediate lobe of the pituitary, as well as Fos-related antigen expression in tyrosine hydroxylase (TH)-immunoreactive (ir) neurones of the arcuate nucleus (ARC) and periventricular nucleus (Pe). During oestrus, the 4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence decreased at 16.00 h, coinciding with the increase in plasma prolactin levels. Similarly, the expression of Fos-related antigen in TH-ir neurones of Pe and rostral-, dorsomedial- and caudal-ARC also decreased at 16.00 h. On dioestrus, 4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence and Fos-related antigen expression in TH-ir neurones of Pe and rostral-ARC decreased at 18.00 h, whereas prolactin levels were unaltered. No variation in dopaminergic activity was found in the neurointermediate lobe of the pituitary on either oestrus or dioestrus. The number of TH-ir neurones in the ARC and parameters of dopaminergic activity were found to be generally lower on oestrus compared to dioestrus. The transitory decrease in the activity of neuroendocrine dopaminergic neurones temporally associated with the prolactin surge on the afternoon of oestrus suggests a role for dopamine in the generation of the oestrous prolactin surge.

Kisspeptin Regulates Prolactin Release through Hypothalamic Dopaminergic Neurons

Prolactin (PRL) is tonically inhibited by dopamine (DA) released from neurons in the arcuate and periventricular nuclei. Kisspeptin plays a pivotal role in LH regulation. In rodents, kisspeptin neurons are found mostly in the anteroventral periventricular and arcuate nuclei, but the physiology of arcuate kisspeptin neurons is not completely understood. We investigated the role of kisspeptin in the control of hypothalamic DA and pituitary PRL secretion in adult rats. Intracerebroventricular kisspeptin-10 (Kp-10) elicited PRL release in a dose-dependent manner in estradiol (E2)-treated ovariectomized rats (OVX+E2), whereas no effect was found in oil-treated ovariectomized rats (OVX). Kp-10 increased PRL release in males and proestrous but not diestrous females. Associated with the increase in PRL release, intracerebroventricular Kp-10 reduced Fos-related antigen expression in tyrosine hydroxylase-immunoreactive (ir) neurons of arcuate and periventricular nuclei in OVX+E2 rats, with no effect in OVX rats. Kp-10 also decreased 3,4-dihydroxyphenylacetic acid concentration and 3,4-dihydroxyphenylacetic acid-DA ratio in the median eminence but not striatum in OVX+E2 rats. Double-label immunofluorescence combined with confocal microscopy revealed kisspeptin-ir fibers in close apposition to and in contact with tyrosine hydroxylase-ir perikarya in the arcuate. In addition, Kp-10 was not found to alter PRL release from anterior pituitary cell cultures regardless of E2 treatment. We provide herein evidence that kisspeptin regulates PRL release through inhibition of hypothalamic dopaminergic neurons, and that this mechanism is E2 dependent in females. These findings suggest a new role for central kisspeptin with possible implications for reproductive physiology. (Endocrinology 151: 3247-3257, 2010)

Noradrenaline Involvement in the Negative-Feedback Effects of Ovarian Steroids on Luteinising Hormone Secretion

Noradrenaline has been shown to modulate the ovarian-steroid feedback on luteinising-hormone (LH) release. However, despite the high amount of evidence accumulated over many years, the role of noradrenaline in LH regulation is still not clearly understood. The present study aimed to further investigate the involvement of noradrenaline in the negative-feedback effect of oestradiol and progesterone on basal LH secretion. In experiment 1, ovariectomised (OVX) rats received a single injection of oil, oestradiol, or progesterone at 09.00-10.00 h and were decapitated 30 or 60 min later. Levels of noradrenaline and its metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), were determined in microdissections of the preoptic area (POA) and medial basal hypothalamus-median eminence (MBH-ME) and correlated with LH secretion. Basal LH levels were decreased 30 and 60 min after oestradiol or progesterone injection, and this hormonal response was significantly correlated with a reduction in POA MHPG levels, which reflect noradrenaline release. In addition, noradrenaline levels in the POA were increased, whereas noradrenaline turnover (MHPG/noradrenaline ratio) was decreased 60 min after the injection of both hormones. No effect was found in the MBH-ME. In experiment 2, i.c.v. administration of noradrenaline (60 nmol), performed 15 min before oestradiol or progesterone injection in jugular vein-cannulated OVX rats, completely prevented the ovarian steroid-induced inhibition of LH secretion. The data obtained provide direct evidence that LH secretion in OVX rats is positively regulated by basal noradrenergic activity in the POA, and its reduction appears to play a role in the negative-feedback effect of ovarian steroids on LH secretion in vivo.

The rate-limiting step for glucose transport into the hypothalamus is across the blood-hypothalamus interface.

Specialized glucosensing neurons are present in the hypothalamus, some of which neighbor the median eminence, where the blood-brain barrier has been reported leaky. A leaky blood-brain barrier implies high tissue glucose levels and obviates a role for endothelial glucose transporters in the control of hypothalamic glucose concentration, important in understanding the mechanisms of glucose sensing We therefore addressed the question of blood-brain barrier integrity at the hypothalamus for glucose transport by examining the brain tissue-to-plasma glucose ratio in the hypothalamus relative to other brain regions. We also examined glycogenolysis in hypothalamus because its occurrence is unlikely in the potential absence of a hypothalamus-blood interface. Across all regions the concentration of glucose was comparable at a given plasma glucose concentration and was a near linear function of plasma glucose. At steady-state, hypothalamic glucose concentration was similar to the extracellular hypothalamic glucose concentration reported by others. Hypothalamic glycogen fell at a rate of approximately 1.5 micromol/g/h and remained present in substantial amounts. We conclude for the hypothalamus, a putative primary site of brain glucose sensing that: the rate-limiting step for glucose transport into brain cells is at the blood-hypothalamus interface, and that glycogenolysis is consistent with a substantial blood -to- intracellular glucose concentration gradient.

Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision.

The elucidation of mechanisms underlying telencephalic neural development has been limited by the lack of knowledge regarding the molecular and cellular aspects of the ganglionic eminence (GE), an embryonic structure that supplies the brain with diverse sets of GABAergic neurons. Here, we report a comprehensive transcriptomic analysis of this structure including its medial (MGE), lateral (LGE) and caudal (CGE) subdivisions and its temporal dynamics in 12.5 to 16 day-old rat embryos. Surprisingly, comparison across subdivisions showed that CGE gene expression was the most unique providing unbiased genetic evidence for its differentiation from MGE and LGE. The molecular signature of the CGE comprised a large set of genes, including Rwdd3, Cyp26b1, Nr2f2, Egr3, Cpta1, Slit3, and Hod, of which several encode cell signaling and migration molecules such as WNT5A, DOCK9, VSNL1 and PRG1. Temporal analysis of the MGE revealed differential expression of unique sets of cell specification and migration genes, with early expression of Hes1, Lhx2, Ctgf and Mdk, and late enrichment of Olfm3, SerpinE2 and Wdr44. These GE profiles reveal new candidate regulators of spatiotemporally governed GABAergic neuronogenesis.

Immunolocalization of GLUTX1 in the testis and to specific brain areas and vasopressin-containing neurons.

GLUTX1 or GLUT8 is a newly characterized glucose transporter isoform that is expressed at high levels in the testis and brain and at lower levels in several other tissues. Its expression was mapped in the testis and brain by using specific antibodies. In the testis, immunoreactivity was expressed in differentiating spermatocytes of type 1 stage but undetectable in mature spermatozoa. In the brain, GLUTX1 distribution was selective and localized to a variety of structures, mainly archi- and paleocortex. It was found in hippocampal and dentate gyrus neurons as well as amygdala and primary olfactory cortex. In these neurons, its location was close to the plasma membrane of cell bodies and sometimes in proximal dendrites. High GLUTX1 levels were detected in the hypothalamus, supraoptic nucleus, median eminence, and the posterior pituitary. Neurons of these areas synthesize and secrete vasopressin and oxytocin. As shown by double immunofluorescence microscopy and immunogold labeling, GLUTX1 was expressed only in vasopressin neurons. By immunogold labeling of ultrathin cryosections microscopy, GLUTX1 was identified in dense core vesicles of synaptic nerve endings of the supraoptic nucleus and secretory granules of the vasopressin positive neurons. This localization suggests an involvement of GLUTX1 both in specific neuron function and endocrine mechanisms.

Formation of a direct arterial blood supply to the anterior pituitary gland following complete or partial interruption of the hypophyseal portal vessels

If regions of the anterior pituitary gland received systemic blood via a direct arterial blood supply these regions would escape hypothalamic regulation and thus be a sequela in endocrine disorders. Since, in the untreated rat, all of the blood supply to the anterior pituitary gland is via the hypophyseal portal vessels, we hypothesized that partial interruption of the portal vessels could provoke the establishment of a direct arterial blood supply (arteriogenesis). We utilized the injection of polystyrene microspheres (15 or 9 micron diameter) into the left ventricle of the heart to test this hypothesis. Microspheres are trapped in the first capillary plexus they reach since they are too large to traverse the capillaries. No microspheres reached the anterior pituitary gland of control rats, a finding consistent with the fact that the anterior pituitary gland receives all of its blood supply via the hypophyseal portal blood vessels. Microspheres were observed in the primary portal capillary plexus in the infundibulum (median eminence), infundibular stalk (pituitary stalk), and infundibular process (pars nervosa), the first capillary plexus which they reached. A lesion of the medial basal hypothalamus (MBH) which destroyed the long portal vessels did not result in arteriogenesis since few, if any, microspheres were observed in the anterior pituitary gland. We confirmed, using vascular casts, that these lesions resulted in the long-term destruction of the primary portal capillaries in the infundibulum and of the long portal vessels. In MBH-lesioned animals it appears that all of the blood supply of the anterior pituitary gland is via short portal vessels arising from the infundibular stem and process.(ABSTRACT TRUNCATED AT 250 WORDS)

Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome.

Individuals with an inherited deficiency in gonadotropin-releasing hormone (GnRH) have impaired sexual reproduction. Previous genetic linkage studies and sequencing of plausible gene candidates have identified mutations associated with inherited GnRH deficiency, but the small number of affected families and limited success in validating candidates have impeded genetic diagnoses for most patients. Using a combination of exome sequencing and computational modeling, we have identified a shared point mutation in semaphorin 3E (SEMA3E) in 2 brothers with Kallmann syndrome (KS), which causes inherited GnRH deficiency. Recombinant wild-type SEMA3E protected maturing GnRH neurons from cell death by triggering a plexin D1-dependent (PLXND1-dependent) activation of PI3K-mediated survival signaling. In contrast, recombinant SEMA3E carrying the KS-associated mutation did not protect GnRH neurons from death. In murine models, lack of either SEMA3E or PLXND1 increased apoptosis of GnRH neurons in the developing brain, reducing innervation of the adult median eminence by GnRH-positive neurites. GnRH neuron deficiency in male mice was accompanied by impaired testes growth, a characteristic feature of KS. Together, these results identify SEMA3E as an essential gene for GnRH neuron development, uncover a neurotrophic function for SEMA3E in the developing brain, and elucidate SEMA3E/PLXND1/PI3K signaling as a mechanism that prevents GnRH neuron deficiency.

Correlations between ANP concentrations in atria, plasma and cerebral structures and sodium chloride preference in Wistar rats

We determined whether ANP (atrial natriuretic peptide) concentrations, measured by radioimmunoassay, in the ANPergic cerebral regions involved in regulation of sodium intake and excretion and pituitary gland correlated with differences in sodium preference among 40 Wistar male rats (180-220 g). Sodium preference was measured as mean spontaneous ingestion of 1.5% NaCl solution during a test period of 12 days. The relevant tissues included the olfactory bulb (OB), the posterior and anterior lobes of the pituitary gland (PP and AP, respectively), the median eminence (ME), the medial basal hypothalamus (MBH), and the region anteroventral to the third ventricle (AV3V). We also measured ANP content in the right (RA) and left atrium (LA) and plasma. The concentrations of ANP in the OB and the AP were correlated with sodium ingestion during the preceding 24 h, since an increase of ANP in these structures was associated with a reduced ingestion and vice-versa (OB: r = -0.3649, P<0.05; AP: r = -0.3291, P<0.05). Moreover, the AP exhibited a correlation between ANP concentration and mean NaCl intake (r = -0.4165, P<0.05), but this was not the case for the OB (r = 0.2422). This suggests that differences in sodium preference among individual male rats can be related to variations of AP ANP level. Earlier studies indicated that the OB is involved in the control of NaCl ingestion. Our data suggest that the OB ANP level may play a role mainly in day-to-day variations of sodium ingestion in the individual rat

Neuroendocrine regulation of salt and water metabolism

Neurons which release atrial natriuretic peptide (ANPergic neurons) have their cell bodies in the paraventricular nucleus and in a region extending rostrally and ventrally to the anteroventral third ventricular (AV3V) region with axons which project to the median eminence and neural lobe of the pituitary gland. These neurons act to inhibit water and salt intake by blocking the action of angiotensin II. They also act, after their release into hypophyseal portal vessels, to inhibit stress-induced ACTH release, to augment prolactin release, and to inhibit the release of LHRH and growth hormone-releasing hormone. Stimulation of neurons in the AV3V region causes natriuresis and an increase in circulating ANP, whereas lesions in the AV3V region and caudally in the median eminence or neural lobe decrease resting ANP release and the response to blood volume expansion. The ANP neurons play a crucial role in blood volume expansion-induced release of ANP and natriuresis since this response can be blocked by intraventricular (3V) injection of antisera directed against the peptide. Blood volume expansion activates baroreceptor input via the carotid, aortic and renal baroreceptors, which provides stimulation of noradrenergic neurons in the locus coeruleus and possibly also serotonergic neurons in the raphe nuclei. These project to the hypothalamus to activate cholinergic neurons which then stimulate the ANPergic neurons. The ANP neurons stimulate the oxytocinergic neurons in the paraventricular and supraoptic nuclei to release oxytocin from the neural lobe which circulates to the atria to stimulate the release of ANP. ANP causes a rapid reduction in effective circulating blood volume by releasing cyclic GMP which dilates peripheral vessels and also acts within the heart to slow its rate and atrial force of contraction. The released ANP circulates to the kidney where it acts through cyclic GMP to produce natriuresis and a return to normal blood volume

Vasopressor mechanisms in acute aortic coarctation hypertension

Angiotensin II (ANG II) and vasopressin (AVP) act together with the mechanical effect of aortic constriction in the onset of acute aortic coarctation hypertension. Blockade of ANG II and AVP V1 receptors demonstrated that ANG II acts on the prompt (5 min) rise in pressure whereas AVP is responsible for the maintenance (30-45 min) of the arterial pressure elevation during aortic coarctation. Hormone assays carried out on blood collected from conscious rats submitted to aortic constriction supported a role for ANG II in the early stage and a combined role for both ANG II and AVP in the maintenance of proximal hypertension. As expected, a role for catecholamines was ruled out in this model of hypertension, presumably due to the inhibitory effect of the sinoaortic baroreceptors. The lack of afferent feedback from the kidneys for AVP release from the central nervous system in rats with previous renal denervation allowed ANG II to play the major role in the onset of the hypertensive response. Median eminence-lesioned rats exhibited a prompt increase in proximal pressure followed by a progressive decline to lower hypertensive levels, revealing a significant role for the integrity of the neuroaxis in the maintenance of the aortic coarctation hypertension through the release of AVP. In conclusion, the important issue raised by this model of hypertension is the likelihood of a link between some vascular territory - probably renal - below the coarctation triggering the release of AVP, with this vasoconstrictor hormone participating with Ang II and the mechanical effect of aortic constriction in the acute aortic coarctation hypertension