A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat

Previous studies indicated that there is a separate hypothalamic control of follicle-stimulating hormone (FSH) release distinct from that of luteinizing hormone (LH). An FSH-releasing factor (FSHRF) was purified from rat and sheep hypothalami, but has not been isolated. We hypothesized that FSHRF might be an analogue of mammalian luteinizing hormone-releasing hormone (m-LHRH) and evaluated the activity of many analogues of m-LHRH and of the known LHRHs found in lower forms. Here we demonstrate that lamprey (l) LHRH-III has a potent, dose-related FSH- but not LH-releasing action on incubated hemipituitaries of male rats. l-LHRH-I on the other hand, had little activity to release either FSH or LH. m-LHRH was equipotent to l-LHRH-III to release FSH, but also had a high potency to release LH in contrast to l-LHRH-III that selectively released FSH. Chicken LHRH-II had considerable potency to release both LH and FSH, but no selectivity in its action. Salmon LHRH had much less potency than the others tested, except for l-LHRH-I, and no selectivity in its action. Because ovariectomized, estrogen, progesterone-treated rats are a sensitive in vivo assay for FSH- and LH-releasing activity, we evaluated l-LHRH-III in this assay and found that it had a completely selective stimulatory effect on FSH release at the two doses tested (10 and 100 pmols). Therefore, l-LHRH-III is a highly potent and specific FSH-releasing peptide that may enhance fertility in animals and humans. It may be the long sought after m-FSHRF.

Peptide analogue studies of the hypothalamic neuropeptide Y receptor mediating pituitary adrenocorticotrophic hormone release

Hypothalamic neuropeptide Y (NPY) is thought to be important in the regulation of feeding and also in the release of Adrenocorticotrophic hormone (ACTH). Intracerebroventricular administration of NPY to male rats significantly increased plasma ACTH 10 min after injection and stimulated 2-h food intake. A series of analogues of NPY that have a greatly reduced affinity for the Y1 [human pancreatic polypeptide (human PP), NPY(3–36)], the Y2 ([Pro34]NPY, human PP), the Y3 (peptide YY), and the Y6 (human PP) receptor, all markedly stimulated ACTH release. Rat PP, which binds with high affinity to the Y4 receptor, was unable to stimulate ACTH release. A novel analogue fragment [Pro34]NPY(13–36) was synthesized as a ligand with low Y1 and Y2 receptor affinity. Interestingly, neither [Pro34]NPY(13–36) nor the selective Y5 receptor agonist [d-Trp32]NPY stimulated food intake, whereas both significantly increased plasma ACTH. Thus the hypothalamic NPY receptor mediating increases in plasma ACTH has a fragment activation profile unlike the Y1–Y4 or Y6 receptors and appears distinct from the NPY receptor controlling food intake.

Multi-responsiveness of single anterior pituitary cells to hypothalamic-releasing hormones: A cellular basis for paradoxical secretion

The classic view for hypothalamic regulation of anterior pituitary (AP) hormone secretion holds that release of each AP hormone is controlled specifically by a corresponding hypothalamic-releasing hormone (HRH). In this scenario, binding of a given HRH (thyrotropin-, growth hormone-, corticotropin-, and luteinizing hormone-releasing hormones) to specific receptors in its target cell increases the concentration of cytosolic Ca2+ ([Ca2+]i), thereby selectively stimulating the release of the appropriate hormone. However, “paradoxical” responses of AP cells to the four well-established HRHs have been observed repeatedly with both in vivo and in vitro systems, raising the possibility of functional overlap between the different AP cell types. To explore this possibility, we evaluated the effects of HRHs on [Ca2+]i in single AP cells identified immunocytochemically by the hormone they stored. We found that each of the five major AP cell types contained discrete subpopulations that were able to respond to several HRHs. The relative abundance of these multi-responsive cells was 59% for lactotropes, 33% for thyrotropes, and in the range of 47–55% for gonadotropes, corticotropes, and somatotropes. Analysis of prolactin release from single living cells revealed that each of the four HRHs tested were able to induce hormone release from a discrete lactotrope subpopulation, the size of which corresponded closely to that in which [Ca2+]i changes were induced by the same secretagogues. When viewed as a whole, our diverse functional measurements of multi-responsiveness suggest that hypothalamic control of pituitary function is more complicated than previously envisioned. Moreover, they provide a cellular basis for the so-called “paradoxical” behavior of pituitary cells to hypothalamic hypophysiotropic agents.

Hypothalamic integration of body fluid regulation.

The progression of animal life from the paleozoic ocean to rivers and diverse econiches on the planet's surface, as well as the subsequent reinvasion of the ocean, involved many different stresses on ionic pattern, osmotic pressure, and volume of the extracellular fluid bathing body cells. The relatively constant ionic pattern of vertebrates reflects a genetic "set" of many regulatory mechanisms--particularly renal regulation. Renal regulation of ionic pattern when loss of fluid from the body is disproportionate relative to the extracellular fluid composition (e.g., gastric juice with vomiting and pancreatic secretion with diarrhea) makes manifest that a mechanism to produce a biologically relatively inactive extracellular anion HCO3- exists, whereas no comparable mechanism to produce a biologically inactive cation has evolved. Life in the ocean, which has three times the sodium concentration of extracellular fluid, involves quite different osmoregulatory stress to that in freshwater. Terrestrial life involves risk of desiccation and, in large areas of the planet, salt deficiency. Mechanisms integrated in the hypothalamus (the evolutionary ancient midbrain) control water retention and facilitate excretion of sodium, and also control the secretion of renin by the kidney. Over and above the multifactorial processes of excretion, hypothalamic sensors reacting to sodium concentration, as well as circumventricular organs sensors reacting to osmotic pressure and angiotensin II, subserve genesis of sodium hunger and thirst. These behaviors spectacularly augment the adaptive capacities of animals. Instinct (genotypic memory) and learning (phenotypic memory) are melded to give specific behavior apt to the metabolic status of the animal. The sensations, compelling emotions, and intentions generated by these vegetative systems focus the issue of the phylogenetic emergence of consciousness and whether primal awareness initially came from the interoreceptors and vegetative systems rather than the distance receptors.

Nitric oxide synthase content of hypothalamic explants: increase by norepinephrine and inactivated by NO and cGMP.

Release of luteinizing hormone (LH)-releasing hormone (LHRH), the hypothalamic peptide that controls release of LH from the adenohypophysis, is controlled by NO. There is a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers in the lateral median eminence, intermingled with terminals of the LHRH neurons. To study relations between NOS and LHRH in this brain region, we measured NOS activity in incubated medial basal hypothalamus (MBH). NOS converts [14C]arginine to equimolar quantities of [14C]citrulline plus NO, which rapidly decomposes. The [14C]citrulline serves as an index of the NO produced. NOS basal activity was suppressed by incubation of the tissue with an inhibitor of NOS, nitroarginine methyl ester (NAME) (10(-5) M). Furthermore, incubation of MBH explants for 30 min with norepinephrine (NE) increased NOS activity and the increase was prevented by prazosine (10(-5) M), an alpha 1-adrenergic receptor blocker; however, direct addition of NE to the tissue homogenate or to a preparation of MBH synaptosomes did not alter enzyme activity, which suggested that NE increased the content of NOS during incubation with the tissue. After purification of NOS, the increase in enzyme content induced by NE was still measurable. This indicates that within 30 min NE increased the synthesis of NOS in vitro. Incubation of MBH or the MBH homogenate with various concentrations of sodium nitroprusside (NP), a releaser of NO, reduced NOS activity at high concentrations (> or = 0.9 mM), which were associated with either a reduction of stimulation or a plateau of LHRH release. Finally, incubation of either MBH or the homogenate with cGMP, a major mediatior of NO action, at concentrations that increased LHRH release also reduced NOS activity. These results indicate that NO at high concentrations can inactivate NOS and that cGMP can also inhibit the enzyme directly. Therefore, the increased NOS activity induced by activation of alpha 1 receptors by NE is inhibited by NO itself and a principal product of its activity, cGMP, providing negative feedback on NOS. In central nervous system (CNS) infections with high concentrations of inducible NOS produced by glial elements, the high concentrations of NO and cGMP produced may suppress LHRH release, resulting in decreased gonadotropin and gonadal steroid release.

Distinct mechanisms underlie activation of hypothalamic neurosecretory neurons and their medullary catecholaminergic afferents in categorically different stress paradigms.

Intermittent electrical footshock induces c-fos expression in parvocellular neurosecretory neurons expressing corticotropin-releasing factor and in other visceromotor cell types of the paraventricular hypothalamic nucleus (PVH). Since catecholaminergic neurons of the nucleus of the solitary tract and ventrolateral medulla make up the dominant loci of footshock-responsive cells that project to the PVH, these were evaluated as candidate afferent mediators of hypothalamic neuroendocrine responses. Rats bearing discrete unilateral transections of this projection system were exposed to a single 30-min footshock session and sacrificed 2 hr later. Despite depletion of the aminergic innervation on the ipsilateral side, shock-induced up-regulation of Fos protein and corticotropin-releasing factor mRNA were comparable in strength and distribution in the PVH on both sides of the brain. This lesion did, however, result in a substantial reduction of Fos expression in medullary aminergic neurons on the ipsilateral side. These results contrast diametrically with those obtained in a systemic cytokine (interleukin 1) challenge paradigm, where similar cuts ablated the Fos response in the ipsilateral PVH but left intact the induction seen in the ipsilateral medulla. We conclude that (i) footshock-induced activation of medullary aminergic neurons is a secondary consequence of stress, mediated via a descending projection transected by our ablation, (ii) stress-induced activation of medullary aminergic neurons is not necessarily predictive of an involvement of these cell groups in driving hypothalamic visceromotor responses to a given stressor, and (iii) despite striking similarities in the complement of hypothalamic effector neurons and their afferents that may be activated by stresses of different types, distinct mechanisms may underlie adaptive hypothalamic responses in each.

Estradiol and the inhibition of hypothalamic gonadotropin-releasing hormone pulse generator activity in the rhesus monkey.

In mammals, gonadal function is controlled by a hypothalamic signal generator that directs the pulsatile release of gonadotropin-releasing hormone (GnRH) and the consequent pulsatile secretion of luteinizing hormone. In female rhesus monkeys, the electrophysiological correlates of GnRH pulse generator activity are abrupt, rhythmic increases in hypothalamic multiunit activity (MUA volleys), which represent the simultaneous increase in firing rate of individual neurons. MUA volleys are arrested by estradiol, either spontaneously at midcycle or after the administration of the steroid. Multiunit recordings, however, provide only a measure of total neuronal activity, leaving the behavior of the individual cells obscure. This study was conducted to determine the mode of action of estradiol at the level of single neurons associated with the GnRH pulse generator. Twenty-three such single units were identified by cluster analysis of multiunit recordings obtained from a total of six electrodes implanted in the mediobasal hypothalamus of three ovariectomized rhesus monkeys, and their activity was monitored before and after estradiol administration. The bursting of all 23 units was arrested within 4 h of estradiol administration although their baseline activity was maintained. The bursts of most units reappeared at the same time as the MUA volleys, the recovery of some was delayed, and one remained inhibited for the duration of the study (43 days). The results indicate that estradiol does not desynchronize the bursting of single units associated with the GnRH pulse generator but that it inhibits this phenomenon. The site and mechanism of action of estradiol in this regard remain to be determined.

Differential subcellular mRNA targeting: deletion of a single nucleotide prevents the transport to axons but not to dendrites of rat hypothalamic magnocellular neurons.

It has previously been shown that mRNA encoding the arginine vasopressin (AVP) precursor is targeted to axons of rat magnocellular neurons of the hypothalamo-neurohypophyseal tract. In the homozygous Brattle-boro rat, which has a G nucleotide deletion in the coding region of the AVP gene, no such targeting is observed although the gene is transcribed. RNase protection and heteroduplex analyses demonstrate that, in heterozygous animals, which express both alleles of the AVP gene, the wild-type but not the mutant transcript is subject to axonal compartmentation. In contrast, wild-type and mutant AVP mRNAs are present in dendrites. These data suggest the existence of different mechanisms for mRNA targeting to the two subcellular compartments. Axonal mRNA localization appears to take place after protein synthesis; the mutant transcript is not available for axonal targeting because it lacks a stop codon preventing its release from ribosomes. Dendritic compartmentation, on the other hand, is likely to precede translation and, thus, would be unable to discriminate between the two mRNAs.

A progesterone metabolite stimulates the release of gonadotropin-releasing hormone from GT1-1 hypothalamic neurons via the gamma-aminobutyric acid type A receptor.

The reduced progesterone metabolite tetrahydroprogesterone (3 alpha-hydroxy-5 alpha-pregnan-20-one; 3 alpha,5 alpha-THP) is a positive modulator of the gamma-aminobutyric acid type A (GABAA) receptor. Experiments performed in vitro with hypothalamic fragments have previously shown that GABA could modulate the release of gonadotropin-releasing hormone (GnRH). Using GT1-1 immortalized GnRH neurons, we investigated the role of GABAA receptor ligands, including 3 alpha,5 alpha-THP, on the release of GnRH. We first characterized the GABAA receptors expressed by these neurons. [3H]Muscimol, but not [3H]flunitrazepam, bound with high affinity to GT1-1 cell membranes (Kd = 10.9 +/- 0.3 nM; Bmax = 979 +/- 12 fmol/mg of protein), and [3H]muscimol binding was enhanced by 3 alpha,5 alpha-THP. mRNAs encoding the alpha 1 and beta 3 subunits of the GABAA receptor were detected by the reverse transcriptase polymerase chain reaction. In agreement with binding data, the benzodiazepine-binding gamma subunit mRNA was absent. GnRH release studies showed a dose-related stimulating action of muscimol. 3 alpha,5 alpha-THP not only modulated muscimol-induced secretion but also stimulated GnRH release when administered alone. Bicuculline and picrotoxin blocked the effects of 3 alpha,5 alpha-THP and muscimol. Finally, we observed that GT1-1 neurons convert progesterone to 3 alpha,5 alpha-THP. We propose that progesterone may increase the release of GnRH by a membrane mechanism, via its reduced metabolite 3 alpha,5 alpha-THP acting at the GABAA receptor.

Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.

Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion

Elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca2+ influx. Increased [Ca2+]i stimulates Ca2+-sensitive K+ channels (IK-Ca), and this, in turn, hyperpolarizes the cell and inhibits Ca2+ influx. However, in some cells expressing IK-Ca the elevation in [Ca2+]i by depletion of intracellular stores facilitates voltage-gated Ca2+ influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca2+)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca2+]i accompanied by transient hyperpolarization, followed by a sustained [Ca2+]i plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca2+]i and was mediated by apamin-sensitive IK-Ca channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca2+]i and were not mediated by inhibition of K+ current, but by facilitation of a voltage-insensitive, Ca2+-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process may also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.

Social stress and the reactivation of latent herpes simplex virus type 1

Psychological stress is thought to contribute to reactivation of latent herpes simplex virus (HSV). Although several animal models have been developed in an effort to reproduce different pathogenic aspects of HSV keratitis or labialis, until now, no good animal model existed in which application of a psychological laboratory stressor results in reliable reactivation of the virus. Reported herein, disruption of the social hierarchy within colonies of mice increased aggression among cohorts, activated the hypothalamic-pituitary-adrenal axis, and caused reactivation of latent HSV type 1 in greater than 40% of latently infected animals. However, activation of the hypothalamic-pituitary-adrenal axis using restraint stress did not activate the latent virus. Thus, the use of social stress in mice provides a good model in which to investigate the neuroendocrine mechanisms that underlie behaviorally mediated reactivation of latent herpesviruses.

Mahogany (mg) stimulates feeding and increases basal metabolic rate independent of its suppression of agouti

The mahogany (mg) locus originally was identified as a recessive suppressor of agouti, a locus encoding a skin peptide that modifies coat color by antagonizing the melanocyte-stimulating hormone receptor or MC1-R. Certain dominant alleles of agouti cause an obesity syndrome when ectopic expression of the peptide aberrantly antagonizes the MC4-R, a related melanocyte-stimulating hormone receptor expressed in hypothalamic circuitry and involved in the regulation of feeding behavior and metabolism. Recent work has demonstrated that mg, when homozygous, blocks not only the ability of agouti to induce a yellow coat color when expressed in the skin of the lethal yellow mouse (AY), but also the obesity resulting from ectopic expression of agouti in the brain. Detailed analysis of mg/mg AY/a animals, presented here, demonstrates that mg/mg blocks the obesity, hyperinsulinemia, and increased linear growth induced by ectopic expression of the agouti peptide. Remarkably, however, mg/mg did not reduce hyperphagia in the AY/a mouse. Furthermore, mg/mg induced hyperphagia and an increase in basal metabolic rate in the C57BL/6J mouse in the absence of AY. Consequently, although mahogany is broadly required for agouti peptide action, it also appears to be involved in the control of metabolic rate and feeding behavior independent of its suppression of agouti.

Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice

Leptin is a circulating protein involved in the long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety signals. We investigated the interaction between leptin and CCK-8 in the short-term regulation of food intake induced by 24-hr fasting in lean mice. Leptin, injected intraperitoneally (i.p.) at low doses (4–120 μg/kg), which did not influence feeding behavior for the first 3 hr postinjection, decreased food intake dose dependently by 47–83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was not observed between leptin and bombesin. The food-reducing effect of leptin injected with CCK was not associated with alterations in gastric emptying or locomotor behavior. Leptin–CCK action was blocked by systemic capsaicin at a dose inducing functional ablation of sensory afferent fibers and by devazepide, a CCK-A receptor antagonist but not by the CCK-B receptor antagonist, L-365,260. The decrease in food intake which occurs 5 hr after i.p. injection of leptin alone was also blunted by devazepide. Coinjection of leptin and CCK enhanced the number of Fos-positive cells in the hypothalamic paraventricular nucleus by 60%, whereas leptin or CCK alone did not modify Fos expression. These results indicate the existence of a functional synergistic interaction between leptin and CCK leading to early suppression of food intake which involves CCK-A receptors and capsaicin-sensitive afferent fibers.