Evidence that Orexin-Containing Neurones Provide Direct Input to Gonadotropin-Releasing Hormone Neurones in the Ovine Hypothalamus

Orexins A and B (ORX) have been added recently to the growing list of neuropeptides implicated in feeding and drinking behaviour as well as neuroendocrine function. In the present study, we have used single and dual labelling immunohistochemistry and a rabbit polyclonal anti-orexin-A antibody, which recognizes both ORX A and B, to examine ORX pathways in the sheep hypothalamus. ORX immunoreactive cells were distributed in the dorsomedial hypothalamic nucleus, lateral hypothalamic area, zona incerta and perifornical area; a few cells were also observed in the anterior hypothalamic area. In contrast to distribution in the rat brain, most of the ORX immunoreactive cells are localized to the dorsomedial hypothalamic nucleus and perifornical area; scattered cells are found in lateral hypothalamic area. ORX immunoreactive fibres were widely distributed throughout the hypothalamus and preoptic area with dense innervation of the medial preoptic area and bed nucleus of stria terminalis. Dual labelling demonstrated widespread expression of the long form of the leptin receptor within all ORX cells that were examined. Thirty percent of the gonadotropin releasing hormone (GnRH) cells that were examined had ORX immunoreactive terminals in close contact with no regional or sex differences. FluoroGold injections into the preoptic area retrogradely labelled a subpopulation of ORX cells in the lateral hypothalamic/perifornical area, showing ORX cells of this region project to the preoptic and could potentially provide input to GnRH cells. These findings suggest an integral role for ORX in the regulation of GnRH cells in the sheep and thus provide evidence of a novel mechanism whereby leptin can influence reproductive neuroendocrine function.     

Electrical Stimulation of the Rat Periventricular Nucleus Influences the Activity of Hypothalamic Arcuate Neurones

In rats, the release of growth hormone (GH) is inhibited during electrical stimulation of the periventricular nucleus but after the end of stimulation, there is a rebound ‘hypersecretion’ of GH. We examined the responses of arcuate neurones in pentobarbitone-anaesthetized male rats, following electrical stimulation of the periventricular nucleus to test the hypothesis that the effects of periventricular nucleus stimulation on GH secretion are mediated via effects upon GH-releasing hormone (GRF) neurones in the arcuate nucleus. The electrical activity of 2 groups of arcuate neurones were analysed before, during and after periventricular nucleus stimulation (10 Hz, 5 min, 0.5 mA biphasic, 0.5/1.0 ms): a) putative neurosecretory cells which were antidromically identified (AD) as projecting to the median eminence (n = 53) and b) non-neurosecretory cells, identified by their spontaneous ‘bursting’ pattern of activity (n = 29). During stimulation predominantly inhibitory responses were observed in both AD and bursting cell groups. Of the 39 AD cells which were spontaneously active, 25 were inhibited during the periventricular nucleus stimulation, and 10 of these showed a rebound hyperactivation following the end of stimulation. Fifteen bursting cells were inhibited during stimulation and 4 of these displayed a rebound hyperactivation following the end of stimulation. Additional evidence was sought for the identity of these cells by testing their response to electrical stimulation of the basolateral amygdala (which has previously been shown to increase plasma GH concentration without influencing the release of other pituitary hormones). Six of the 10 AD cells which displayed the inhibition/rebound response to periventricular nucleus stimulation were also excited following electrical stimulation of the basolateral amygdala. We conclude that 1) electrical stimulation of the periventricular nucleus and the basolateral amygdala exert predominantly inhibitory and excitatory effects respectively upon the activity of arcuate neurones but for neither site were the effects of stimulation exclusively upon GRF neurones, and 2) the rebound hypersecretion of GH following PeN stimulation is likely to involve the rebound activation of arcuate neurones.     

The Role of Leptin in the Regulation of Energy Balance and Adiposity

Since its discovery, leptin (a 167-amino acid product of the OB gene) has quickly moved to the forefront as an important hormone for regulation of energy balance. It closes a feedback loop from adipose tissue to hypothalamic neuropeptide-containing neural circuitry involved in regulation of food intake and neuroendocrine/autonomic outflow. While increased central leptin signalling reduces adiposity via a reduction in food intake, it also has remarkable metabolic effects that promote leanness, independent of food intake. These include: (i) increased energy expenditure, (ii) in-place degradation of fat, and (iii) increased thermogenesis. Hypothalamic neurones that synthesize corticotropin releasing hormone and melanocortins (i.e. alpha-melanocyte-stimulating hormone and agouti-related protein) are likely effector pathways that mediate the anorexigenic and metabolic effects of leptin. Activation of sympathetic outflow (via neuropeptidergic effector pathways of central leptin) to a number of tissues that store fat might be an important mechanism through which these peripheral metabolic effects are elicited. It is proposed that these peripheral metabolic effects contribute to the satiating properties of leptin.     

Somatostatin-lmmunoreactive Neurones in the Hypothalamic Ventromedial Nucleus Possess Oestrogen Receptors in the Male and Female Rat

Neurones containing oestrogen receptors (ERs) in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) are believed to play an important role in mediating oestrogen's regulatory influence on reproductive behaviour. As somatostatin (SOM)-immunoreactive neurones are found exclusively within the ventrolateral division of VMM, this study has used double-labelling immunocytochemistry techniques to evaluate whether ER-immunoreactive cells in the VMNvl synthesize SOM in both the male and female rat. Rats gonadecto-mized 1 week earlier were perfused and brain sections through the mediobasal hypothalamus processed for ER and SOM immunoreactivity using the H222 monoclonal rat ER antibody and a polyclonal rabbit SOM antiserum. Within the VMN, cells immunoreactive for SOM were found predominantly in the rostral portion of the VMNvl while ER-immunoreactive cells were distributed throughout the VMNvl. Sequential double-labelling studies revealed that many ER-containing cells in the rostral VMNvl were also immunoreactive for SOM. A semi-quantitative analysis of double-labelled cells in the rostral VMNvl of male and female rats, respectively, estimated that 52 ± 2% and 55 ±8% of SOM-IR cells possess ERs while 35 ± 1% and 28 ±3% of ER-IR cells synthesise SOM in the rostral VMNvl. No sex differences were detected at this level. These results show that approximately half of the SOM-synthesizing neurones in the rostral VMN possess ERs and indicate that SOM should now be considered alongside enkephalin and Substance P as a putative mediator of oestrogen's regulatory influence on reproduction through the VMN of the rat.     

Insulin Does Not Target CamkIIα Neurones to Critically Regulate the Neuroendocrine Reproductive Axis in Mice

Insulin signalling in the brain plays an important role in the central regulation of energy homeostasis and fertility, such that mice exhibiting widespread deletion of insulin receptors (InsR) throughout the brain and peripheral nervous system display diet sensitive obesity and hypothalamic hypogonadism. However, the specific cell types mediating the central effects of insulin on fertility remain largely unidentified. To date, the targeted deletion of InsR from individual neuronal populations implicated in the metabolic control of fertility has failed to recapitulate the hypogonadic and subfertile phenotype observed in brain‐specific InsR knockout mice. Because insulin and leptin share similar roles as centrally‐acting metabolic regulators of fertility, we used the Cre‐loxP system to generate mice with a selective inactivation of the Insr gene from the same widespread neuronal population previously shown to mediate the central effects of leptin on fertility by crossing Insr‐flox mice with calcium/calmodulin‐dependent protein kinase type IIα (CamkIIα)‐Cre mice. Multiple reproductive and metabolic parameters were then compared between male and female Insr‐flox/Cre‐positive (CamK‐IRKO) and Insr‐flox/Cre‐negative control mice. Consistent with brain‐specific InsR knockout mice, CamK‐IRKO mice exhibited a mild but significant obesogenic phenotype. Unexpectedly, CamK‐IRKO mice exhibited normal reproductive maturation and function compared to controls. No differences in the age of puberty onset, oestrous cyclicity or fecundity were observed between CamK‐IRKO and control mice. We conclude that the central effects of insulin on the neuroendocrine reproductive axis are not critically mediated via the same neuronal populations targeted by leptin.     

Systemic Leptin Increases the Electrical Activity of Supraoptic Nucleus Oxytocin Neurones in Virgin and Late Pregnant Rats

In the rat hypothalamus, fasting attenuates the expression of oxytocin and this can be reversed by exogenous leptin administration. In the present study, we investigated the effects of systemically administered leptin on the electrical activity of magnocellular neurones in the supraoptic nucleus of urethane‐anaesthetised rats. In virgin female rats, systemic leptin significantly excited identified oxytocin neurones with no detected effects on the patterning of activity, as reflected by hazard function analyses. The lowest dose that was consistently effective was 100 μg/i.v., and this dose had no significant effect on vasopressin neurones. In virgin rats fasted overnight, the spontaneous firing rate of oxytocin neurones was significantly lower than in unfasted rats, although leptin had a similar excitatory effect as in unfasted rats. In late pregnant rats (days 19–21 of pregnancy), spontaneous firing rates of oxytocin neurones were higher than in virgins, and the initial response to leptin was similar to that in virgin rats, although the increase in activity was more persistent. In fasted pregnant rats, the mean spontaneous firing rate of oxytocin neurones was again lower than in unfasted rats, although leptin had no significant effect even at the higher dose of 1 mg/rat. Thus, fasting reduced the spontaneous firing rates of oxytocin neurones in nonpregnant rats, and this effect could be reversed by the excitatory effects of leptin. Pregnant rats showed some evidence of leptin resistance but only after an overnight fast.     

Des‐Acyl Ghrelin Directly Targets the Arcuate Nucleus in a Ghrelin‐Receptor Independent Manner and Impairs the Orexigenic Effect of Ghrelin

Ghrelin is a stomach‐derived octanoylated peptide hormone that plays a variety of well‐established biological roles acting via its specific receptor known as growth hormone secretagogue receptor (GHSR). In plasma, a des‐octanoylated form of ghrelin, named des‐acyl ghrelin (DAG), also exists. DAG is suggested to be a signalling molecule that has specific targets, including the brain, and regulates some physiological functions. However, no specific receptor for DAG has been reported until now, and, consequently, the potential role of DAG as a hormone has remained a matter of debate. In the present study, we show that DAG specifically binds to and acts on a subset of arcuate nucleus (ARC) cells in a GHSR‐independent manner. ARC cells labelled by a DAG fluorescent tracer include the neuropeptide Y (NPY) and non‐NPY neurones. Given the well‐established role of the ARC in appetite regulation, we tested the effect of centrally administered DAG on food intake. We found that DAG failed to affect dark phase feeding, as well as food intake, after a starvation period; however, it impaired the orexigenic actions of peripherally administered ghrelin. Thus, we conclude that DAG directly targets ARC neurones and antagonises the orexigenic effects of peripherally administered ghrelin.     

Abnormal cholinergic and GABAergic vascular innervation in the hypothalamic arcuate nucleus of obese tub/tub mice

Tubby and tubby-like proteins (TULPs) are encoded by members of a small gene family. An autosomal recessive mutation in the mouse tub gene leads to blindness, deafness, and maturity-onset obesity. The mechanisms by which the mutation causes the obesity syndrome has not been established. We compared obese tub/tub mice and their lean littermates in order to find abnormalities within the mediobasal hypothalamus, a region intimately associated with the regulation of body weight. Using an antiserum to the vesicular acetylcholine transporter (VAChT), a marker for cholinergic neurons, many unusually large VAChT-immunoreactive (-ir) nerve terminals, identified by colocalization with the synaptic vesicle protein synaptophysin, were demonstrated in the hypothalamic arcuate nucleus of obese tub/tub mice. Double-labeling showed that VAChT-ir nerve endings also contained glutamic acid decarboxylase (GAD), a marker for gamma-aminobutyric acid (GABA) neurons. The VAChT- and GAD-ir nerve terminals were in close contact with blood vessels, identified with antisera to platelet endothelial cell adhesion molecule-1 (PECAM; also called CD31), laminin, smooth muscle actin (SMA), and glucose transporter-1 (GLUT1). Such large cholinergic and GABAergic nerve terminals surrounding blood vessels were not seen in the arcuate nucleus of lean tub/+ mice. The presence of abnormal cholinergic/GABAergic vascular innervation in the arcuate nucleus suggests that alterations in this region, which contains neurons that receive information from the periphery and which relays information about the energy status to other parts of the brain, may be central in the development of the obese phenotype in animals with an autosomal recessive mutation in the tub gene.     

Corticosterone Exerts Site-Specific and State-Dependent Effects in Prefrontal Cortex and Amygdala on Regulation of Adrenocorticotropic Hormone, Insulin and Fat Depots

Chronic stress stimulates corticosterone secretion and recruits brain pathways that regulate energy balance (caloric acquisition and deposition) and facilitate hypothalamic-pituitary-adrenal responsiveness to new stressors. We implanted corticosterone or cholesterol bilaterally either near the central nucleus of the amygdala (CeA) or in the prefrontal cortex to determine whether high concentrations of the steroid act at either site, with or without chronic stress. Rats were adrenalectomized and treated systemically with low doses of corticosterone. Half were maintained at room temperature and the other half were exposed to 5 degrees C cold for 5 days before all rats were restrained. There was limited diffusion of corticosterone from brain implants. Corticosterone in prefrontal cortex, but not CeA, decreased plasma insulin and adrenocorticotropic hormone (ACTH) responses to acute restraint in both control and chronically cold stressed rats. Corticosterone implants near CeA decreased the weight of fat depots only in cold; corticosterone implants in prefrontal cortex were ineffective. We conclude that (i) corticosterone inhibits insulin and ACTH secretion by an action in prefrontal cortex but not CeA; (ii) high concentrations of corticosterone secreted during chronic stress alter metabolism through (autonomic) outputs of the CeA and prefrontal cortex in site- and variable-specific fashion; and (iii) the amygdala is a component of a stress-recruited, state-dependent pathway.     

Sexually Dimorphic Expression of Androgen Receptor Immunoreactivity by Somatostatin Neurones in Rat Hypothalamic Periventricular Nucleus and Bed Nucleus of the Stria Terminalis

Gonadal steroids exert important regulatory actions on the hypothalamic neurones regulating growth hormone secretion and are believed to play a role in generating its sexually dimorphic pattern of secretion. Recent evidence indicates that estrogen actions on one of these neural populations, the periventricular somatostatin (SOM) neurones, are likely to be indirect as they do not possess nuclear estrogen receptors in either sex although androgen receptors (ARs) have been reported within these cells in male rats. The present study has used double-labelling immunocytochemistry procedures to examine whether sex differences exist in AR expression by SOM neurones located in the periventricular nucleus and bed nucleus of the stria terminalis (BNST). Within the hypothalamus, SOM-immunoreactive neurones were found concentrated in the periventricular nucleus while both anterior and posterior divisions of the BNST contained scattered populations of SOM cells. Cells immunoreactive for the AR were detected in all of these areas. Although the intensity of AR cell nuclei staining was equivalent in males and females in regions such as the lateral septum, the intensity of AR staining in many individual cells of the periventricular nucleus and posterior BNST of the female was reduced when compared with the male. Double-labelling experiments revealed that approximately 40% of periventricular SOM neurones expressed AR immunoreactivity in the male compared with significantly (P<0.01) fewer cells in the female (～7%). In the BNST, double-labelled cells were only detected within the principle encapsulated, interfascicular and transverse nuclei of its posterior division. Approximately 60% of SOM cells in these nuclei expressed AR immunoreactivity in the male while significantly (P<0.01) fewer did so in the female (～25%). These results indicate that substantial sex differences exist in AR expression by SOM neurones in both the periventricular nucleus and BNST. Such differences in AR expression by periventricular SOM cells may contribute to their sexually dimorphic nature and, consequently, sex differences in growth hormone secretion.     

Elevated nocturnal profiles of serum leptin in patients with depression

Leptin, the protein product of the obese (ob) gene, has been suggested to play a role in the regulation of food intake. As depressive episodes are frequently characterized by loss of appetite, reduced food intake and weight loss, altered leptin secretion might also be expected in patients with depression. Therefore, we examined nocturnal (10.00 p.m. to 7.00 a.m.) secretion of leptin, cortisol, ACTH and growth hormone (GH) in a group of 15 patients with depression and age- and sex-matched controls (age range 23–71 years). In addition, the effects of pulsatile administration of growth hormone-releasing hormone (GHRH), thought to be an endogenous antagonist of corticotropin-releasing hormone (CRH), which in turn is believed to play a critical role for the pathophysiology of depression, on nocturnal hormone secretion were assessed. Patients with depression showed a trend towards elevated nocturnal cortisol secretion (F=3.8, p<0.05). Nocturnal serum leptin was significantly higher in patients, despite a reported weight loss (F=8, p<0.05), but showed the same sexual dimorphism as in controls (F=20.9, p<0.01). No significant differences were seen between patients and controls with regard to plasma GH and ACTH. GHRH treatment increased GH secretion in both patients and controls, while the other hormones were not affected. Furthermore, serum leptin was correlated with body mass index (BMI) in controls, but not in patients with depression, supporting an altered regulation of leptin secretion in depressive illness. Finally, we provide some evidence that in young female patients the normal nocturnal leptin surge is blunted. As glucocorticoids can prevent the fasting-induced decline in serum leptin, we propose that hypercortisolism in depression might counteract the reduction in leptin secretion caused by decreased food intake and weight loss. Elevated serum leptin in depression might in turn further promote CRH release, as shown in animals and, hence, contribute to HPA system hyperactivity seen in depression.     

Characterisation of Kiss1r (Gpr54)‐Expressing Neurones in the Arcuate Nucleus of the Female Rat Hypothalamus

Kisspeptin is essential in reproduction and acts by stimulating neurones expressing gonadotrophin‐releasing hormone (GnRH). Recent studies suggest that kisspeptin has multiple roles in the modulation of neuronal circuits in systems outside the hypothalamic‐pituitary‐gonadal axis. Our recent research using in situ hybridisation (ISH) clarified the histological distribution of Kiss1r (Gpr54)‐expressing neurones in the rat brain that were presumed to be putative targets of kisspeptin. The arcuate nucleus (ARN) of the hypothalamus is one of the brain regions in which Kiss1r expression in non‐GnRH neurones is prominent. However, the characteristics of Kiss1r‐expressing neurones in the ARN remain unclear. The present study aimed to determine the neurochemical characteristics of Kiss1r‐expressing neurones in the ARN using ISH and immunofluorescence. We revealed that the majority (approximately 63%) of Kiss1r‐expressing neurones in the ARN were pro‐opiomelanocortin (POMC) neurones, which have an anorexic effect in mammals. Additionally, a few Kiss1r‐expressing neurones in the dorsal ARN are tuberoinfundibular dopamine (TIDA) neurones, which control milk production by inhibiting prolactin secretion from the anterior pituitary. TIDA neurones showed a relatively weak Kiss1r ISH signal compared to POMC neurones, as well as low co‐expression of Kiss1r (approximately 15%). We also examined the expression of Kiss1r in neuropeptide Y and kisspeptin neurones, which are reported to arise from POMC‐expressing progenitor cells during development. However, the vast majority of neuropeptide Y and kisspeptin neurones in the ARN did not express Kiss1r. These results suggest that kisspeptin may directly regulate energy homeostasis and milk production by modulating the activity of POMC and TIDA neurones, respectively. Our results provide an insight into the wide variety of roles that kisspeptin plays in homeostatic and neuroendocrine functions.