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1.  Endogenous angiotensin II-induced p44/42 MAPK activation mediates sodium appetite but not thirst or neurohypophysial secretion in male rats 
Journal of neuroendocrinology  2013;25(2):97-106.
The renin-angiotensin-aldosterone system makes a critical contribution to body fluid homeostasis, and abnormalities in this endocrine system have been implicated in certain forms of hypertension. The peptide hormone angiotensin II (AngII) regulates hydromineral homeostasis and blood pressure by acting on both peripheral and brain targets. In the brain, AngII binds to the angiotensin type 1 receptor (AT1R) to stimulate thirst, sodium appetite and both arginine vasopressin (AVP) and oxytocin (OT) secretion. The present work used an experimental model of endogenous AngII to examine the role of p44/42 mitogen-activated protein kinase (MAPK) as a signalling mechanism to mediate these responses. Animals were given a combined treatment of furosemide and a low dose of captopril (furo/cap), a diuretic and an angiotensin converting enzyme inhibitor, respectively, to elevate endogenous AngII levels in the brain. Furo/cap induced p44/42 MAPK activation in key brain areas that express AT1R, and this effect was reduced with either a centrally administered AT1R antagonist (irbesartan) or a p44/42 MAPK inhibitor (U0126). Additionally, furo/cap treatment elicited water and sodium intake, and irbesartan markedly reduced both of these behaviors. Central injection of U0126markedly attenuated furo/cap-induced sodium intake but not water intake. Furthermore, p44/42 MAPK signalling was not necessary for either furo/cap- or exogenous AngII-induced AVP or OT release. Taken together, these results indicate that p44/42 MAPK is required for AngII-induced sodium appetite, but not thirst or neurohypophysial secretion. This result may allow for discovery of more specific downstream targets of p44/42 MAPK to curb sodium appetite, known to exacerbate hypertension, while leaving thirst and neurohypophysial hormone secretion undisturbed.
PMCID: PMC4084568  PMID: 22913624
Fluid Homeostasis; Diuresis; Circumventricular Organs; Vasopressin; Oxytocin
2.  Intranasal application of vasopressin fails to elicit changes in brain immediate early gene expression, neural activity and behavioral performance of rats 
Journal of neuroendocrinology  2013;25(7):655-667.
Intranasal administration has been widely used to investigate effects of the neuropeptides vasopressin and oxytocin on human behaviors and neurological disorders, but exactly what happens when these neuropeptides are administered intranasally is far from clear. In particular, it is not clear whether a physiological significant amount of peptide enters the brain to account for the observed effects. Here, we investigated whether intranasal administration of vasopressin and oxytocin to rats induces expression of the immediate-early gene product Fos in brain areas that are sensitive to centrally administered peptide, whether it alters neuronal activity in the way that centrally administered peptide does, and whether it affects behavior in ways expected from studies of centrally administered peptide. We found that, whereas intracerebroventricular (icv) injection of very low doses of vasopressin or oxytocin increased Fos expression in several distinct brain regions, intranasal administration of large doses of the peptides had no significant effect. In contrast to the effects of vasopressin applied topically to the main olfactory bulb, we saw no changes in the electrical activity of olfactory bulb mitral cells after intranasal vasopressin administration. In addition, vasopressin given intranasally had no significant effects on social recognition or short-term recognition memory. Finally, intranasal infusions of vasopressin had no significant effects on the parameters monitored on the elevated plus maze, a rodent model of anxiety. Our data in rats suggest that, after intranasal administration, significant amounts of vasopressin and oxytocin do not reach areas in the brain at levels sufficient to change immediate early gene expression, neural activity or behavior in the ways described for central administration of the peptides.
PMCID: PMC3697072  PMID: 23656518
vasopressin; oxytocin; olfactory bulb intranasal; mitral cells; c-Fos; blood pressure; behavior
3.  Sex steroid levels and AD-like pathology in 3xTgAD mice 
Journal of neuroendocrinology  2013;25(2):131-144.
Decreases in testosterone (T) and 17β-oestradiol (E2) are associated with an increased risk for Alzheimer's disease (AD), which has been attributed to an increase in beta amyloid (Aβ) and tau pathologic lesions. While recent studies have used transgenic animal models to test the effects of sex steroid manipulations on AD-like pathology, virtually none have systematically characterised the associations between AD lesions and sex steroid levels in the blood or brain in any mutant model. The present study evaluated age-related changes in T and E2 concentrations, as well as androgen receptor (AR) and oestrogen receptor (ER) α and β expression, in brain regions displaying AD pathology in intact male and female 3xTgAD and non-transgenic (ntg) mice. We report for the first time that circulating and brain T levels significantly increase in male 3xTgAD mice with age, but without changes in AR-immunoreactive (ir) cell number in either the hippocampal CA1 or medial amygdala. The age-related increase in hippocampal T levels correlated positively with increases in the conformational tau isoform, Alz50. These data suggest that the over-expression of human tau may up regulate the hypothalamic-pituitary-gonadal axis in these mice. Although circulating and brain E2 levels remained stable with age in both male and female 3xTgAD and ntg mice, ER-ir cell number in the hippocampus and medial amygdala decreased with age in female transgenic mice. Further, E2 levels were significantly higher in the hippocampus than in serum, suggesting local production of E2. Although triple transgenic mice mimic AD-like pathology, they do not fully replicate changes in human sex steroid levels, and may not be the best model for studying the effects of sex steroids on AD lesions.
PMCID: PMC4065422  PMID: 22889357
Alzheimer's disease; androgen receptor; hippocampus; transgenic; testosterone; oestradiol
4.  Photoperiodic Changes in Endocannabinoid Levels and Energetic Responses to Altered Signalling at CB1 Receptors in Siberian Hamsters 
Journal of neuroendocrinology  2012;24(7):1030-1039.
Siberian hamsters (Phodopus sungorus) adapt to seasonal environmental conditions with marked changes in body mass, primarily in the form of adiposity. Winter-like conditions (e.g. short days) are sufficient to decrease body mass by approximately 30% in part via reductions in food intake. The neuroendocrine mechanisms responsible for these changes are not well understood, and homeostatic orexigenic/anorexigenic systems of the hypothalamus provide little explanation. We investigated the potential role of endocannabinoids, which are known modulators of appetite and metabolism, in mediating seasonal changes in energy balance. Specifically, we housed hamsters in long or short days for 0, 3, or 9 weeks and measured endocannabinoid levels in the hypothalamus, brainstem, liver and retroperitoneal white adipose tissue (RWAT). An additional group of males housed in short days for 25 weeks were also compared with long-day controls. Following 9 weeks in short days, levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) were significantly elevated in RWAT and reduced in brainstem, although they returned to long-day levels by week 25 in short-day males that had cycled back to summer-like energy balance. Endocannabinoid levels in these tissues correlated significantly with adiposity and change in body mass. No photoperiodic changes were observed in the hypothalamus or liver; however, sex differences in 2-AG levels were found in the liver (males > females). We further tested the effects of CB1 receptor signalling on ingestive behaviour. Five daily injections of CB1 antagonist SR141716 significantly reduced food intake and body mass but not food hoarding. Although the CB1 agonist arachidonyl-2-chloroethylamide did not appreciably affect either ingestive behaviour, body mass was significantly elevated following 2 days of injections. Taken altogether, these findings demonstrate that endocannabinoid levels vary with sex and photoperiod in a site-specific manner, and that altered signalling at CB1 receptors affects energy balance in Siberian hamsters.
PMCID: PMC4060156  PMID: 22420341
energy balance; adiposity; seasonality; sex differences; 2-AG
5.  Maternal Contact Differentially Modulates Central and Peripheral Oxytocin in Rat Pups During a Brief Regime of Mother–Pup Interaction that Induces a Filial Huddling Preference 
Journal of neuroendocrinology  2012;24(5):831-840.
Central oxytocin mediates the acquisition of a filial preference for maternal odour in rat pups, manifested by their huddling preferences. The present study was designed to examine whether maternal care modulates oxytocin concentrations in rat pups and, if so, how different types of maternal contact are associated with the pups’ oxytocin concentrations. Pairs of 14-day-old littermates were removed from their home cage for 1 h and then placed with a lactating foster mother for 2 h, or they remained isolated at room temperature. Enzyme immunoassays revealed that maternal care and maternal separation can differentially modulate pups’ oxytocin concentrations. Both hypothalamic and serum oxytocin increased during the 1-h separation. Pups placed with a foster mother after the separation maintained the same concentrations in the hypothalamus and serum through the fostering period. By contrast, pups placed with no mother showed a further increase in hypothalamic oxytocin but serum oxytocin decreased. Behavioural analyses revealed that skin-to-skin contact with the mother, but not simple physical contact or maternal licking / grooming, was positively correlated with the pups’ hypothalamic oxytocin concentrations. These neuroendocrine data match previous findings showing that skin-to-skin contact with mother facilitates the acquisition of the pups’ huddling preference for a maternally-associated odour. Taken together, the present study suggests that maternal skin-to-skin contact stimulates pups’ central oxytocin, at the same time as creating the conditions for inducing a preference for maternal odour and establishing a social affiliation in rat pups; the natural schedule of maternal separation and reunion may modulate pups’ oxytocin concentrations, providing scaffolding for the acquisition of their filial huddling preference.
PMCID: PMC4060530  PMID: 22260655
oxytocin; maternal care; maternal separation; skin-to-skin contact; rat pup; affiliative behaviour; attachment; associative learning
6.  Oestradiol decreases melanin-concentrating hormone (MCH) and MCH receptor expression in the hypothalamus of female rats 
Journal of neuroendocrinology  2013;25(6):570-579.
Previous studies have shown that oestradiol (E2) decreases the orexigenic effect of melanin-concentrating hormone (MCH). Here, we examined whether this action of E2 is mediated by its ability to decrease the expression of MCH or its receptor (MCHR1). Using immunocytochemistry and Western blotting, we examined whether E2 decreases MCH-immunoreactive neurones or MCHR1 protein content in the hypothalamus of female rats. We found that both MCH and MCHR1 protein expression was decreased by acute E2 treatment in OVX rats, and by the peri-ovulatory increase in circulating E2 in pro-oestrous rats, relative to rats at other cycle stages. To determine whether these changes in MCH/MCHR1 protein expression may be mediated by E2’s ability to directly regulate the transcription of MCH and MCHR1 genes, the effect of E2 treatment on MCH and MCHR1 mRNA expression in a neuronal hypothalamic cell line was examined using real time RT-PCR. We also determined whether MCH and oestrogen receptor alpha (ERα) are co-expressed in the hypothalamus of female rats. E2 treatment did not decrease MCH or MCHR1 mRNA expression in vitro, and no hypothalamic neurones were identified that co-expressed MCH and ERα. We conclude that E2-dependent decreases in hypothalamic MCH/MCHR1 protein expression mediate E2’s ability to decrease MCH-induced feeding. The current findings suggest, however, that E2 exerts these actions indirectly, likely though interactions with other neuronal systems that provide afferent input to MCH and MCHR1 neurones.
PMCID: PMC3668853  PMID: 23414264
Food Intake; Oestrogen; Lateral Hypothalamus; Oestrogen Receptor α; PPT
7.  Rapid non-genomic effects of oestradiol on GnRH neurons 
Journal of neuroendocrinology  2012;24(1):117-121.
That oestradiol can have both negative and positive feedback actions upon the release of gonadotropin-releasing hormone (GnRH) has been understood for decades. The vast majority of studies have investigated the effects of in vivo oestrogen administration. In the past decade, evidence has accumulated in many neuronal and non-neuronal systems that in addition to traditional genomic action via transcription factor receptors, steroids can also initiate effects rapidly via signaling cascades typically associated with the cell membrane. Here we review work examining the rapid actions of oestradiol on GnRH neurons, addressing the questions of dose dependence, receptor subtypes, signaling cascade and intrinsic and synaptic properties that are rapidly modulated by this steroid.
PMCID: PMC3983560  PMID: 21496126
8.  Social stress and the polymorphic region of the serotonin reuptake transporter gene modify estradiol-induced changes on central monoamine concentrations in female rhesus monkeys 
Journal of neuroendocrinology  2013;25(4):321-328.
Psychosocial stress exposure is linked to a disruption of emotional regulation that can manifest as anxiety and depression. Women are more likely to suffer from such psychopathologies than men, indicating that gender-based differences in gonadal steroids may be a key factor in the etiology of stress-induced adverse health outcomes. Estradiol (E2) positively influences mood and cognition in females, an effect likely related to E2’s ability to modulate the serotonin and dopamine neurotransmitter systems. Furthermore, genetic variation due to the polymorphism in the promoter region of the gene (SLC6A4) encoding the serotonin transporter (5HTTLPR) also can influence E2’s ability to modulate behavior and physiology. However, it remains uncertain whether exposure to social stress interacts with the 5HTTLPR to influence E2-induced changes in behavior and physiology. The present study used ovariectomized adult female rhesus monkeys to investigate acute and chronic effects of E2 on central monoamine metabolite concentrations using CSF sampling. We further assessed how E2-induced changes in monoamine metabolite levels are modified by the unpredictable stress of social subordination and the 5HTTLPR polymorphism. Levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5HIAA) decreased significantly during chronic E2 treatment only in dominant females with the long promoter length of SLC6A4. Chronic administration of E2 decreased levels of the dopamine metabolite dihydrophenylacetic acid (DOPAC) in a manner independent of the social status, 5HTTLPR genotype, or their interactions. Overall levels of dopamine and serotonin metabolites were increased in subordinate females but this effect of social stress was not influenced by 5HTTLPR genotype. Together, these data emphasize how E2 can modulate central neurotransmitter systems and indicate that social subordination in female monkeys is a valid model for examining how chronic psychosocial stress alters sensitivity to E2. Future studies are necessary to elaborate how changes in central neurotransmitter metabolism due to E2 and prolonged exposure to stress affect behavior and physiology.
PMCID: PMC3605214  PMID: 23253112
Psychosocial stress; estradiol; serotonin reuptake polymorphism; monkeys
9.  Prenatal Programming by Testosterone of Hypothalamic Metabolic Control Neurones in the Ewe 
Journal of neuroendocrinology  2011;23(5):401-411.
Ewes treated prenatally with testosterone (T) develop metabolic deficits, including insulin resistance, in addition to reproductive dysfunctions that collectively mimic polycystic ovarian syndrome (PCOS), a common endocrine disease in women. We hypothesised that metabolic deficits associated with prenatal T excess involve alterations in arcuate nucleus (ARC) neurones that contain either agouti-related peptide (AgRP) or proopiomelanocortin (POMC). Characterization of these neurones in the ewe showed that immunoreactive AgRP and POMC neurones were present in separate populations in the ARC, that AgRP and POMC neurones co-expressed either neuropeptide Y or cocaine- and amphetamine-regulated transcript, respectively, and that each population had a high degree of colocalization with androgen receptors. Examination of the effect of prenatal T exposure on the number of AgRP and POMC neurones in adult ewes showed that prenatal T excess significantly increased the number of AgRP but, not POMC neurones compared to controls; this increase was restricted to the middle division of the ARC, was mimicked by prenatal treatment with dihydrotestosterone, a non-aromatizable androgen, and was blocked by co-treatment of prenatal T with the anti-androgen, flutamide. The density of AgRP fibre immunoreactivity in the preoptic area, paraventricular nucleus, lateral hypothalamus and dorsomedial hypothalamic nucleus was also increased by prenatal T exposure. Thus, ewes that were exposed to androgens during foetal life showed alterations in the number of AgRP-immunoreactive neurones and the density of fibre immunoreactivity in their projection areas, suggestive of permanent prenatal programming of metabolic circuitry that may, in turn, contribute to insulin resistance and increased risk of obesity in this model of PCOS.
PMCID: PMC3939689  PMID: 21418339
Sheep; Androgens; AgRP; POMC; PCOS
10.  Ovarian regulation of kisspeptin neurones in the arcuate nucleus of the rhesus monkey (Macaca mulatta) 
Journal of neuroendocrinology  2013;25(5):488-496.
Tonic gonadotrophin secretion throughout the menstrual cycle is regulated by the negative feedback actions of ovarian oestradiol (E2) and progesterone (P). While kisspeptin neurones in the arcuate nucleus (ARC) of the hypothalamus appear to play a major role in mediating these feedback actions of the steroids in non-primate species, this issue has been less well studied in the monkey. Here, we used immunohistochemistry (IHC) and in situ hybridization (ISH) to examine kisspeptin and KISS1 expression, respectively, in the mediobasal hypothalamus (MBH) of adult ovariectomised (OVX) rhesus monkeys. We also examined kisspeptin expression in the MBH of ovarian intact females, and the effect of E2, P and E2+P replacement on KISS1 expression in OVX animals. Kisspeptin or KISS1 expressing neurons and pronounced kisspeptin fibres were readily identified throughout the ARC of ovariectomised monkeys, but in intact animals on the other hand kisspeptin cell bodies were small in size and number and only fine fibers were observed. Replacement of OVX monkeys with physiologic levels of E2, either alone or with luteal phase levels of P, abolished KISS1 expression in the ARC. Interestingly, P replacement alone for 14 days also resulted in a significant downregulation of KISS1 expression. These findings support the view that, in primates, as in rodents and sheep, kisspeptin signaling in ARC neurones appears to play an important role in mediating the negative feedback action of E2 on gonadotrophin secretion, and indicate a need to further study their regulation by P.
PMCID: PMC3928808  PMID: 23331967
kisspeptin; KISS1; negative feedback; monkey; gonadotrophin
11.  Social opportunity causes rapid transcriptional changes in the social behaviour network of the brain in an African cichlid fish 
Journal of neuroendocrinology  2013;25(2):145-157.
Animals constantly integrate external stimuli with their own internal physiological state to make appropriate behavioural decisions. Little is known, however, about where in the brain the salience of these signals is evaluated, or which neural and transcriptional mechanisms link this integration to adaptive behaviours. We used an African cichlid fish Astatotilapia burtoni to test the hypothesis that a new social opportunity activates the conserved ‘social behaviour network’ (SBN), a collection of brain nuclei known to regulate social behaviours across vertebrates. We measured mRNA levels of immediate early genes (IEGs) in microdissected brain regions as a proxy for neuronal activation, and discovered that IEGs were higher in all SBN nuclei in males that were given an opportunity to rise in social rank compared to control stable subordinate and dominant individuals. Further, since the presence of sex-steroid receptors is one defining criteria of SBN nuclei, we also tested whether social opportunity or status influenced androgen and oestrogen receptor mRNA levels within these same regions. There were several rapid region-specific changes in receptor mRNA levels induced by social opportunity, most notably in oestrogen receptor subtypes in areas that regulate social aggression and reproduction, suggesting that oestrogenic signalling pathways play an important role in regulating male status. Several receptor mRNA changes occurred in regions with putative homologies to the mammalian septum and extended amygdala, two regions shared by SBN and reward circuits, suggesting an important role in integration of social salience, stressors, hormonal state, and adaptive behaviours. We also show increases in plasma sex- and stress-steroids at 30-min after a rise in social rank. This rapid endocrine and transcriptional response suggests that the SBN is involved in the integration of social inputs with internal hormonal state to facilitate the transition to dominant status, which ultimately leads to improved fitness for the previously reproductively-suppressed individual.
PMCID: PMC3537875  PMID: 22958303
Astatotilapia burtoni; dominance; immediate early gene; SBN; steroid receptor; teleost
12.  PI3K is an upstream regulator of the PDE3B pathway of leptin signaling that may not involve activation of Akt in the rat hypothalamus 
Journal of neuroendocrinology  2013;25(2):168-179.
Leptin, the product of the obese gene, regulates energy homeostasis by acting primarily at the level of the hypothalamus. Leptin action through its receptor involves various pathways including the signal transducer and activator of transcription (STAT3), phosphatidylinositol 3-kinase (PI3K), and phosphodiesterase 3B (PDE3B)-cAMP signaling in the CNS and peripheral tissues. In the hypothalamus, leptin stimulates STAT3 activation, and induces PI3K and PDE3B activities, among others. We have previously demonstrated that PDE3B activation in the hypothalamus is critical for transducing anorectic and body weight reducing effects of leptin. Similarly, PI3K has been implicated toplay a critical role in leptin signaling in the hypothalamus. Whereas in insulin signaling pathway, PI3K is known to be an upstream regulator of PDE3B in non-neuronal tissues, it is still unknown whether this is also the case for leptin signaling in the hypothalamus. To address this possibility, the effect of wortmannin, a specific PI3K inhibitor, was examined on the leptin-induced PDE3B activity in the hypothalamus of male rats. Intracerebroventricular (icv) injection of leptin (4 μg) significantly increased PDE3B activity by 2-fold in the hypothalamus as expected. However, prior administration of wortmannin completely reversed the stimulatory effect of leptin on PDE3B activity in the hypothalamus. To demonstrate whether leptin stimulates p-Akt levels and there by a possible upstream regulator of PDE3B, we examined the effects of icv leptin on p-Akt levels in the hypothalamus and compared that with the known stimulatory effect of insulin on p-Akt. We observed that insulin increased p-Akt levels but leptin failed to do so although it increased p-STAT3 levels in the rat hypothalamus. Immunocytochemistry confirmed the biochemical finding in that leptin failed but insulin increased the number of p-Akt positive cells in various hypothalamic nuclei. Altogether these results implicate PI3K but not Akt as an upstream regulator of the PDE3B pathway of leptin signaling in the rat hypothalamus.
PMCID: PMC3549038  PMID: 22967108
leptin; insulin; PI3K; p-Akt; PDE3B; p-STAT3; hypothalamus
13.  A novel reproductive peptide, phoenixin 
Journal of neuroendocrinology  2013;25(2):206-215.
Normal anterior pituitary function is essential for fertility. Release from the gland of the reproductive hormones LH and FSH is regulated primarily by hypothalamically-derived gonadotropin releasing hormone (GnRH), although other releasing factors have been postulated to exist. Using a bioinformatic approach, we have identified a novel peptide, phoenixin, that regulates pituitary gonadotropin secretion by modulating expression of the GnRH receptor, an action with physiologically relevant consequences. Compromise of phoenixin in vivo using siRNA resulted in the delayed appearance of oestrus and a reduction in GnRH receptor expression in the pituitary. Phoenixin may represent a new class of hypothalamically-derived pituitary priming factors (PFs) that sensitise the pituitary to the action of other RFs, rather than directly stimulating the fusion of secretory vesicles to pituitary membranes.
PMCID: PMC3556183  PMID: 22963497
gonadotrophins; GnRH receptor; siRNA; bioinformatics; pituitary; reproduction
14.  Neuropeptide W has cell phenotype-specific effects on the excitability of different subpopulations of paraventricular nucleus neurons 
Journal of neuroendocrinology  2009;21(10):10.1111/j.1365-2826.2009.01904.x.
Administration of the neuropeptides NPW and NPB in rodents has been shown to influence the activity of a variety of autonomic and neuroendocrine systems. The paraventricular nucleus is a major autonomic and neuroendocrine integration site in the hypothalamus and neurons within this nucleus express the receptor for these ligands, neuropeptide B/W receptor 1 (NPBWR1). Therefore we used whole cell patch clamp recordings coupled with single cell RT-PCR to examine the effects of neuropeptide W-23 (NPW-23) on the excitability of identified paraventricular nucleus neurons. Oxytocin, vasopressin and thyrotropin releasing hormone neurons were all found to be responsive to 10 nM NPW-23, although both depolarizing and hyperpolarizing effects were observed in each of these cell groups. In contrast corticotropin releasing hormone cells were unaffected. Further subdivision of chemically phenotyped cell groups into magnocellular, neuroendocrine or pre-autonomic neurons, using their electrophysiological fingerprints, revealed that neurons projecting to medullary and spinal targets were predominantly inhibited by NPW-23 while those that projected to median eminence or neural lobe showed nearly equivalent numbers of depolarizing and hyperpolarizing cells. The demonstration of particular phenotypic populations of paraventricular nucleus neurons showing NPW-induced effects on excitability reinforces the importance of the NPB/NPW neuropeptide system as a regulator of autonomic function.
PMCID: PMC3861898  PMID: 19686447
Single cell RT-PCR; hypothalamus; oxytocin; vasopressin; thyrotropin releasing hormone; corticotropin releasing hormone
15.  Intact Catecholamine Inputs to the Forebrain Are Required for Appropriate Regulation of CRH and Vasopressin Gene Expression by Corticosterone in the Rat Paraventricular Nucleus 
Journal of neuroendocrinology  2012;24(12):1517-1526.
CRH neuroendocrine neurones in the paraventricular nucleus of the hypothalamus (PVH) drive ACTH and thereby glucocorticoid release from pituitary corticotrophs and adrenal cortex respectively. Glucocorticoids suppress the ability of neuroendocrine CRH neurons to synthesise and release ACTH secretogogues. Despite the importance of glucocorticoids as regulatory signals to CRH neurones in the extended time domain, how and where they act in this capacity is still not fully understood. Ascending catecholamine projections encode important cardiovascular, metabolic, and other visceral information to the rat PVH and surrounding hypothalamus. These afferents have previously been implicated as targets for glucocorticoid action, including a role in the feedback regulation of PVH neuroendocrine neurones. To determine the contribution of these neurones to corticosterone’s long-term actions on CRH and vasopressin (AVP) gene expression in the PVH we used an immunocytotoxin (a conjugate of the cytotoxin saporin and an antibody against dopamine-β-hydroxylase) that specifically ablates adrenergic and noradrenergic neurones. Lesions were administered to intact animals and to adrenalectomized animals with either no corticosterone or corticosterone replacement that provided levels above those required to normalise Crh expression. The ability of elevated levels of corticosterone to suppress Crh expression was abolished in animals lacking catecholaminergic innervation of the PVH. No effect was seen in the absence of corticosterone or in animals with intact adrenals. Furthermore, Avp expression, which is increased in CRH neurons following adrenalectomy, was suppressed in adrenalectomized catecholaminergic lesioned animals. Interactions between corticosterone and catecholaminergic projections to the hypothalamus therefore make significant contributions to the regulation of Crh and Avp expression. However, the importance of catecholamine inputs is only apparent when circulating corticosterone concentrations are maintained either below or above those required to maintain the activity of the hypothalamo-pituitary-adrenal axis that is seen in intact animals.
PMCID: PMC3502639  PMID: 22831701
Hypothalamus; ACTH secretogogue; Body weight; Metabolism; Stress; Amygdala
16.  Selective up-regulation of JunD transcript and protein expression in vasopressinergic supraoptic nucleus (SON) neurones in water deprived rats 
Journal of neuroendocrinology  2012;24(12):1542-1552.
The magnocellular neurones (MCN) of the supraoptic nucleus (SON) undergo reversible changes during dehydration. We hypothesise that alterations in steady-state transcript levels might be partially responsible for this plasticity. In turn, regulation of transcript abundance might be mediated by transcription factors. We have previously used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation. We observed down-regulation of 11 and up-regulation of 31 transcription factor transcripts, including members of the AP-1 gene family, namely c-fos, c-jun, fosl1 and junD. As JunD expression and regulation within the SON has not been previously described, we have used in situ hybridisation and quantitative RT-PCR to confirm the array results, demonstrating a significant increase in JunD mRNA levels following 24-hours and 72-hours of water deprivation. Western blot and immunohistochemistry revealed a significant increase in JunD protein expression following dehydration. Double staining fluorescence immunohistochemistry with a neurone specific marker (NeuN) demonstrated that JunD staining is predominantly neuronal. Additionally, JunD immunoreactivity is observed primarily in vasopressin containing neurones with markedly less staining seen in oxytocin containing MCNs. Furthermore, JunD is highly co-expressed with c-Fos in MCNs of the SON following dehydration. These results suggest that JunD plays a role in the regulation of gene expression within MCNs of the SON in association with other Fos and Jun family members.
PMCID: PMC3499652  PMID: 22827527
hypothalamus; osmotic regulation; plasticity; microarray; RT-PCR; in situ hybridisation
17.  Brain aromatase and circulating corticosterone are rapidly regulated by combined acute stress and sexual interaction in a sex specific manner 
Journal of neuroendocrinology  2012;24(10):1322-1334.
Neural production of 17β-oestradiol via aromatisation of testosterone may play a critical role in rapid, non-genomic regulation of physiological and behavioural processes. In brain nuclei implicated in the control of sexual behaviour, sexual or stressfull stimuli induce respectively a rapid inhibition or increase in preoptic aromatase activity (AA). Here, we tested quail that were either non-stressed or acutely stressed (15 min restraint) immediately prior to sexual interaction (5 min) with stressed or non-stressed partners. We measured nuclei-specific AA changes, corresponding behavioural output, fertilisation rates and corticosterone (CORT) concentrations. In males, sexual interaction rapidly reversed stress-induced increases of AA in the medial preoptic nucleus (POM). This time scale (<5min) highlights the dynamic potential of the aromatase system to integrate input from stimuli that drive AA in opposing directions. Moreover, acute stress had minimal effects on male behaviour suggesting that the input from the sexual stimuli on POM AA may actively preserve sexual behaviour despite stress exposure. We also found distinct sex differences in contextual physiological responses: while males did not show any effect of partner status, females responded to both their stress exposure and the male partner’s stress exposure at the level of circulating CORT and AA. In addition, fertilisation rates and female CORT correlated with the male partner’s exhibition of sexually aggressive behaviour suggesting that female perception of the male can affect their physiology as much as direct stress. Overall, male reproduction appears relatively simple – sexual stimuli, irrespective of stress, drives major neural changes including rapid reversal of stress-induced changes of AA. In contrast, female reproduction appears more nuanced and context specific, with subjects responding physiologically and behaviourally to stress, the male partner’s stress exposure, and female-directed male behaviour.
PMCID: PMC3510384  PMID: 22612582
Oestrogens; acute stress; sexual behavior; sex differences; medial preoptic nucleus
18.  Characterisation of Human Oestrogen Receptor beta (ERβ) Splice Variants in Neuronal Cells 
Journal of neuroendocrinology  2012;24(10):1311-1321.
Oestrogen receptor (ER)α and ERβ are members of the ligand-activated superfamily of nuclear receptors and mediate most facets of oestrogen signalling. Several naturally occurring splice variants of each ER have been identified in the human brain, yet the biological significance of these splice variants in the brain remains unknown. In the present study, we exploit the unique structural differences of the human ERβ splice variants to determine the functional significance of individual ER domains in the brain. We previously established that full-length rodent ERβ (i.e. rERβ1) has constitutive transcriptional activity in neuronal cells in the absence of ligand. By contrast to the rodent splice variants, the human ERβ splice variants used in the present study contain varying length truncations of exon 8, which encodes for the E/F domains. Our results reveal that, in neuronal cells, each human-specific ERβ splice variant constitutively activated promoters mediated by a canonical oestrogen response element and repressed promoters mediated by activator protein-1 sites via p38 activity. From these data, we conclude that the C-terminus, encoding the AF-2 region and F domain, is not essential for the constitutive properties of human ERβ. Taken together, these studies show that human-specific ERβ variants are constitutively active and also provide novel insight into the contributions of the functional domains of ERβ towards mediating constitutive transcription at various promoters in neuronal cells.
PMCID: PMC3727901  PMID: 22577883
oestrogen receptor β; splice variants; ERE; AP-1; vasopressin; neurones
19.  Dependence of the Excitability of Pituitary Cells on Cyclic Nucleotides 
Journal of neuroendocrinology  2012;24(9):1183-1200.
Cyclic 3′,5′-adenosine monophosphate and cyclic 3′,5′-guanosine monophosphate are intracellular (second) messengers that are produced from the nucleotide triphosphates by a family of enzymes consisting of adenylyl and guanylyl cyclases. These enzymes are involved in a broad array of signal transduction pathways mediated by the cyclic nucleotide monophosphates and their kinases, which control multiple aspects of cell function through the phosphorylation of protein substrates. Here, we review the findings and working hypotheses on the role of the cyclic nucleotides and their kinases in the control of electrical activity of the endocrine pituitary cells and the plasma membrane channels involved in this process.
PMCID: PMC3421050  PMID: 22564128
cAMP; cGMP; protein kinase A; protein kinase G; voltage-gated channels
20.  Diet-induced obesity resistance of Kv1.3−/− mice is olfactory bulb dependent 
Journal of Neuroendocrinology  2012;24(8):1087-1095.
Gene-targeted deletion of the voltage-gated potassium channel, Kv1.3 (Kv1.3−/−), increases olfactory sensitivity and discriminatory ability, and causes resistance to diet-induced obesity (DIO) in mice. The objective of the present study was to determine if the enhanced olfactory ability of the Kv1.3−/− mouse contributes to the resistance to DIO. Kv1.3+/+ and Kv1.3−/− mice were subject to bilateral olfactory bulbectomy (OBX) or sham surgery at 9 weeks of age and placed on either a control chow diet (CF) or a 32% moderately high-fat diet (MHF). Caloric and water intake, locomotor activity, and oxygen consumption were monitored after 5 weeks of diet treatment. At the end of 26 weeks of diet treatment, fat pad weight and blood chemistry were evaluated. Kv1.3+/+ mice exhibited a significant increase in weight, adiposity, fasting glucose and fasting leptin in response to the MHF-diet, with or without OBX. When treated with a MHF-diet, Kv1.3−/− mice gained significantly less weight than Kv1.3 +/+ mice and exhibited a significant increase in light phase metabolism. OBX of Kv1.3−/− mice prevented the resistance to DIO and concomitant upregulation of light phase metabolism while decreasing dark phase metabolism and total energy expenditure. These findings suggest that pathways activated in Kv1.3 −/− that increased energy expenditure and led to resistance to DIO are olfactory bulb dependent. Thus, these findings add to a growing body of evidence suggesting that the olfactory system can modulate pathways involved in the regulation of energy balance.
PMCID: PMC3391345  PMID: 22435906
Olfactory bulbectomy; diet-induced obesity; super-smeller; potassium channel; oxygen consumption
21.  Differential and Interactive Effects of Ligand-Bound Progesterone Receptor A and B Isoforms on Tyrosine Hydroxylase Promoter Activity 
Journal of neuroendocrinology  2011;23(10):915-925.
The classical progesterone receptors (PRs) are expressed in some hypothalamic dopaminergic and brainstem noradrenergic neurons. Progesterone influences prolactin and luteinising hormone release from the anterior pituitary gland, in part by regulating the activity of these catecholaminergic neurons. The aim of this study was to determine the effects of PRs on tyrosine hydroxylase (TH) promoter activity. When CAD, SK-N-SH and CV-1 cells were transfected with TH promoter constructs and PR-A or PR-B expression vectors, progesterone treatment caused three- to six-fold increases in TH-9.0kb promoter activity in PR-B expressing cells, but a modest increase or no change in PR-A expressing cells. Using CAD cells, deletional analysis mapped the site of PR action to the −1403 to −1304 bp region of the TH promoter. Mutational analysis of putative regulatory sequences in this region indicated multiple DNA elements are required for complete PR-B transactivation. Electrophoretic mobility shift assays were unable to demonstrate direct PR-B binding to TH promoter DNA sequences. However, chromatin immunoprecipitation (ChIP) analysis indicated PR-B was recruited to the TH promoter. Two different PR-B DNA binding domain mutants had opposite effects on PR-B mediated TH promoter activation. A GS to AA mutation located in the p-box of the first zinc finger of PR-B inhibited progesterone transactivation of the TH promoter, whereas a C to A mutation in the zinc finger increased transactivation. PR-A was able to inhibit PR-B transactivation in a dose-dependent manner, although the degree of PR-A inhibition was dependent on the TH promoter deletion construct. These data indicate that ligand-bound PR-B is recruited to DNA elements in the TH promoter and acts as a transcriptional activator of the TH gene and that changes in the ratio of PR-A to PR-B may affect the ability of progesterone to increase TH expression.
PMCID: PMC3721978  PMID: 21815951
catecholamine; tyrosine hydroxylase; progesterone receptor; gene expression; transcriptional regulation; steroid hormones
22.  Maternal deprivation in rats is associated with corticotropin releasing hormone (CRH) promoter hypomethylation and enhances CRH transcriptional responses to stress in adulthood 
Journal of Neuroendocrinology  2012;24(7):1055-1064.
Exposure to stress during early development causes long-lasting alterations in behavior and hypothalamic pituitary adrenal (HPA) axis activity, including increased expression of corticotropin releasing hormone (CRH). To determine whether early life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription, 8-week old female and male rats, subjected to maternal deprivation (MD) between days 2 and 13 post-birth, were studied for HPA axis responses to stress and CRH promoter methylation in the hypothalamic paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA). Plasma corticosterone and PVN CRH hnRNA responses to acute restraint stress were higher in MD rats of both sexes. DNA methylation analysis of the CRH promoter revealed a significantly lower percent of methylation in 2 CpGs preceding (CpG1) and inside (CpG2) the cyclic AMP-responsive element (CRE) at −230 bp in the CRH promoter in the PVN but not the CeA of MD rats. Gel-shift assays, using nuclear proteins from forskolin treated hypothalamic 4B cells and CRH promoter CRE oligonucleotides, unmethylated or methylated at CpG1, revealed a strong band which was supershifted by phospho-CREB antibody. This band was 50% weaker using oligonucleotides methylated at CpG2 (intra-CRE), or methylated at both CpG1 and CpG2. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress causes long-lasting enhanced CRH transcriptional activity in the PVN of both sexes. Hypomethylation of the CRH promoter CRE, a region critical for CRH transcriptional activation, could serve as a mechanism for the increased transcriptional responses to stress observed in MD rats.
PMCID: PMC3380160  PMID: 22375940
maternal deprivation; corticotropin releasing hormone; DNA methylation; HPA axis; CRH
23.  Two types of burst firing in gonadotropin-releasing hormone (GnRH) neurones 
Journal of Neuroendocrinology  2012;24(7):1065-1077.
GnRH neurones fire spontaneous bursts of action potentials, but little is understood about the underlying mechanisms. Here we show evidence for two types of bursting/oscillation driven by different mechanisms. Properties of these different types are clarified using mathematical modeling and a recently developed active-phase/silent-phase correlation technique. The first type of GnRH neurone (1–2%) exhibits slow (~0.05Hz) spontaneous oscillations in membrane potential. Action potential bursts are often observed during oscillation depolarization, but some oscillations were entirely subthreshold. Oscillations persist after blockade of fast sodium channels with TTX and blocking receptors for ionotropic fast synaptic transmission, indicating they are intrinsically generated. In the second type of GnRH neurone, bursts were irregular and TTX caused a stable membrane potential. The two types of bursting cells exhibited distinct active-phase/silent-phase correlation patterns, which is suggestive of distinct mechanisms underlying the rhythms. Further studies of type 1 oscillating cells revealed that the oscillation period was not affected by current or voltage steps, although amplitude was sometimes damped. Oestradiol, an important feedback regulator of GnRH neuronal activity, acutely and markedly altered oscillations, specifically depolarizing the oscillation nadir and initiating or increasing firing. Blocking calcium-activated potassium channels, which are rapidly reduced by oestradiol, had a similar effect on oscillations. Kisspeptin, a potent activator of GnRH neurones, translated the oscillation to more depolarised potentials, without altering period or amplitude. These data show that there are at least two distinct types of GnRH neurone bursting patterns with different underlying mechanisms.
PMCID: PMC3380170  PMID: 22435872
burst; oscillation; hypothalamus; neuroendocrine; parabolic
24.  TTF1, a homeodomain containing transcription factor, contributes to regulating periodic oscillations in GnRH gene expression 
Journal of Neuroendocrinology  2012;24(6):916-929.
Thyroid transcription factor 1 (TTF1), a member of the NK family of transcription factors required for basal forebrain morphogenesis, functions in the postnatal hypothalamus as a transcriptional regulator of genes encoding neuromodulators and hypophysiotrophic peptides. One of these peptides is gonadotropin-releasing hormone (GnRH). Here we show that Ttf1 mRNA abundance vary in a diurnal and melatonin-dependent fashion in the preoptic area (POA) of the rat, with maximal Ttf1 expression attained during the dark phase of the light/dark cycle, preceding the nocturnal peak in GnRH mRNA content. GnRH promoter activity oscillates in a circadian manner in GT1-7 cells, and this pattern is enhanced by TTF1 and blunted by siRNA-mediated Ttf1 gene silencing. TTF1 trans-activates GnRH transcription by binding to two sites in the GnRH promoter. Rat GnRH neurons in situ contain key proteins components of the positive (BMAL1, CLOCK) and negative (PER1) limbs of the circadian oscillator, and these proteins repress Ttf1 promoter activity in vitro. In contrast, Ttf1 transcription is activated by CRY1, a clock component required for circadian rhythmicity. In turn, TTF1 represses transcription of Rev-erbα, a heme receptor that controls circadian transcription within the positive limb of the circadian oscillator. These findings suggest that TTF1 is a component of the molecular machinery controlling circadian oscillations in GnRH gene transcription.
PMCID: PMC3350608  PMID: 22356123
circadian rhythm; GnRH gene expression; clock genes; TTF1; transcriptional control
25.  Dopamine agonist signalling in the hypothalamus of female rats is independent of calcium-dependent kinases 
Journal of neuroendocrinology  2009;21(11):954-960.
We have previously has demonstrated that dopamine agonist, SKF38396 (SKF), can substitute for progesterone (P) in the facilitation of female reproductive behavior in oestradiol benzoate (EB)-primed female rats and mice. We also reported that both P- and SKF-initiated signalling was mediated by cAMP-dependent PKA signal transduction cascade. As the rapid effects of P are also mediated by calcium-dependent kinases, calcium and calmodulin-dependent kinase (CaMKII) and protein kinase (PKC), we sought to determine whether SKF-initiated signalling also recruited calcium as a second messenger. We measured the changes in the activation of CaMKII and PKCin the ventromedial nucleus (VMN) of the hypothalamus and preoptic area (POA) of the rat brain, the two regions implicated in the regulation of female reproductive behavior in rodents. We measured the basal activities representing the activation of the kinases by in vivo treatments as well as the total kinase activities assayed in the presence of exogenous cofactors in vitro. We report that in contrast to P-initiated signalling, there was no recruitment of calcium by SKF in the hypothalamus as shown by the absence of changes in CaMKII activities in the VMN and POA. Furthermore, SKF-treatment resulted in a rapid increase in calcium-independent basal PKC activity in the VMN but not the POA. These rapid changes were not due to changes in PKC protein levels or phosphorylation status. These data indicate that P and SKF-recruit distinct signalling molecules within the same regions of the brain to activate region-specific signal transduction pathways.
PMCID: PMC3655436  PMID: 19732294

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