Impact of chronic variable stress on neuroendocrine hypothalamus and pituitary in male and female C57BL/6J mice.

Chronic stress exerts multiple negative effects on the physiology and health of an individual. In the present study, we examined hypothalamic, pituitary and endocrine responses to 14 days of chronic variable stress (CVS) in male and female C57BL/6J mice. In both sexes, CVS induced a significant decrease in body weight and enhanced the acute corticosterone stress response, which was accompanied by a reduction in thymus weight only in females. However, single-point blood measurements of basal prolactin, thyroid-stimulating hormone, luteinising hormone, growth hormone and corticosterone levels taken at the end of the CVS were not different from those of controls. Similarly, pituitary mRNA expression of Fshb, Lhb, Prl and Gh was unchanged by CVS, although Pomc and Tsh were significantly elevated. Within the adrenal medulla, mRNA for Th, Vip and Gal were elevated following CVS. Avp transcript levels within the paraventricular nucleus of the hypothalamus were increased by CVS; however, levels of Gnrh1, Crh, Oxt, Sst, Trh, Ghrh, Th and Kiss1 remained unchanged. Oestrous cycles were lengthened slightly by CVS and ovarian histology revealed a reduction in the number of preovulatory follicles and corpora lutea. Taken together, these observations indicate that 14 days of CVS induces an up-regulation of the neuroendocrine stress axis and creates a mild disruption of female reproductive function. However, the lack of changes in other neuroendocrine axes controlling anterior and posterior pituitary secretion suggest that most neuroendocrine axes are relatively resilient to CVS.

Altered aspects of anxiety-related behavior in kisspeptin receptor-deleted male mice.

The roles of kisspeptin signaling outside the hypothalamus in the brain are unknown. We examined here the impact of Kiss1r-deletion on hippocampus-related behaviors of anxiety and spatial learning in adult male mice using two mouse models. In the first, global Kiss1r-null and control mice were gonadectomized (GDX KISS1R-KO). In the second, KISS1R signalling was rescued selectively in gonadotropin-releasing hormone neurons to generate Kiss1r-null mice with normal testosterone levels (intact KISS1R-KO). Intact KISS1R-KO rescue mice were found to spend twice as much time in the open arms of the elevated plus maze (EPM) compared to controls (P < 0.01). GDX KISS1R-KO mice showed a similar but less pronounced trend. No differences were detected between intact KISS1R-KO mice and controls in the open field test (OFT), although a marked reduction in time spent in the centre quadrant was observed for all GDX mice (P < 0.001). No effects of KISS1R deletion or gonadectomy were detected in the Morris water maze. These observations demonstrate that KISS1R signalling impacts upon anxiogenic neural circuits operative in the EPM, while gonadal steroids appear important for anxiety behaviour observed in the OFT. The potential anxiogenic role of kisspeptin may need to be considered in the development of kisspeptin analogs for the clinic.

Pulse and Surge Profiles of Luteinizing Hormone Secretion in the Mouse.

Using a new tail-tip bleeding procedure and a sensitive ELISA, we describe here the patterns of LH secretion throughout the mouse estrous cycle; in ovariectomized mice; in ovariectomized, estradiol-treated mice that model estrogen-negative and -positive feedback; and in transgenic GNR23 mice that exhibit allele-dependent reductions in GnRH neuron number. Pulsatile LH secretion was evident at all stages of the estrous cycle, with LH pulse frequency being approximately one pulse per hour in metestrous, diestrous, and proestrous mice but much less frequent at estrus (less than one pulse per 4 h). Ovariectomy resulted in substantial increases in basal and pulsatile LH secretion with pulses occurring approximately every 21 minutes. Chronic treatment with negative-feedback, estradiol-filled capsules returned LH pulse frequency to intact follicular phase levels, although pulse amplitude remained elevated. On the afternoon of proestrus, the LH surge was found to begin in a highly variable manner over a 4-hour range, lasting for more than 3 hours. In contrast, ovariectomized, estradiol-treated, positive-feedback mice exhibited a relatively uniform surge onset at approximately 0.5 hour prior to lights out. Gonadectomized wild-type and heterozygous GNR23 (∼200 GnRH neurons) male mice exhibited an LH pulse every 60 minutes. Homozygous GNR23 mice (∼80 GnRH neurons) had very low basal LH concentrations but continued to exhibit small amplitude LH pulses every 90 minutes. These studies provide the first characterization in mice of pulse and surge modes of LH secretion across the estrous cycle and demonstrate that very few GnRH neurons are required for pulsatile LH secretion.

Hypothalamic control of the male neonatal testosterone surge.

Sex differences in brain neuroanatomy and neurophysiology underpin considerable physiological and behavioural differences between females and males. Sexual differentiation of the brain is regulated by testosterone secreted by the testes predominantly during embryogenesis in humans and the neonatal period in rodents. Despite huge advances in understanding how testosterone, and its metabolite oestradiol, sexually differentiate the brain, little is known about the mechanism that actually generates the male-specific neonatal testosterone surge. This review examines the evidence for the role of the hypothalamus, and particularly the gonadotropin-releasing hormone (GnRH) neurons, in generating the neonatal testosterone surge in rodents and primates. Kisspeptin-GPR54 signalling is well established as a potent and critical regulator of GnRH neuron activity during puberty and adulthood, and we argue here for an equally important role at birth in driving the male-specific neonatal testosterone surge in rodents. The presence of a male-specific population of preoptic area kisspeptin neurons that appear transiently in the perinatal period provide one possible source of kisspeptin drive to neonatal GnRH neurons in the mouse.

Novel role for anti-Müllerian hormone in the regulation of GnRH neuron excitability and hormone secretion.

Anti-Müllerian hormone (AMH) plays crucial roles in sexual differentiation and gonadal functions. However, the possible extragonadal effects of AMH on the hypothalamic-pituitary-gonadal axis remain unexplored. Here we demonstrate that a significant subset of GnRH neurons both in mice and humans express the AMH receptor, and that AMH potently activates the GnRH neuron firing in mice. Combining in vivo and in vitro experiments, we show that AMH increases GnRH-dependent LH pulsatility and secretion, supporting a central action of AMH on GnRH neurons. Increased LH pulsatility is an important pathophysiological feature in many cases of polycystic ovary syndrome (PCOS), the most common cause of female infertility, in which circulating AMH levels are also often elevated. However, the origin of this dysregulation remains unknown. Our findings raise the intriguing hypothesis that AMH-dependent regulation of GnRH release could be involved in the pathophysiology of fertility and could hold therapeutic potential for treating PCOS.

Conditional Viral Tract Tracing Delineates the Projections of the Distinct Kisspeptin Neuron Populations to Gonadotropin-Releasing Hormone (GnRH) Neurons in the Mouse.

Kisspeptin neurons play an essential role in the regulation of fertility through direct regulation of the GnRH neurons. However, the relative contributions of the two functionally distinct kisspeptin neuron subpopulations to this critical regulation are not fully understood. Here we analyzed the specific projection patterns of kisspeptin neurons originating from either the rostral periventricular nucleus of the third ventricle (RP3V) or the arcuate nucleus (ARN) using a cell-specific, viral-mediated tract-tracing approach. We stereotaxically injected a Cre-dependent recombinant adenovirus encoding farnesylated enhanced green fluorescent protein into the ARN or RP3V of adult male and female mice expressing Cre recombinase in kisspeptin neurons. Fibers from ARN kisspeptin neurons projected widely; however, we did not find any evidence for direct contact with GnRH neuron somata or proximal dendrites in either sex. In contrast, we identified RP3V kisspeptin fibers in close contact with GnRH neuron somata and dendrites in both sexes. Fibers originating from both the RP3V and ARN were observed in close contact with distal GnRH neuron processes in the ARN and in the lateral and internal aspects of the median eminence. Furthermore, GnRH nerve terminals were found in close contact with the proximal dendrites of ARN kisspeptin neurons in the ARN, and ARN kisspeptin fibers were found contacting RP3V kisspeptin neurons in both sexes. Together these data delineate selective zones of kisspeptin neuron inputs to GnRH neurons and demonstrate complex interconnections between the distinct kisspeptin populations and GnRH neurons.

Gonadal steroid neuromodulation of developing and mature hypothalamic neuronal networks.

This review highlights recent findings regarding the diverse mechanisms through which gonadal steroid hormones modulate neuronal structure and function. In males, estradiol aromatized from testosterone in the perinatal period permanently masculinizes several components of mammalian brain circuitry. There is growing appreciation, however, that these 'organizational' actions also occur outside the perinatal period and may exist in females as well. In the mature brain, fluctuating levels of plasma estradiol modulate the activity of many neuronal circuits. It is clear that estradiol modulates hypothalamic kisspeptin neurons to drive cyclical changes in their gene expression and excitability to modulate brain control of fertility. Neuronal circuits controlling metabolism and body weight are another network modulated by fluctuating estradiol levels, with studies now beginning to delineate the estradiol-sensitive components of this circuitry.

Dependence of fertility on kisspeptin-Gpr54 signaling at the GnRH neuron.

Signaling between kisspeptin and its receptor, G-protein-coupled receptor 54 (Gpr54), is now recognized as being essential for normal fertility. However, the key cellular location of kisspeptin-Gpr54 signaling is unknown. Here we create a mouse with a GnRH neuron-specific deletion of Gpr54 to assess the role of gonadotropin-releasing hormone (GnRH) neurons. Mutant mice are infertile, fail to go through puberty and exhibit markedly reduced gonadal size and follicle-stimulating hormone levels alongside GnRH neurons that are unresponsive to kisspeptin. In an attempt to rescue the infertile phenotype of global Gpr54⁻/⁻ mutants, we use BAC transgenesis to target Gpr54 to the GnRH neurons. This results in mice with normal puberty onset, estrous cyclicity, fecundity and a recovery of kisspeptin's stimulatory action upon GnRH neurons. Using complimentary cell-specific knockout and knockin approaches we demonstrate here that the GnRH neuron is the key site of kisspeptin-Gpr54 signaling for fertility.

The role of cAMP response element-binding protein in estrogen negative feedback control of gonadotropin-releasing hormone neurons.

The mechanisms through which estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) neurons to control fertility are unclear. Previous studies have demonstrated that E2 rapidly phosphorylates cAMP response element-binding protein (CREB) in GnRH neurons in vivo. In the present study, we used GnRH neuron-specific CREB-deleted mutant mice [GnRH-CREB knock-outs (KOs)] with and without global cAMP response element modulator (CREM) deletion (global-CREM KOs) to investigate the role of CREB in estrogen negative feedback on GnRH neurons. Evaluation of GnRH-CREB KO mice with and without global CREM deletion revealed normal puberty onset. Although estrus cycle length in adults was the same in controls and knock-out mice, cycles in mutant mice consisted of significantly longer periods of diestrus and less estrus. In GnRH-CREB KO mice, basal levels of luteinizing hormone (LH) and the postovariectomy increment in LH were normal, but the ability of E2 to rapidly suppress LH was significantly blunted. In contrast, basal and postovariectomy LH levels were abnormal in GnRH-CREB KO/global-CREM KO mice. Fecundity studies showed that GnRH-CREB KO with and without global CREM deletion were normal up to ∼9 months of age, at which time they became prematurely reproductively senescent. Morphological analysis of GnRH neurons revealed a significant reduction (p < 0.01) in GnRH somatic spine density of GnRH-CREB KO mice compared to control females. These observations implicate CREB within the GnRH neuron as an important target for E2's negative feedback actions. They also indicate that the rapid modulation of CREB by E2 is of physiological significance in the CNS.

Kisspeptin neurons co-express met-enkephalin and galanin in the rostral periventricular region of the female mouse hypothalamus.

It is now well established that the kisspeptin neurons of the hypothalamus play a key role in regulating the activity of gonadotropin-releasing hormone (GnRH) neurons. The population of kisspeptin neurons residing in the rostral periventricular region of the third ventricle (RP3V), encompassing the anteroventral periventricular (AVPV) and periventricular preoptic nuclei (PVpo), are implicated in the generation of the preovulatory GnRH surge mechanism and puberty onset in female rodents. The present study examined whether these kisspeptin neurons may express other neuropeptides in the adult female mouse. Initially, the distribution of galanin, neurotensin, met-enkephalin (mENK), and cholecystokinin (CCK)-immunoreactive cells was determined within the RP3V of colchicine-treated mice. Subsequent experiments, using a new kisspeptin-10 antibody raised in sheep, examined the relationship of these neuropeptides to kisspeptin neurons. No evidence was found for expression of neurotensin or CCK by RP3V kisspeptin neurons, but subpopulations of kisspeptin neurons were observed to express galanin and mENK. Dual-labeled RP3V kisspeptin/galanin cells represented 7% of all kisspeptin and 21% of all galanin neurons whereas dual-labeled kisspeptin/mENK cells represented 28-38% of kisspeptin neurons and 58-68% of the mENK population, depending on location within the AVPV or PVpo. Kisspeptin neurons in the arcuate nucleus were also found to express galanin but not mENK. These observations indicate that, like the kisspeptin population of the arcuate nucleus, kisspeptin neurons in the RP3V also co-express a range of neuropeptides. This pattern of co-expression should greatly increase the dynamic range with which kisspeptin neurons can modulate the activity of their afferent neurons.

Gonadotropin-releasing hormone neurons extend complex highly branched dendritic trees outside the blood-brain barrier.

GnRH neurons project axons to the median eminence to control pituitary release of gonadotropins and, as such, represent the principal output neurons of the neuronal network controlling fertility. It is well established that the GnRH neurons exhibit a simple bipolar morphology with one or two long dendrites. Using adult GnRH-green fluorescent protein transgenic mice and juxtacellular cell filling, we report here that a subpopulation of GnRH neurons located in the rostral preoptic area exhibit extremely complex branching dendritic trees that fill the organum vasculosum of the lamina terminalis (OVLT). The dendritic nature of these processes was demonstrated at both light and electron microscopic levels by the presence of spines, dendritic ultrastructure, and synapses. Further, electrophysiological recordings showed that GnRH neurons were excited by glutamate as well as kisspeptin puffed onto their dendrites located within the OVLT. Using iv injection of horseradish peroxidase, a molecule unable to penetrate the blood-brain barrier (BBB), we show that GnRH neuron cell bodies and dendrites within 100 µm of the OVLT reside outside the BBB. Approximately 85% of GnRH neurons in this area express c-Fos at the time of the GnRH surge. These observations demonstrate that GnRH neurons extend complex, highly branched dendritic trees beyond the BBB into the OVLT, where they will be able to sense directly molecules circulating in the bloodstream. This indicates a new mechanism for the modulation of GnRH neurons that extends considerably the range of factors that are integrated by these neurons for the control of fertility.

Rapid actions of oestrogen on gonadotropin-releasing hormone neurons; from fantasy to physiology?

Oestradiol (E2) exerts critical homeostatic feedback effects upon gonadotropin-releasing hormone (GnRH) neurons to maintain fertility. In the female, E2 has both negative and positive feedback actions to suppress and stimulate GnRH neuron activity at different times of the ovarian cycle. This review summarizes reported rapid E2 effects on native embryonic and adult GnRH neurons and attempts to put them into a physiological perspective. Oestrogen has been shown to rapidly modulate multiple processes in embryonic and adult GnRH neurons including intracellular calcium levels, electrical activity and specific second messenger pathways, as well as GnRH secretion itself. Evaluation of in vivo data suggests that there is no essential role for rapid E2 actions in the positive feedback mechanism but that they may comprise part of the negative feedback pathway. Adult GnRH neurons are only likely to be exposed to E2 from the gonads via the circulation with appropriate physiological E2 concentrations in the rodent being 10-50 pM for negative feedback ranging up to 400 pM for positive feedback. Although most studies to date have examined the effects of supraphysiological E2 levels on GnRH neurons, there is accumulating evidence that rapid E2 actions may have a physiological role in suppressing GnRH neuron activity.

Dendritic action potential initiation in hypothalamic gonadotropin-releasing hormone neurons.

It is dogma that action potentials are initiated at the soma/axon hillock of neurons. However, dendrites often exhibit conductances necessary for spike generation and represent functionally independent processing compartments within neurons. GnRH neurons provide an interesting neuronal phenotype with simple, relatively unbranched, unipolar or bipolar dendrites of extensive lengths (>1000 microm) covered in spines. These neurons control fertility and must integrate a variety of internal homeostatic and external environmental cues. We used imaging, electrophysiological, and modeling studies to understand how they integrate and process information along dendrites. Simultaneous recordings from distal dendrites and somata of individual GnRH neurons indicate distal dendrites are the primary site of spike initiation in these cells. Compartmental modeling indicates that sites of spike initiation depend upon location of excitatory input and dendrite geometry. Together, these studies demonstrate a novel pattern of spike generation in mammalian neurons and indicate that afferent inputs within distal dendritic microdomains directly initiate action potentials.

Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons.

The neuropeptide kisspeptin has recently been implicated as having a critical role in the activation of the GnRH neurons to bring about puberty. We examined here the postnatal development of kisspeptin neuronal populations and their projections to GnRH neurons in the mouse. Three populations of kisspeptin neurons located in the 1) anteroventral periventricular nucleus (AVPV) and the preoptic periventricular nucleus (PeN), 2) dorsomedial hypothalamus, and 3) arcuate nucleus were identified using an antisera raised against mouse kisspeptin-10. A marked 10-fold (P<0.01), female-dominant sex difference in the numbers of kisspeptin neurons existed in the AVPV/PeN but not elsewhere. Kisspeptin neurons in the AVPV/PeN of both sexes displayed a similar pattern of postnatal development with no cells detected at postnatal day (P) 10, followed by increases from P25 to reach adult levels by puberty onset (P<0.01; P31 females and P45 males). This pattern was not found in the dorsomedial hypothalamus or arcuate nucleus. Dual immunofluorescence experiments demonstrated close appositions between kisspeptin fibers and GnRH neuron cell bodies that were first apparent at P25 and increased across postnatal development in both sexes. These studies demonstrate kisspeptin peptide expression in the mouse hypothalamus and reveal the postnatal development of a sexually dimorphic continuum of kisspeptin neurons within the AVPV and PeN. This periventricular population of kisspeptin neurons reaches adult-like proportions at the time of puberty onset and is the likely source of the kisspeptin inputs to GnRH neurons.