Allan E. Herbison

Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty

We examined the role of kisspeptin and its receptor, the G-protein-coupled receptor GPR54, in governing the onset of puberty in the mouse. In the adult male and female mouse, kisspeptin (10–100 nm) evoked a remarkably potent, long-lasting depolarization of >90% of gonadotropin-releasing hormone (GnRH)–green fluorescent protein neurons in situ. In contrast, in juvenile [postnatal day 8 (P8) to P19] and prepubertal (P26–P33) male mice, kisspeptin activated only 27 and 44% of GnRH neurons, respectively. This developmental recruitment of GnRH neurons into a kisspeptin-responsive pool was paralleled by an increase in the ability of centrally administered kisspeptin to evoke luteinizing hormone secretion in vivo. To learn more about the mechanisms through which kisspeptin–GPR54 signaling at the GnRH neuron may change over postnatal development, we performed quantitative in situ hybridization for kisspeptin and GPR54 transcripts. Approximately 90% of GnRH neurons were found to express GPR54 mRNA in both juvenile and adult mice, without a detectable difference in the mRNA content between the age groups. In contrast, the expression of KiSS-1 mRNA increased dramatically across the transition from juvenile to adult life in the anteroventral periventricular nucleus (AVPV; p < 0.001). These results demonstrate that kisspeptin exerts a potent depolarizing effect on the excitability of almost all adult GnRH neurons and that the responsiveness of GnRH neurons to kisspeptin increases over postnatal development. Together, these observations suggest that activation of GnRH neurons by kisspeptin at puberty reflects a dual process involving an increase in kisspeptin input from the AVPV and a post-transcriptional change in GPR54 signaling within the GnRH neuron.

Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons and fertility

The mechanisms through which estrogen regulates gonadotropin-releasing hormone (GnRH) neurons to control mammalian ovulation are unknown. We found that estrogen positive feedback to generate the preovulatory gonadotropin surge was normal in estrogen receptor beta knockout (ERbeta) mutant mice, but absent in ERalpha mutant mice. An ERalpha-selective compound was sufficient to generate positive feedback in wild-type mice. As GnRH neurons do not express ERalpha, estrogen positive feedback upon GnRH neurons must be indirect in nature. To establish the cell type responsible, we generated a neuron-specific ERalpha mutant mouse line. These mice failed to exhibit estrogen positive feedback, demonstrating that neurons expressing ERalpha are critical. We then used a GnRH neuron-specific Pseudorabies virus (PRV) tracing approach to show that the ERalpha-expressing neurons innervating GnRH neurons are located within rostral periventricular regions of the hypothalamus. These studies demonstrate that ovulation is driven by estrogen actions upon ERalpha-expressing neuronal afferents to GnRH neurons.

Kisspeptin–GPR54 Signaling Is Essential for Preovulatory Gonadotropin-Releasing Hormone Neuron Activation and the Luteinizing Hormone Surge

Kisspeptin and its receptor GPR54 have recently been identified as key signaling partners in the neural control of fertility in animal models and humans. The gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons of the neural network controlling fertility and are suspected to be the primary locus of kisspeptin–GPR54 signaling. Using mouse models, the present study addressed whether kisspeptin and GPR54 have a key role in the activation of GnRH neurons to generate the luteinizing hormone (LH) surge responsible for ovulation. Dual-label immunocytochemistry experiments showed that 40–60% of kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) expressed estrogen receptor α and progesterone receptors. Using an ovariectomized, gonadal steroid-replacement regimen, which reliably generates an LH surge, ∼30% of RP3V kisspeptin neurons were found to express c-FOS in surging mice compared with 0% in nonsurging controls. A strong correlation was found between the percentage of c-FOS-positive kisspeptin neurons and the percentage of c-FOS-positive GnRH neurons. To evaluate whether kisspeptin and/or GPR54 were essential for GnRH neuron activation and the LH surge, Gpr54- and Kiss1-null mice were examined. Whereas wild-type littermates all exhibited LH surges and c-FOS in ∼50% of their GnRH neurons, none of the mutant mice from either line showed an LH surge or any GnRH neurons with c-FOS. These observations provide the first evidence that kisspeptin–GPR54 signaling is essential for GnRH neuron activation that initiates ovulation. This broadens considerably the potential roles and therapeutic possibilities for kisspeptin and GPR54 in fertility regulation.

Relationship of neuronal nitric oxide synthase immunoreactivity to GnRH neurons in the ovariectomized and intact female rat.

The present study has used a rat neuronal nitric oxide synthase (nNOS) antibody to examine the relationship of nNOS immunoreactivity to GnRH neurons in the ovariectomized and intact diestrous and proestrous rat. A striking band of nNOS‐immunoreactive cells was identified in the rostral preoptic area which began in the median preoptic nucleus and organum vasculosum of the lamina terminalis and formed an inverted Y‐type distribution above the rostral third ventricle at the level of the anteroventral periventricular nucleus. Another band of nNOS‐immunoreactivity was found extending through the internal zone of the median eminence into the arcuate nucleus. Although nNOS immunoreactivity was not detected within GnRH neuronal cell bodies in any of the experimental groups, GnRH perikarya located in the rostral preoptic area, but not elsewhere, were found to be surrounded by nNOS‐containing cells. In the median eminence, nNOS and GnRH immunoreactivities were distributed separately in the internal and external zones, respectively.

Plasticity in Fast Synaptic Inhibition of Adult Oxytocin Neurons Caused by Switch in GABAA Receptor Subunit Expression

We found that magnocellular oxytocin neurons in adult female rats exhibit an endogenous GABA(A) receptor subunit switch around parturition: a decrease in alpha1:alpha2 subunit mRNA ratio correlated with a decrease in allopregnanolone potentiation and increase in decay time constant of the GABA(A) receptor-mediated IPSCs in these cells. The causal relationship between changes in alpha1:alpha2 mRNA ratio and the ion channel kinetics was confirmed using in vitro antisense deletion. Further, GABA(A) receptors exhibited a tonic inhibitory influence upon oxytocin release in vivo, and allopregnanolone helped to restrain oxytocin neuron in vitro firing only before parturition, when the alpha1:alpha2 subunit mRNA ratio was still high. Such observations provide evidence for the physiological significance of GABA(A) receptor subunit heterogeneity and plasticity in the adult brain.

Localization of oestrogen receptors in preoptic neurons containing neurotensin but not tyrosine hydroxylase, cholecystokinin or luteinizing hormone-releasing hormone in the male and female rat

The neurochemical identity of preoptic neurons containing oestrogen receptors was investigated in the male and female rat using a sequential double-staining immunocytochemistry procedure. Single-immunostaining revealed large populations of cells with nuclear immunoreactivity to the oestrogen receptor in the medial preoptic area of the male and female rat. Optimal double-staining of sections for the oestrogen receptor and one of several neuropeptides or tyrosine hydroxylase, was achieved with short-term (two- to four-day) gonadectomized rats treated with colchicine where necessary. Neurotensin-immunoreactive cells were distributed in a sexually dimorphic manner in the region of the anteroventral preoptic nucleus and exhibited oestrogen receptor immunoreactivity in both sexes. Double-labelled cells in this area of the female rat comprised 50% and 11% of the total neurotensin- and oestrogen receptor-containing cell populations, respectively, compared with 25% and 4% in the male (P less than 0.01). The numbers of neurotensin-immunoreactive cells in the region of the medial preoptic nucleus were similar in male and female rats with double-labelled cells making up 20-38% and 3-5% of the total numbers of cells containing neurotensin and oestrogen receptors, respectively, in both sexes. Neurons immunoreactive for tyrosine hydroxylase were distributed in a gender-specific manner within the anterior periventricular area but were not immunoreactive for the oestrogen receptor in either sex. Following colchicine treatment, cholecystokinin-immunoreactive cells were identified predominantly within periventricular regions of the preoptic area and similarly, did not possess immunoreactivity to the oestrogen receptor in either the male or the female rat. Neurons containing luteinizing hormone-releasing hormone were found immediately lateral to the cell populations containing oestrogen receptors and immunoreactivity to the oestrogen receptor was not identified within any neurons containing luteinizing hormone-releasing hormone in either the male or female rat. The absence of oestrogen receptor immunoreactivity in neurons containing tyrosine hydroxylase, cholecystokinin or luteinizing hormone-releasing hormone suggests that gonadal steroids acting through this receptor do not influence these cells directly in either sex. In particular, it appears that gender-specific patterns of luteinizing hormone secretion cannot be attributed to sex differences in oestrogen receptor localization within luteinizing hormone-releasing hormone neurons. These experiments also show that the sexually dimorphic neurotensin neurons in the preoptic area possess oestrogen receptors and that female rats have larger number of neurons co-localizing neurotensin and oestrogen receptors. As such, these neurons may be involved in mediating sex-specific actions of the gonadal steroids in the preoptic area.

Detection of Estrogen Receptor α and β Messenger Ribonucleic Acids in Adult Gonadotropin-Releasing Hormone Neurons1

The behavior of the gonadotropin-releasing hormones (GnRH) neurons controlling fertility is dependent upon cyclic fluctuations in circulating concentrations of estrogen. However, the nature of estrogen action upon these cells has remained controversial due to their dispersed distribution within the brain, and evidence indicating that they do not express nuclear estrogen receptors (ERs) in vivo. We report here an acute brain slice preparation that enables individual living GnRH neurons to be identified within the mouse brain and show, using single cell multiplex RT-PCR, that the greater than 50% of GnRH neurons in adult and prepubertal females contain ERα messenger RNA. Approximately 10% of GnRH neurons contained ERβ transcripts that were always coexistent with ERα. Single cell RT-PCR analysis of nonGnRH cells located in the medial preoptic area revealed a similar coexpression pattern of ERα and ERβ transcripts. In contrast, single striatal cells were not found to contain ERβ despite ERα being present in a...

Distribution of Estrogen Receptor-Immunoreactive Cells in the Preoptic Area of the Ewe: Co-Localization with Glutamic Acid Decarboxylase but Not Luteinizing Hormone-Releasing Hormone

Using immunocytochemical techniques we have examined the distribution of cells containing estrogen receptors (ERs) in the preoptic and anterior hypothalamic regions of short-term (1 week) ovariectomized ewes. Subsequent double-labelling experiments examined the co-localization patterns of ER and luteinizing hormone-releasing hormone (LHRH) or glutamic acid decarboxylase (GAD) immunoreactivities. ER-immunoreactive (-IR) cells were identified throughout the central and medial aspects of the preoptic area in a continuum which begins at the level of the organum vasculosum of the lamina terminalis and terminates in the caudal anterior hypothalamic area. A conspicuous sub-population of densely clustered ER-IR cells was identified within this distribution extending from the central region of the preoptic area into the bed nucleus of the stria terminalis. ER-IR cells were also identified in the ventrolateral septum and supraoptic nuclei. Double-labelling experiments showed that although rostral LHRH neurons were surrounded by ER-IR cells, they did not themselves possess ER immunoreactivity. In marked contrast, we estimate that approximately 40% of GAD-IR cells in the central aspect of the medial preoptic area are immunoreactive for the ER and that these cells account for nearly 30% of all ER-IR cells in this region. These results indicate that, in common with other species, LHRH neurons in the ewe do not possess ERs and suggest therefore, that these neurons are unlikely to be modulated directly by circulating estrogens. However, large numbers of adjacent GABA neurons possess ERs and may comprise a major neuronal population mediating gonadal steroid input to LHRH neurons.

New evidence for estrogen receptors in gonadotropin-releasing hormone neurons.

Estrogen exerts a critical regulatory influence upon the biosynthetic and secretory activity of the gonadotropin-releasing hormone (GnRH) neurons. It seems likely that estrogen regulates the behavior of the GnRH neuron through multiple transsynaptic, neuronal-glial, and direct membrane modes of action. Advances in our understanding of these mechanisms over the last 3 years are highlighted. In addition, very recent studies have begun to provide evidence for the expression of estrogen receptors (ERs) in GnRH neurons in the rodent. Although not yet firmly established, the current consensus supports the hypothesis that GnRH neurons express ERbeta. Evidence exists for ERbeta mRNA expression by GnRH neurons throughout development and ERbeta immunoreactivity has now also been detected in these cells. Murine GnRH neurons have further been shown to express estrogen receptor-related receptor-alpha, an orphan receptor thought to constitutively activate estrogen response elements. Together, these findings provide a cornerstone for the reassessment of the role of ERs and related receptors in the direct genomic and potential nontranscriptional actions of estrogen upon the GnRH neuron.

Leptin Indirectly Regulates Gonadotropin-Releasing Hormone Neuronal Function

The adipose-derived hormone leptin communicates information about metabolic status to the hypothalamic GnRH neuronal system. It is unclear whether leptin can act directly on GnRH neurons. To examine this, we used three approaches. First, the presence of leptin-induced signal transducer and activator of transcription-3 activation was examined in GnRH neurons in male and female rats. Intracerebroventricular treatment with 4 mug leptin-induced robust signal transducer and activator of transcription-3 expression within the anteroventral periventricular nucleus but not in GnRH neurons. Second, fertility was assessed in male and female CRE-loxP transgenic mice with conditional leptin receptor (Lepr) deletion from either all forebrain neurons or GnRH neurons only. Forebrain neuron LEPR deletion prevented the onset of puberty resulting in infertility in males and females and blocked estradiol-induced LH surge. However, mice with GnRH neuron-selective Lepr deletion exhibited normal fertility apart from a slight puberty delay in males. Lastly, the highly sensitive technique of single-cell nested PCR was used to test for Lepr transcript presence in individual GnRH neurons, identified in situ using GnRH-green fluorescent protein transgenics. Whereas 75% of positive control (proopiomelanocortin) neurons contained Lepr mRNA, no (none of 18) GnRH neurons were Lepr mRNA positive. Collectively, these results show that leptin does not act directly on GnRH neurons in rats and mice. Leptin appears to regulate GnRH function via forebrain neurons that are afferent to GnRH because forebrain neuronal LEPR deletion caused infertility. The location and phenotype of these leptin-responsive neurons remains to be elucidated.