Functional GnRH receptor signaling regulates striatal cholinergic neurons in neonatal but not in adult mice.

Reproduction in mammals is controlled by hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Recent studies from our laboratory established that the basal ganglia of the human brain contain additional large populations of GnRH synthesizing neurons which are absent in adult mice. Such extrahypothalamic GnRH neurons mostly occur in the putamen where they correspond to subsets of the striatal cholinergic interneurons (ChINs) and express GnRHR autoreceptors. In an effort to establish a mouse model for functional studies of striatal GnRH/GnRHR signaling, we carried out electrophysiological experiments on acute brain slices from male transgenic mice. Using PN4-7 neonatal mice, half of striatal ChINs responded with transient hyperpolarization and decreased firing rate to 1.2 µM GnRH, whereas medium spiny projection neurons remained unaffected. GnRH acted on its specific receptor because no response was observed in the presence of the GnRHR antagonist Antide. Addition of the membrane-impermeable G protein-coupled receptor inhibitor GDP-ß-S to the internal electrode solution eliminated the effect of GnRH. Further, GnRH was able to inhibit ChINs in presence of tetrodotoxin which blocked action potential mediated events. Collectively, these data indicated that the receptor underlying the effects of GnRH in neonatal mice is localized within ChINs. GnRH responsiveness of ChINs was transient and entirely disappeared in adult mice. These results raise the possibility to use neonatal transgenic mice as a functional model to investigate the role of GnRH/GnRHR signaling discovered earlier in adult human ChINs.

Dissecting the KNDy hypothesis: KNDy neuron-derived kisspeptins are dispensable for puberty but essential for preserved female fertility and gonadotropin pulsatility.

BACKGROUND: Kiss1 neurons in the hypothalamic arcuate-nucleus (ARC) play key roles in the control of GnRH pulsatility and fertility. A fraction of ARC Kiss1 neurons, termed KNDy, co-express neurokinin B (NKB; encoded by Tac2). Yet, NKB- and Kiss1-only neurons are also found in the ARC, while a second major Kiss1-neuronal population is present in the rostral hypothalamus. The specific contribution of different Kiss1 neuron sub-sets and kisspeptins originating from them to the control of reproduction and eventually other bodily functions remains to be fully determined. METHODS: To tease apart the physiological roles of KNDy-born kisspeptins, conditional ablation of Kiss1 in Tac2-expressing cells was implemented in vivo. To this end, mice with Tac2 cell-specific Kiss1 KO (TaKKO) were generated and subjected to extensive reproductive and metabolic characterization. RESULTS: TaKKO mice displayed reduced ARC kisspeptin content and Kiss1 expression, with greater suppression in females, which was detectable at infantile-pubertal age. In contrast, Tac2/NKB levels were fully preserved. Despite the drop of ARC Kiss1/kisspeptin, pubertal timing was normal in TaKKO mice of both sexes. However, young-adult TaKKO females displayed disturbed LH pulsatility and sex steroid levels, with suppressed basal LH and pre-ovulatory LH surges, early-onset subfertility and premature ovarian insufficiency. Conversely, testicular histology and fertility were grossly conserved in TaKKO males. Ablation of Kiss1 in Tac2-cells led also to sex-dependent alterations in body composition, glucose homeostasis, especially in males, and locomotor activity, specifically in females. CONCLUSIONS: Our data document that KNDy-born kisspeptins are dispensable/compensable for puberty in both sexes, but required for maintenance of female gonadotropin pulsatility and fertility, as well as for adult metabolic homeostasis. SIGNIFICANCE STATEMENT: Neurons in the hypothalamic arcuate nucleus (ARC) co-expressing kisspeptins and NKB, named KNDy, have been recently suggested to play a key role in pulsatile secretion of gonadotropins, and hence reproduction. However, the relative contribution of this Kiss1 neuronal-subset, vs. ARC Kiss1-only and NKB-only neurons, as well as other Kiss1 neuronal populations, has not been assessed in physiological settings. We report here findings in a novel mouse-model with elimination of KNDy-born kisspeptins, without altering other kisspeptin compartments. Our data highlights the heterogeneity of ARC Kiss1 populations and document that, while dispensable/compensable for puberty, KNDy-born kisspeptins are required for proper gonadotropin pulsatility and fertility, specifically in females, and adult metabolic homeostasis. Characterization of this functional diversity is especially relevant, considering the potential of kisspeptin-based therapies for management of human reproductive disorders.

NOS1 mutations cause hypogonadotropic hypogonadism with sensory and cognitive deficits that can be reversed in infantile mice.

The nitric oxide (NO) signaling pathway in hypothalamic neurons plays a key role in the regulation of the secretion of gonadotropin-releasing hormone (GnRH), which is crucial for reproduction. We hypothesized that a disruption of neuronal NO synthase (NOS1) activity underlies some forms of hypogonadotropic hypogonadism. Whole-exome sequencing was performed on a cohort of 341 probands with congenital hypogonadotropic hypogonadism to identify ultrarare variants in NOS1. The activity of the identified NOS1 mutant proteins was assessed by their ability to promote nitrite and cGMP production in vitro. In addition, physiological and pharmacological characterization was carried out in a Nos1-deficient mouse model. We identified five heterozygous NOS1 loss-of-function mutations in six probands with congenital hypogonadotropic hypogonadism (2%), who displayed additional phenotypes including anosmia, hearing loss, and intellectual disability. NOS1 was found to be transiently expressed by GnRH neurons in the nose of both humans and mice, and Nos1 deficiency in mice resulted in dose-dependent defects in sexual maturation as well as in olfaction, hearing, and cognition. The pharmacological inhibition of NO production in postnatal mice revealed a critical time window during which Nos1 activity shaped minipuberty and sexual maturation. Inhaled NO treatment at minipuberty rescued both reproductive and behavioral phenotypes in Nos1-deficient mice. In summary, lack of NOS1 activity led to GnRH deficiency associated with sensory and intellectual comorbidities in humans and mice. NO treatment during minipuberty reversed deficits in sexual maturation, olfaction, and cognition in Nos1 mutant mice, suggesting a potential therapy for humans with NO deficiency.

Estrogen differentially regulates transcriptional landscapes of preoptic and arcuate kisspeptin neuron populations.

Kisspeptin neurons residing in the rostral periventricular area of the third ventricle (KPRP3V) and the arcuate nucleus (KPARC) mediate positive and negative estrogen feedback, respectively. Here, we aim to compare transcriptional responses of KPRP3V and KPARC neurons to estrogen. Transgenic mice were ovariectomized and supplemented with either 17ß-estradiol (E2) or vehicle. Fluorescently tagged KPRP3V neurons collected by laser-capture microdissection were subjected to RNA-seq. Bioinformatics identified 222 E2-dependent genes. Four genes encoding neuropeptide precursors (Nmb, Kiss1, Nts, Penk) were robustly, and Cartpt was subsignificantly upregulated, suggesting putative contribution of multiple neuropeptides to estrogen feedback mechanisms. Using overrepresentation analysis, the most affected KEGG pathways were neuroactive ligand-receptor interaction and dopaminergic synapse. Next, we re-analyzed our previously obtained KPARC neuron RNA-seq data from the same animals using identical bioinformatic criteria. The identified 1583 E2-induced changes included suppression of many neuropeptide precursors, granins, protein processing enzymes, and other genes related to the secretory pathway. In addition to distinct regulatory responses, KPRP3V and KPARC neurons exhibited sixty-two common changes in genes encoding three hormone receptors (Ghsr, Pgr, Npr2), GAD-65 (Gad2), calmodulin and its regulator (Calm1, Pcp4), among others. Thirty-four oppositely regulated genes (Kiss1, Vgf, Chrna7, Tmem35a) were also identified. The strikingly different transcriptional responses in the two neuron populations prompted us to explore the transcriptional mechanism further. We identified ten E2-dependent transcription factors in KPRP3V and seventy in KPARC neurons. While none of the ten transcription factors interacted with estrogen receptor-α, eight of the seventy did. We propose that an intricate, multi-layered transcriptional mechanism exists in KPARC neurons and a less complex one in KPRP3V neurons. These results shed new light on the complexity of estrogen-dependent regulatory mechanisms acting in the two functionally distinct kisspeptin neuron populations and implicate additional neuropeptides and mechanisms in estrogen feedback.

Transcriptome profiling of kisspeptin neurons from the mouse arcuate nucleus reveals new mechanisms in estrogenic control of fertility.

Kisspeptin neurons in the mediobasal hypothalamus (MBH) are critical targets of ovarian estrogen feedback regulating mammalian fertility. To reveal molecular mechanisms underlying this signaling, we thoroughly characterized the estrogen-regulated transcriptome of kisspeptin cells from ovariectomized transgenic mice substituted with 17ß-estradiol or vehicle. MBH kisspeptin neurons were harvested using laser-capture microdissection, pooled, and subjected to RNA sequencing. Estrogen treatment significantly (p.adj. < 0.05) up-regulated 1,190 and down-regulated 1,139 transcripts, including transcription factors, neuropeptides, ribosomal and mitochondrial proteins, ion channels, transporters, receptors, and regulatory RNAs. Reduced expression of the excitatory serotonin receptor-4 transcript (Htr4) diminished kisspeptin neuron responsiveness to serotonergic stimulation. Many estrogen-regulated transcripts have been implicated in puberty/fertility disorders. Patients (n = 337) with congenital hypogonadotropic hypogonadism (CHH) showed enrichment of rare variants in putative CHH-candidate genes (e.g., LRP1B, CACNA1G, FNDC3A). Comprehensive characterization of the estrogen-dependent kisspeptin neuron transcriptome sheds light on the molecular mechanisms of ovary-brain communication and informs genetic research on human fertility disorders.

Highlights of neuroanatomical discoveries of the mammalian gonadotropin-releasing hormone system.

The anatomy and morphology of gonadotropin-releasing hormone (GnRH) neurons makes them both a joy and a challenge to investigate. They are a highly unique population of neurons given their developmental migration into the brain from the olfactory placode, their relatively small number, their largely scattered distribution within the rostral forebrain, and, in some species, their highly varied individual anatomical characteristics. These unique features have posed technological hurdles to overcome and promoted fertile ground for the establishment and use of creative approaches. Historical and more contemporary discoveries defining GnRH neuron anatomy remain critical in shaping and challenging our views of GnRH neuron function in the regulation of reproductive function. We begin this review with a historical overview of anatomical discoveries and developing methodologies that have shaped our understanding of the reproductive axis. We then highlight significant discoveries across specific groups of mammalian species to address some of the important comparative aspects of GnRH neuroanatomy. Lastly, we touch on unresolved questions and opportunities for future neuroanatomical research on this fascinating and important population of neurons.

Sexually Dimorphic Neurosteroid Synthesis Regulates Neuronal Activity in the Murine Brain.

Sex steroid hormones act on hypothalamic kisspeptin neurons to regulate reproductive neural circuits in the brain. Kisspeptin neurons start to express estrogen receptors in utero, suggesting steroid hormone action on these cells early during development. Whether neurosteroids are locally produced in the embryonic brain and impinge onto kisspeptin/reproductive neural circuitry is not known. To address this question, we analyzed aromatase expression, a key enzyme in estrogen synthesis, in male and female mouse embryos. We identified an aromatase neuronal network comprising ∼6000 neurons in the hypothalamus and amygdala. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male arcuate aromatase neurons convert testosterone to estrogen to regulate kisspeptin neuron activity. We provide spatiotemporal information on aromatase neuronal network development and highlight a novel mechanism whereby aromatase neurons regulate the activity of distinct neuronal populations expressing estrogen receptors.SIGNIFICANCE STATEMENT Sex steroid hormones, such as estradiol, are important regulators of neural circuits controlling reproductive physiology in the brain. Embryonic kisspeptin neurons in the hypothalamus express steroid hormone receptors, suggesting hormone action on these cells in utero Whether neurosteroids are locally produced in the brain and impinge onto reproductive neural circuitry is insufficiently understood. To address this question, we analyzed aromatase expression, a key enzyme in estradiol synthesis, in mouse embryos and identified a network comprising ∼6000 neurons in the brain. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male aromatase neurons convert testosterone to estradiol to regulate kisspeptin neuron activity.

The human hypothalamic kisspeptin system: Functional neuroanatomy and clinical perspectives.

In mammals, kisspeptin neurons are the key components of the hypothalamic neuronal networks that regulate the onset of puberty, account for the pulsatile secretion of gonadotropin-releasing hormone (GnRH) and mediate negative and positive estrogen feedback signals to GnRH neurons. Being directly connected anatomically and functionally to the hypophysiotropic GnRH system, the major kisspeptin cell groups of the preoptic area/rostral hypothalamus and the arcuate (or infundibular) nucleus, respectively, are ideally positioned to serve as key nodes which integrate various types of environmental, endocrine, and metabolic signals that can influence fertility. This chapter provides an overview of the current state of knowledge on the anatomy, functions, and plasticity of brain kisspeptin systems based on the wide literature available from different laboratory and domestic species. Then, the species-specific features of human hypothalamic kisspeptin neurons are described, covering their topography, morphology, unique neuropeptide content, plasticity, and connectivity to hypophysiotropic GnRH neurons. Some newly emerging roles of central kisspeptin signaling in behavior and finally, clinical perspectives, are discussed.

The cryptic gonadotropin-releasing hormone neuronal system of human basal ganglia.

Human reproduction is controlled by ~2000 hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Here, we report the discovery and characterization of additional ~150,000-200,000 GnRH-synthesizing cells in the human basal ganglia and basal forebrain. Nearly all extrahypothalamic GnRH neurons expressed the cholinergic marker enzyme choline acetyltransferase. Similarly, hypothalamic GnRH neurons were also cholinergic both in embryonic and adult human brains. Whole-transcriptome analysis of cholinergic interneurons and medium spiny projection neurons laser-microdissected from the human putamen showed selective expression of GNRH1 and GNRHR1 autoreceptors in the cholinergic cell population and uncovered the detailed transcriptome profile and molecular connectome of these two cell types. Higher-order non-reproductive functions regulated by GnRH under physiological conditions in the human basal ganglia and basal forebrain require clarification. The role and changes of GnRH/GnRHR1 signaling in neurodegenerative disorders affecting cholinergic neurocircuitries, including Parkinson's and Alzheimer's diseases, need to be explored.

Characterization of Kisspeptin Neurons in the Human Rostral Hypothalamus.

BACKGROUND: Kisspeptin (KP) neurons in the rostral periventricular region of the 3rd ventricle (RP3V) of female rodents mediate positive estrogen feedback to gonadotropin-releasing hormone neurons and, thus, play a fundamental role in the mid-cycle luteinizing hormone (LH) surge. The RP3V is sexually dimorphic, and male rodents with lower KP cell numbers are unable to mount estrogen-induced LH surges. OBJECTIVE: To find and characterize the homologous KP neurons in the human brain, we studied formalin-fixed post-mortem hypothalami. METHODS: Immunohistochemical techniques were used. RESULTS: The distribution of KP neurons in the rostral hypothalamus overlapped with distinct subdivisions of the paraventricular nucleus. The cell numbers decreased after menopause, indicating that estrogens positively regulate KP gene expression in the rostral hypothalamus in humans, similarly to several other species. Young adult women and men had similar cell numbers, as opposed to rodents reported to have more KP neurons in the RP3V of females. Human KP neurons differed from the homologous rodent cells as well, in that they were devoid of enkephalins, galanin and tyrosine hydroxylase. Further, they did not contain known KP neuron markers of the human infundibular nucleus, neurokinin B, substance P and cocaine- and amphetamine-regulated transcript, while they received afferent input from these KP neurons. CONCLUSIONS: The identification and positive estrogenic regulation of KP neurons in the human rostral hypothalamus challenge the long-held view that positive estrogen feedback may be restricted to the mediobasal part of the hypothalamus in primates and point to the need of further anatomical, molecular and functional studies of rostral hypothalamic KP neurons.

New Perspectives for Anatomical and Molecular Studies of Kisspeptin Neurons in the Aging Human Brain.

The human infundibular nucleus (corresponding to the rodent arcuate nucleus) serves as an important integration center for neuronal signals and hormones released by peripheral endocrine organs. Kisspeptin (KP)-producing neurons of this anatomical site, many of which also synthesize neurokinin B (NKB), are critically involved in sex hormone signaling to gonadotropin-releasing hormone (GnRH) neurons. In recent years, the basic topography, morphology, neuropeptide content, and connectivity of human KP neurons have been investigated with in situ hybridization and immunohistochemistry on postmortem tissues. These studies revealed that human KP neurons differ neurochemically from their rodent counterparts and show robust aging-related plasticity. Earlier immunohistochemical experiments also provided evidence for temporal changes in the hypothalamus of aging men whose NKB and KP neurons undergo hypertrophy, increase in number, exhibit increased neuropeptide mRNA expression and immunoreactivity and give rise to higher numbers of immunoreactive fibers and afferent contacts onto GnRH neurons. Increasing percentages of KP-expressing NKB perikarya, NKB axons, and NKB inputs to GnRH neurons raise the intriguing possibility that a significant subset of NKB neurons begins to cosynthesize KP as aging advances. Although use of postmortem tissues is technically challenging, recently available single-cell anatomical and molecular approaches discussed in this review provide promising new tools to investigate the aging-related anatomical and functional plasticity of the human KP neuronal system.

Chronic Amyloid ß Oligomer Infusion Evokes Sustained Inflammation and Microglial Changes in the Rat Hippocampus via NLRP3.

Microglia are instrumental for recognition and elimination of amyloid ß1-42 oligomers (AßOs), but the long-term consequences of AßO-induced inflammatory changes in the brain are unclear. Here, we explored microglial responses and transciptome-level inflammatory signatures in the rat hippocampus after chronic AßO challenge. Middle-aged Long Evans rats received intracerebroventricular infusion of AßO or vehicle for 4 weeks, followed by treatment with artificial CSF or MCC950 for the subsequent 4 weeks. AßO infusion evoked a sustained inflammatory response including activation of NF-κB, triggered microglia activation and increased the expression of pattern recognition and phagocytic receptors. Aß1-42 plaques were not detectable likely due to microglial elimination of infused oligomers. In addition, we found upregulation of neuronal inhibitory ligands and their cognate microglial receptors, while downregulation of Esr1 and Scn1a, encoding estrogen receptor alpha and voltage-gated sodium-channel Na(v)1.1, respectively, was observed. These changes were associated with impaired hippocampus-dependent spatial memory and resembled early neurological changes seen in Alzheimer's disease. To investigate the role of inflammatory actions in memory deterioration, we performed MCC950 infusion, which specifically blocks the NLRP3 inflammasome. MCC950 attenuated AßO-evoked microglia reactivity, restored expression of neuronal inhibitory ligands, reversed downregulation of ERα, and abolished memory impairments. Furthermore, MCC950 abrogated AßO-invoked reduction of serum IL-10. These findings provide evidence that in response to AßO infusion microglia change their phenotype, but the resulting inflammatory changes are sustained for at least one month after the end of AßO challenge. Lasting NLRP3-driven inflammatory alterations and altered hippocampal gene expression contribute to spatial memory decline.

Post mortem single-cell labeling with DiI and immunoelectron microscopy unveil the fine structure of kisspeptin neurons in humans.

Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility; these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1 spines/100 µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.

Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus.

Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS-expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS-immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα-immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFPVglut2 , EYFPVgat , and GFPGad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.

Developing and evaluating rare disease educational materials co-created by expert clinicians and patients: the paradigm of congenital hypogonadotropic hypogonadism.

BACKGROUND: Patients with rare diseases face health disparities and are often challenged to find accurate information about their condition. We aimed to use the best available evidence and community partnerships to produce patient education materials for congenital hypogonadotropic hypogonadism (CHH) and the olfacto-genital (Kallmann) syndrome (i.e., CHH and defective sense of smell), and to evaluate end-user acceptability. Expert clinicians, researchers and patients co-created the materials in a multi-step process. Six validated algorithms were used to assess reading level of the final product. Comprehensibility and actionability were measured using the Patient Education Materials Assessment Tool via web-based data collection. Descriptive statistics were employed to summarize data and thematic analysis for analyzing open-ended responses. Subsequently, translation and cultural adaption were conducted by clinicians and patients who are native speakers. RESULTS: Co-created patient education materials reached the target 6th grade reading level according to 2/6 (33%) algorithms (range: grade 5.9-9.7). The online survey received 164 hits in 2 months and 63/159 (40%) of eligible patients completed the evaluation. Patients ranged in age from 18 to 66 years (median 36, mean 39 ± 11) and 52/63 (83%), had adequate health literacy. Patients scored understandability at 94.2% and actionability at 90.5%. The patient education materials were culturally adapted and translated into 20 languages (available in Additional file 1). CONCLUSIONS: Partnering with patients enabled us to create patient education materials that met patient- identified needs as evidenced by high end-user acceptability, understandability and actionability. The web-based evaluation was effective for reaching dispersed rare disease patients. Combining dissemination via traditional healthcare professional platforms as well as patient-centric sites can facilitate broad uptake of culturally adapted translations. This process may serve as a roadmap for creating patient education materials for other rare diseases.

Ovariectomy Alters Gene Expression of the Hippocampal Formation in Middle-Aged Rats.

Ovarian hormones regulate the transcriptome of the hippocampus and modulate its functions. During menopause this complex signaling declines, leading to impaired learning and memory. This study was undertaken to clarify the effects of long-term, surgical ovariectomy (OVX) on the rat hippocampal transcriptome. At age of 13 months, intact control and ovariectomized groups were formed. All animals were killed 5 weeks after gonadectomy; hippocampal formations were dissected and processed for transcriptome analysis. Microarray and polymerase chain reaction studies identified 252 and 61 genes, respectively, whose expression was altered in the lack of ovarian hormones. Pathway analysis revealed impact on neuroactive ligand-receptor interaction, endocannabinoid, and estrogen signaling, among others. Network and interaction analyses of proteins encoded by OVX-regulated genes revealed upregulation of growth/troph/transcription factor signaling assembly (Mdk, Fgf1, Igf2, Ngf, Ngfr, Ntf3, Ntrk1, Otx2, Hif1a, Esr1, Nr4a3), peptides/peptide receptors (Cartpt, Kl, Ttr, Gnrhr), neurotransmission (Grm1, Gria4, Gls, Slc18a2, Kcnj6), and genes serving immune functions (C3, Ccl2, Itgam, Il1b). Downregulated clusters included neuropeptides and their receptors (Adcyap1, Cbln2, Cck, Cckbr, Crhr1 and 2, Oprd1, Nts, Penk, Sstr1, Vip), neurotransmitter signaling (Htr2c, Chrna3, Chrm4, Grm8, Hrh3, Slc17a6), and potassium channels (Kcnk9, Kcnj9, Kcnma1, Kcnc2). Several transcription factors (Rxra, Thrb), solute carriers and defense molecules (Apitd1, Bcl2, C1ql3, Ilr3a, Sod1, Sncb) also underwent downregulation. The findings indicate that surgical gonadectomy carried out at middle-age robustly changes the hippocampal transcriptome that alters neurogenesis, synaptic plasticity, immune modulation, causing cognitive dysfunctions.

Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains.

Fertility in mammals is controlled by hypothalamic neurons that secrete gonadotropin-releasing hormone (GnRH). These neurons differentiate in the olfactory placodes during embryogenesis and migrate from the nose to the hypothalamus before birth. Information regarding this process in humans is sparse. Here, we adapted new tissue-clearing and whole-mount immunohistochemical techniques to entire human embryos/fetuses to meticulously study this system during the first trimester of gestation in the largest series of human fetuses examined to date. Combining these cutting-edge techniques with conventional immunohistochemistry, we provide the first chronological and quantitative analysis of GnRH neuron origins, differentiation and migration, as well as a 3D atlas of their distribution in the fetal brain. We reveal not only that the number of GnRH-immunoreactive neurons in humans is significantly higher than previously thought, but that GnRH cells migrate into several extrahypothalamic brain regions in addition to the hypothalamus. Their presence in these areas raises the possibility that GnRH has non-reproductive roles, creating new avenues for research on GnRH functions in cognitive, behavioral and physiological processes.

Long-Term Estrogen Receptor Beta Agonist Treatment Modifies the Hippocampal Transcriptome in Middle-Aged Ovariectomized Rats.

Estradiol (E2) robustly activates transcription of a broad array of genes in the hippocampal formation of middle-aged ovariectomized rats via estrogen receptors (ERα, ERß, and G protein-coupled ER). Selective ERß agonists also influence hippocampal functions, although their downstream molecular targets and mechanisms are not known. In this study, we explored the effects of long-term treatment with ERß agonist diarylpropionitrile (DPN, 0.05 mg/kg/day, sc.) on the hippocampal transcriptome in ovariectomized, middle-aged (13 month) rats. Isolated hippocampal formations were analyzed by Affymetrix oligonucleotide microarray and quantitative real-time PCR. Four hundred ninety-seven genes fulfilled the absolute fold change higher than 2 (FC > 2) selection criterion. Among them 370 genes were activated. Pathway analysis identified terms including glutamatergic and cholinergic synapse, RNA transport, endocytosis, thyroid hormone signaling, RNA degradation, retrograde endocannabinoid signaling, and mRNA surveillance. PCR studies showed transcriptional regulation of 58 genes encoding growth factors (Igf2, Igfb2, Igf1r, Fgf1, Mdk, Ntf3, Bdnf), transcription factors (Otx2, Msx1), potassium channels (Kcne2), neuropeptides (Cck, Pdyn), peptide receptors (Crhr2, Oprm1, Gnrhr, Galr2, Sstr1, Sstr3), neurotransmitter receptors (Htr1a, Htr2c, Htr2a, Gria2, Gria3, Grm5, Gabra1, Chrm5, Adrb1), and vesicular neurotransmitter transporters (Slc32a1, Slc17a7). Protein-protein interaction analysis revealed networking of clusters associated with the regulation of growth/troph factor signaling, transcription, translation, neurotransmitter and neurohormone signaling mechanisms and potassium channels. Collectively, the results reveal the contribution of ERß-mediated processes to the regulation of transcription, translation, neurogenesis, neuromodulation, and neuroprotection in the hippocampal formation of ovariectomized, middle-aged rats and elucidate regulatory channels responsible for DPN-altered functional patterns. These findings support the notion that selective activation of ERß may be a viable approach for treating the neural symptoms of E2 deficiency in menopause.

Altered Gene Expression Profiles of the Hypothalamic Arcuate Nucleus of Male Mice Suggest Profound Developmental Changes in Peptidergic Signaling.

Neuropeptides of the hypothalamic arcuate nucleus (ARC) regulate important homeostatic and endocrine functions and also play critical roles in pubertal development. The altered peptidergic and aminoacidergic neurotransmission accompanying pubertal maturation of the ARC is not fully understood. Here we studied the developmental shift in the gene expression profile of the ARC of male mice. RNA samples for quantitative RT-PCR studies were isolated from the ARC of 14-day-old infantile and 60-day-old adult male mice with laser capture microdissection. The expression of 18 neuropeptide, 15 neuropeptide receptor, 4 sex steroid receptor and 6 classic neurotransmitter marker mRNAs was compared between the two time points. The adult animals showed increased mRNA levels encoding cocaine- and amphetamine-regulated transcripts, galanin-like peptide, dynorphin, kisspeptin, proopiomelanocortin, proenkephalin and galanin and a reduced expression of mRNAs for pituitary adenylate cyclase-activating peptide, calcitonin gene-related peptide, neuropeptide Y, substance P, agouti-related protein, neurotensin and growth hormone-releasing hormone. From the neuropeptide receptors tested, melanocortin receptor-4 showed the most striking increase (5-fold). Melanocortin receptor-3 and the Y1 and Y5 neuropeptide Y receptors increased 1.5- to 1.8-fold, whereas δ-opioid receptor and neurotensin receptor-1 transcripts were reduced by 27 and 21%, respectively. Androgen receptor, progesterone receptor and α-estrogen receptor transcripts increased by 54-72%. The mRNAs of glutamic acid decarboxylases-65 and -67, vesicular GABA transporter and choline acetyltransferase remained unchanged. Tyrosine hydroxylase mRNA increased by 44%, whereas type-2 vesicular glutamate transporter mRNA decreased by 43% by adulthood. Many of the developmental changes we revealed in this study suggest a reduced inhibitory and/or enhanced excitatory neuropeptidergic drive on fertility in adult animals.