RESUMO
Understanding the hypothalamic factors regulating reproduction facilitates maximising the reproductive success of breeding programmes and in the management and conservation of threatened species, including African lions. To provide insight into the physiology and pathophysiology of the hypothalamic-pituitary-gonadal reproductive axis in lions, we studied the luteinising hormone (LH) and steroid hormone responses to gonadotropin-releasing hormone (GnRH) and its upstream regulator, kisspeptin. Six young (13.3 ± 1.7 months, 56.2 ± 4.3 kg) and four adult (40.2 ± 1.4 months, 174 ± 6 kg) male lions (Ukutula Conservation Centre, South Africa) were used in this study. Lions were immobilised with a combination of medetomidine and ketamine and an intravenous catheter was placed in a jugular, cephalic or medial saphenous vein for blood sampling at 10-min intervals for 220 min. The ten-amino acid kisspeptin which has full intrinsic activity (KP-10, 1 µg/kg) and GnRH (1 µg/kg) were administered intravenously to study their effects on LH and steroid hormone plasma concentrations, measured subsequently by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. Basal LH levels were similarly low between the age groups, but testosterone and its precursor levels were higher in the adult animals. Adult lions showed a significant LH response to KP-10 (10-fold) and GnRH (11-fold) administration (p < 0.05 and P < 0.001, respectively) whereas in young lions LH increased significantly only in response to GnRH. In adults alone, testosterone and its precursors steadily increased in response to KP-10, with no significant further increase in response to GnRH. Plasma levels of glucocorticoids in response to KP-10 remained unchanged. We suggest that provocative testing of LH and steroid stimulation with kisspeptin provides a new and sensitive tool for determining reproductive status and possibly an index of exposure to stress, environmental insults such as disease, endocrine disruptors and nutritional status. 272 words.
Assuntos
Kisspeptinas , Leões , Animais , Masculino , Hormônio Liberador de Gonadotropina , Cromatografia Líquida , Status Social , Espectrometria de Massas em Tandem , Hormônio Luteinizante , Reprodução , Testosterona , Meio AmbienteRESUMO
Fertility critically depends on the gonadotropin-releasing hormone (GnRH) pulse generator, a neural construct comprised of hypothalamic neurons coexpressing kisspeptin, neurokoinin-B and dynorphin. Here, using mathematical modeling and in vivo optogenetics we reveal for the first time how this neural construct initiates and sustains the appropriate ultradian frequency essential for reproduction. Prompted by mathematical modeling, we show experimentally using female estrous mice that robust pulsatile release of luteinizing hormone, a proxy for GnRH, emerges abruptly as we increase the basal activity of the neuronal network using continuous low-frequency optogenetic stimulation. Further increase in basal activity markedly increases pulse frequency and eventually leads to pulse termination. Additional model predictions that pulsatile dynamics emerge from nonlinear positive and negative feedback interactions mediated through neurokinin-B and dynorphin signaling respectively are confirmed neuropharmacologically. Our results shed light on the long-elusive GnRH pulse generator offering new horizons for reproductive health and wellbeing.SIGNIFICANCE STATEMENT The gonadotropin-releasing hormone (GnRH) pulse generator controls the pulsatile secretion of the gonadotropic hormones LH and FSH and is critical for fertility. The hypothalamic arcuate kisspeptin neurons are thought to represent the GnRH pulse generator, since their oscillatory activity is coincident with LH pulses in the blood; a proxy for GnRH pulses. However, the mechanisms underlying GnRH pulse generation remain elusive. We developed a mathematical model of the kisspeptin neuronal network and confirmed its predictions experimentally, showing how LH secretion is frequency-modulated as we increase the basal activity of the arcuate kisspeptin neurons in vivo using continuous optogenetic stimulation. Our model provides a quantitative framework for understanding the reproductive neuroendocrine system and opens new horizons for fertility regulation.
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Hormônio Liberador de Gonadotropina/fisiologia , Animais , Dinorfinas/fisiologia , Ciclo Estral/fisiologia , Retroalimentação Fisiológica , Feminino , Kisspeptinas/fisiologia , Hormônio Luteinizante/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Teóricos , Neurocinina B/fisiologia , Neurônios/fisiologia , Optogenética , Gravidez , Reprodução/fisiologia , Ritmo Ultradiano/fisiologiaRESUMO
RFamide-related peptide (RFRP)-3 reduces luteinising hormone (LH) secretion in rodents. Stress has been shown to upregulate the expression of the RFRP gene (Rfrp) with a concomitant reduction in LH secretion, but an effect on expression of the gonadotrophin-releasing hormone (GnRH) gene (Gnrh1) has not been shown. We hypothesised that lipopolysaccharide (LPS)-induced stress affects expression of Rfrp, the gene for kisspeptin (Kiss1) and/or Gnrh1, leading to suppression of LH levels in rats. Intracerebroventricular injections of RFRP-3 (0.1, 1, 5 nmol) or i.v. LPS (15µgkg-1) reduced LH levels. Doses of 1 and 5 nmol RFRP-3 were then administered to analyse gene expression by in situ hybridisation. RFRP-3 (5 nmol) had no effect on Gnrh1 or Kiss1 expression. LPS stress reduced GnRH and Kiss1 expression, without affecting Rfrp1 expression. These data indicate that LPS stress directly or indirectly reduces Gnrh1 expression, but this is unlikely to be due to a change in Rfrp1 expression.
Assuntos
Expressão Gênica/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/efeitos dos fármacos , Kisspeptinas/metabolismo , Lipopolissacarídeos/farmacologia , Neuropeptídeos/farmacologia , Animais , Hormônio Liberador de Gonadotropina/genética , Humanos , Hipotálamo/metabolismo , Kisspeptinas/genética , Hormônio Luteinizante/sangue , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
The exact neural construct underlying the dynamic secretion of gonadotrophin-releasing hormone (GnRH) has only recently been identified despite the detection of multiunit electrical activity volleys associated with pulsatile luteinising hormone (LH) secretion four decades ago. Since the discovery of kisspeptin/neurokinin B/dynorphin neurons in the mammalian hypothalamus, there has been much research into the role of this neuronal network in controlling the oscillatory secretion of gonadotrophin hormones. In this review, we provide an update of the progressive application of cutting-edge techniques combined with mathematical modelling by the neuroendocrine community, which are transforming the functional investigation of the GnRH pulse generator. Understanding the nature and function of the GnRH pulse generator can greatly inform a wide range of clinical studies investigating infertility treatments.
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Hormônio Liberador de Gonadotropina , Hormônio Luteinizante , Animais , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Neurocinina B/metabolismo , Dinorfinas/metabolismo , Kisspeptinas/metabolismo , Núcleo Arqueado do Hipotálamo/metabolismo , Mamíferos/metabolismoRESUMO
Psychosocial stress negatively impacts reproductive function by inhibiting pulsatile luteinizing hormone (LH) secretion. The posterodorsal medial amygdala (MePD) is responsible in part for processing stress and modulating the reproductive axis. Activation of the neurokinin 3 receptor (NK3R) suppresses the gonadotropin-releasing hormone (GnRH) pulse generator, under hypoestrogenic conditions, and NK3R activity in the amygdala has been documented to play a role in stress and anxiety. We investigate whether NK3R activation in the MePD is involved in mediating the inhibitory effect of psychosocial stress on LH pulsatility in ovariectomised female mice. First, we administered senktide, an NK3R agonist, into the MePD and monitored the effect on pulsatile LH secretion. We then delivered SB222200, a selective NK3R antagonist, intra-MePD in the presence of predator odour, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Senktide administration into the MePD dose-dependently suppresses pulsatile LH secretion. Moreover, NK3R signalling in the MePD mediates TMT-induced suppression of the GnRH pulse generator, which we verified using a mathematical model. The model verifies our experimental findings: (i) predator odour exposure inhibits LH pulses, (ii) activation of NK3R in the MePD inhibits LH pulses and (iii) NK3R antagonism in the MePD blocks stressor-induced inhibition of LH pulse frequency in the absence of ovarian steroids. These results demonstrate for the first time that NK3R neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator.
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Hormônio Luteinizante , Quinolinas , Receptores da Neurocinina-3 , Transdução de Sinais , Estresse Psicológico , Substância P/análogos & derivados , Animais , Feminino , Receptores da Neurocinina-3/metabolismo , Receptores da Neurocinina-3/antagonistas & inibidores , Receptores da Neurocinina-3/agonistas , Hormônio Luteinizante/metabolismo , Estresse Psicológico/metabolismo , Camundongos , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos dos fármacos , Complexo Nuclear Corticomedial/metabolismo , Complexo Nuclear Corticomedial/efeitos dos fármacos , Complexo Nuclear Corticomedial/fisiologia , Fragmentos de Peptídeos/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Camundongos Endogâmicos C57BL , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/efeitos dos fármacosRESUMO
Hypothalamic kisspeptin (Kiss1) neurons are vital for pubertal development and reproduction. Arcuate nucleus Kiss1 (Kiss1ARH) neurons are responsible for the pulsatile release of Gonadotropin-releasing Hormone (GnRH). In females, the behavior of Kiss1ARH neurons, expressing Kiss1, Neurokinin B (NKB), and Dynorphin (Dyn), varies throughout the ovarian cycle. Studies indicate that 17ß-estradiol (E2) reduces peptide expression but increases Vglut2 mRNA and glutamate neurotransmission in these neurons, suggesting a shift from peptidergic to glutamatergic signaling. To investigate this shift, we combined transcriptomics, electrophysiology, and mathematical modeling. Our results demonstrate that E2 treatment upregulates the mRNA expression of voltage-activated calcium channels, elevating the whole-cell calcium current and that contribute to high-frequency burst firing. Additionally, E2 treatment decreased the mRNA levels of Canonical Transient Receptor Potential (TPRC) 5 and G protein-coupled K+ (GIRK) channels. When TRPC5 channels in Kiss1ARH neurons were deleted using CRISPR, the slow excitatory postsynaptic potential (sEPSP) was eliminated. Our data enabled us to formulate a biophysically realistic mathematical model of the Kiss1ARH neuron, suggesting that E2 modifies ionic conductances in Kiss1ARH neurons, enabling the transition from high frequency synchronous firing through NKB-driven activation of TRPC5 channels to a short bursting mode facilitating glutamate release. In a low E2 milieu, synchronous firing of Kiss1ARH neurons drives pulsatile release of GnRH, while the transition to burst firing with high, preovulatory levels of E2 would facilitate the GnRH surge through its glutamatergic synaptic connection to preoptic Kiss1 neurons.
RESUMO
Stressful stimuli abound in modern society and have shaped evolution through altering reproductive development, behavior, and physiology. The recent identification of kisspeptin as an important component of the hypothalamic regulatory circuits involved in reproductive homeostasis sparked a great deal of research interest that subsequently implicated kisspeptin signaling in the relay of metabolic, environmental, and physiological cues to the hypothalamo-pituitary-gonadal axis. However, although it is widely recognized that exposure to stress profoundly impacts on reproductive function, the roles of kisspeptin within the complex mechanisms underlying stress regulation of reproduction remain poorly understood. We and others have recently demonstrated that a variety of experimental stress paradigms downregulate the expression of kisspeptin ligand and receptor within the reproductive brain. Coincidently, these stressors also inhibit gonadotropin secretion and delay pubertal onset-processes that rely on kisspeptin signaling. However, a modest literature is inconsistent with an exclusively suppressive influence of stress on the reproductive axis and suggests that complicated neural interactions and signaling mechanisms translate the stress response into reproductive perturbations. The purpose of this chapter is to review the evidence for a novel role of kisspeptin signaling in the modulation of reproductive function by stress and to broaden the understanding of this timely phenomenon.
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Gônadas/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Kisspeptinas/metabolismo , Reprodução/fisiologia , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Animais , Feminino , Humanos , Masculino , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1RESUMO
Optical systems and genetic engineering technologies have made it possible to control neurons and unravel neuronal circuit behavior with high temporal and spatial resolution. The application of optogenetic strategies to understand the physiology of kisspeptin neuronal circuits has evolved in recent years among the neuroendocrine community. Kisspeptin neurons are fundamentally involved in controlling mammalian reproduction but also are implicated in numerous other physiological processes, including but not limited to feeding, energy expenditure, core body temperature and behavior. We conducted a review aiming to shed light on the novel findings obtained from in vitro and in vivo optogenetic studies interrogating kisspeptin neuronal circuits to date. Understanding the function of kisspeptin networks in the brain can greatly inform a wide range of clinical studies investigating infertility treatments, gender identity, metabolic disorders, hot flushes and psychosexual disorders.
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Kisspeptinas , Optogenética , Humanos , Animais , Feminino , Masculino , Kisspeptinas/metabolismo , Identidade de Gênero , Neurônios/metabolismo , Encéfalo/metabolismo , MamíferosRESUMO
Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) are central to the stress response. Chemogenetic activation of PVN CRH neurons decreases LH pulse frequency but the mechanism is unknown. In the present study, optogenetic stimulation of PVN CRH neurons suppressed LH pulse frequency in estradiol-replaced ovariectomized CRH-cre mice, and this effect was augmented or attenuated by intra-PVN GABAA or GABAB receptor antagonism, respectively. PVN CRH neurons signal to local GABA neurons, which may provide a possible indirect mechanism by which PVN CRH neurons suppress LH pulse frequency. Optogenetic stimulation of potential PVN GABAergic projection terminals in the hypothalamic arcuate nucleus in ovariectomized estradiol-replaced Vgat-cre-tdTomato mice via an optic fiber implanted in the arcuate nucleus suppressed LH pulse frequency. To further determine whether PVN CRH neurons signal through PVN GABA neurons to suppress LH pulsatility, we combined recombinase mice with intersectional vectors to selectively target these neurons. CRH-cre::Vgat-FlpO mice expressing the stimulatory opsin ChRmine in non-GABAergic CRH neurons alone or in combination with the inhibitory opsin NpHR3.3 in non-CRH-expressing GABA neurons in the PVN were used. Optogenetic stimulation of non-GABAergic CRH neurons suppressed pulsatile LH secretion; however, LH pulse frequency was not affected when CRH neurons were stimulated and PVN GABA neurons were simultaneously inhibited. Together, these studies demonstrate that suppression of LH pulse frequency in response to PVN CRH neuronal activation is mediated by GABAergic signalling intrinsic to the PVN and may incorporate PVN GABAergic projection to the hypothalamic GnRH pulse generator.
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Hormônio Liberador da Corticotropina , Hormônio Liberador de Gonadotropina , Camundongos , Feminino , Animais , Hormônio Liberador da Corticotropina/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Hormônios Liberadores de Hormônios Hipofisários/farmacologia , Hipotálamo/metabolismo , Neurônios GABAérgicos/metabolismo , Estradiol/farmacologiaRESUMO
Introduction: The impact of stress on reproductive function is significant. Hypothalamic paraventricular nucleus (PVN) corticotrophin-releasing hormone (CRH) plays a major role in regulating the stress response. Understanding how the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis interact is crucial for comprehending how stress can lead to reproductive dysfunction. However, whether stress influences reproductive function via modulating PVN CRH or HPA sequelae is not fully elucidated. Methods: In this study, we investigated the impact of chemogenetic activation of PVN CRH neurons on reproductive function. We chronically and selectively stimulated PVN CRH neurons in female CRH-Cre mice using excitatory designer receptor exclusively activated by designer drugs (DREADDs) viral constructs, which were bilaterally injected into the PVN. The agonist compound-21 (C21) was delivered through the drinking water. We determined the effects of DREADDs activation of PVN CRH neurons on the estrous cycles, LH pulse frequency in diestrus and metestrus and LH surge in proestrus mice. The effect of long-term C21 administration on basal corticosterone secretion and the response to acute restraint stress during metestrus was also examined. Additionally, computer simulations of a mathematical model were used to determine the effects of DREADDs activation of PVN CRH neurons, simulating chronic stress, on the physiological parameters examined experimentally. Results: As a result, and consistent with our mathematical model predictions, the length of the estrous cycle was extended, with an increase in the time spent in estrus and metestrus, and a decrease in proestrus and diestrus. Additionally, the frequency of LH pulses during metestrus was decreased, but unaffected during diestrus. The occurrence of the preovulatory LH surge during proestrus was disrupted. The basal level of corticosterone during metestrus was not affected, but the response to acute restraint stress was diminished after long-term C21 application. Discussion: These data suggest that PVN CRH neurons play a functional role in disrupting ovarian cyclicity and the preovulatory LH surge, and that the activity of the GnRH pulse generator remains relatively robust during diestrus but not during metestrus under chronic stress exposure in accordance with our mathematical model predictions.
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Hormônio Liberador da Corticotropina , Imidazóis , Núcleo Hipotalâmico Paraventricular , Sulfonamidas , Tiofenos , Feminino , Animais , Camundongos , Corticosterona , Ciclo EstralRESUMO
The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes. Inhibition of MePD urocortin-3 (Ucn3) neurons prevents psychological stress-induced suppression of luteinizing hormone (LH) pulsatility while blocking the stress-induced elevations in corticosterone (CORT) secretion in female mice. We explore the neurotransmission and neural circuitry suppressing the gonadotropin-releasing hormone (GnRH) pulse generator by MePD Ucn3 neurons and we further investigate whether MePD Ucn3 efferent projections to the hypothalamic paraventricular nucleus (PVN) control CORT secretion and LH pulsatility. Ucn3-cre-tdTomato female ovariectomized (OVX) mice were unilaterally injected with adeno-associated virus (AAV)-channelrhodopsin 2 (ChR2) and implanted with optofluid cannulae targeting the MePD. We optically activated Ucn3 neurons in the MePD with blue light at 10 Hz and monitored the effect on LH pulses. Next, we combined optogenetic stimulation of MePD Ucn3 neurons with pharmacological antagonism of GABAA or GABAB receptors with bicuculline or CGP-35348, respectively, as well as a combination of NMDA and AMPA receptor antagonists, AP5 and CNQX, respectively, and observed the effect on pulsatile LH secretion. A separate group of Ucn3-cre-tdTomato OVX mice with 17ß-estradiol replacement were unilaterally injected with AAV-ChR2 in the MePD and implanted with fiber-optic cannulae targeting the PVN. We optically stimulated the MePD Ucn3 efferent projections in the PVN with blue light at 20 Hz and monitored the effect on CORT secretion and LH pulses. We reveal for the first time that activation of Ucn3 neurons in the MePD inhibits GnRH pulse generator frequency via GABA and glutamate signaling within the MePD, while MePD Ucn3 projections to the PVN modulate the HPG and HPA axes.
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Complexo Nuclear Corticomedial , Hormônio Luteinizante , Urocortinas , Animais , Feminino , Camundongos , Complexo Nuclear Corticomedial/metabolismo , Ácido gama-Aminobutírico/metabolismo , Ácido Glutâmico/metabolismo , Hormônio Liberador de Gonadotropina/metabolismoRESUMO
Psychological stress is linked to infertility by suppressing the hypothalamic GnRH pulse generator. The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream regulator of GnRH pulse generator activity and displays increased neuronal activation during psychological stress. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centers; however, their functional significance has not been determined. We hypothesize that MePD GABAergic signalling mediates psychological stress-induced suppression of pulsatile LH secretion. We selectively inhibited MePD GABA neurons during psychological stress in ovariectomized (OVX) Vgat-cre-tdTomato mice to determine the effect on stress-induced suppression of pulsatile LH secretion. MePD GABA neurons were virally infected with inhibitory hM4DGi-designer receptor exclusively activated by designer drugs (DREADDs) to selectively inhibit MePD GABA neurons. Furthermore, we optogenetically stimulated potential MePD GABAergic projection terminals in the hypothalamic arcuate nucleus (ARC) and determined the effect on pulsatile LH secretion. MePD GABA neurons in OVX female Vgat-cre-tdTomato mice were virally infected to express channelrhodopsin-2 and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via an optic fiber implanted in the ARC. DREADD-mediated inhibition of MePD GABA neurons blocked predator odor and restraint stress-induced suppression of LH pulse frequency. Furthermore, sustained optogenetic stimulation at 10 and 20â Hz of MePD GABAergic terminals in the ARC suppressed pulsatile LH secretion. These results show for the first time that GABAergic signalling in the MePD mediates psychological stress-induced suppression of pulsatile LH secretion and suggest a functionally significant MePD GABAergic projection to the hypothalamic GnRH pulse generator.
Assuntos
Complexo Nuclear Corticomedial , Neurônios GABAérgicos , Hormônio Luteinizante , Animais , Feminino , Camundongos , Complexo Nuclear Corticomedial/metabolismo , Neurônios GABAérgicos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Luteinizante/metabolismo , Estresse Psicológico/metabolismoRESUMO
Post-traumatic stress disorder impedes pubertal development and disrupts pulsatile LH secretion in humans and rodents. The posterodorsal sub-nucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, pubertal timing, as well as emotional processing and anxiety. Psychosocial stress exposure alters neuronal activity within the MePD increasing the expression of Urocortin3 (Ucn3) and its receptor corticotropin-releasing factor type-2 receptor (CRFR2) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. We test the hypothesis that psychosocial stress, processed by the MePD, is relayed to the hypothalamic GnRH pulse generator to delay puberty in female mice. We exposed C57Bl6/J female mice to the predator odor, 2,4,5-Trimethylthiazole (TMT), during pubertal transition and examined the effect on pubertal timing, pre-pubertal LH pulses and anxiety-like behaviour. Subsequently, we virally infected Ucn3-cre-tdTomato female mice with stimulatory DREADDs targeting MePD Ucn3 neurons and determined the effect on pubertal timing and pre-pubertal LH pulse frequency. Exposure to TMT during pubertal development delayed puberty, suppressed pre-pubertal LH pulsatility and enhanced anxiety-like behaviour, while activation of MePD Ucn3 neurons reduced LH pulse frequency and delayed puberty. Early psychosocial stress exposure decreases GnRH pulse generator frequency delaying puberty while inducing anxiety-behaviour in female mice, an effect potentially involving Ucn3 neurons in the MePD.
Assuntos
Hormônio Luteinizante , Urocortinas , Tonsila do Cerebelo/metabolismo , Animais , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Luteinizante/metabolismo , Camundongos , Maturidade Sexual , Urocortinas/metabolismo , Urocortinas/farmacologiaRESUMO
Kisspeptin neurons are mainly located in the arcuate (Kiss1ARC, vis-à-vis the GnRH pulse generator) and anteroventral periventricular nucleus (Kiss1AVPV, vis-à-vis the GnRH surge generator). Kiss1ARC send fibre projections that connect with Kiss1AVPV somata. However, studies focused on the role of Kiss1ARC neurons in the LH surge are limited, and the role of Kiss1ARC projections to AVPV (Kiss1ARCâAVPV) in the preovulatory LH surge is still unknown. To investigate its function, this study used optogenetics to selectively stimulate Kiss1ARCâAVPV and measured changes in circulating LH levels. Kiss1ARC in Kiss-Cre-tdTomato mice were virally infected to express channelrhodopsin-2 proteins, and optical stimulation was applied selectively via a fibre optic cannula in the AVPV. Sustained 20 Hz optical stimulation of Kiss1ARCâAVPV from 15:30 to 16:30 h on proestrus effectively induced an immediate increase in LH reaching peak surge-like levels of around 8 ng/ml within 10 min, followed by a gradual decline to baseline over about 40 min. Stimulation at 10 Hz resulted in a non-significant increase in LH levels and 5 Hz stimulation had no effect in proestrous animals. The 20 Hz stimulation induced significantly higher circulating LH levels on proestrus compared with diestrus or estrus, which suggested that the effect of terminal stimulation is modulated by the sex steroid milieu. Additionally, intra-AVPV infusion of glutamate antagonists, AP5+CNQX, completely blocked the increase on LH levels induced by Kiss1ARCâAVPV terminal photostimulation in proestrous animals. These results demonstrate for the first time that optical stimulation of Kiss1ARCâAVPV induces an LH surge-like secretion via glutamatergic mechanisms. In conclusion, Kiss1ARC may participate in LH surge generation by glutamate release from terminal projections in the AVPV.
Assuntos
Kisspeptinas , Optogenética , Feminino , Animais , Camundongos , Kisspeptinas/metabolismo , Ácido Glutâmico , Hormônio Luteinizante/metabolismo , Estradiol/farmacologia , Hormônio Liberador de Gonadotropina/metabolismoRESUMO
Mathematical modelling is an indispensable tool in modern biosciences, enabling quantitative analysis and integration of biological data, transparent formulation of our understanding of complex biological systems, and efficient experimental design based on model predictions. This review article provides an overview of the impact that mathematical models had on GnRH research. Indeed, over the last 20 years mathematical modelling has been used to describe and explore the physiology of the GnRH neuron, the mechanisms underlying GnRH pulsatile secretion, and GnRH signalling to the pituitary. Importantly, these models have contributed to GnRH research via novel hypotheses and predictions regarding the bursting behaviour of the GnRH neuron, the role of kisspeptin neurons in the emergence of pulsatile GnRH dynamics, and the decoding of GnRH signals by biochemical signalling networks. We envisage that with the advent of novel experimental technologies, mathematical modelling will have an even greater role to play in our endeavour to understand the complex spatiotemporal dynamics underlying the reproductive neuroendocrine system.
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Hormônio Liberador de Gonadotropina , Kisspeptinas , Hormônio Liberador de Gonadotropina/fisiologia , Kisspeptinas/fisiologia , Modelos Teóricos , Neurônios/fisiologia , Reprodução/fisiologiaRESUMO
Kisspeptin neurons in the arcuate nucleus of the hypothalamus generate gonadotrophin-releasing hormone (GnRH) pulses, and act as critical initiators of functional gonadotrophin secretion and reproductive competency. However, kisspeptin in other brain regions, most notably the posterodorsal subnucleus of the medial amygdala (MePD), plays a significant modulatory role over the hypothalamic kisspeptin population; our recent studies using optogenetics have shown that low-frequency light stimulation of MePD kisspeptin results in increased luteinsing hormone pulse frequency. Nonetheless, the neurochemical pathways that underpin this regulatory function remain unknown. To study this, we have utilised an optofluid technology, precisely combining optogenetic stimulation with intra-nuclear pharmacological receptor antagonism, to investigate the neurotransmission involved in this circuitry. We have shown experimentally and verified using a mathematical model that functional neurotransmission of both GABA and glutamate is a requirement for effective modulation of the GnRH pulse generator by amygdala kisspeptin neurons.
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Hormônio Liberador de Gonadotropina , Kisspeptinas , Feminino , Camundongos , Animais , Kisspeptinas/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Ácido Glutâmico/metabolismo , Hormônio Luteinizante/metabolismo , Núcleo Arqueado do Hipotálamo/metabolismo , Tonsila do Cerebelo/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Neonatal exposure to an immunological challenge (lipopolysaccharide, LPS) increases the activity of hypothalamo-pituitary-adrenal axis and sensitises the GNRH pulse generator to the inhibitory influence of stress in adult rats. We investigated the effects of neonatal exposure to LPS on various reproductive parameters during puberty and into adulthood in female rats. LPS (50âµg/kg, i.p.) or saline was administered on postnatal days 3 and 5. Vaginal opening was recorded, and oestrous cyclicity was monitored immediately post puberty and again at 8-9 weeks of age. At 10 weeks of age, the ovaries were removed and the number of follicles was counted, together with the thickness of the theca interna of the largest antral follicles. Ovarian sympathetic nerve activity was assessed immunohistochemically by measurement of the levels of ovarian low-affinity receptor of nerve growth factor (p75NGFR). In rats exposed to LPS in early life, there was a significant delay in puberty and disruption of oestrous cyclicity immediately post puberty, which persisted into adulthood. The follicle reserve was decreased, the thickness of the theca interna increased and the expression profile of ovarian p75NGFR increased in the neonatal LPS-treated animals. These data suggest that exposure to LPS during early neonatal life can have long-term dysfunctional effects on the female reproductive system, which might involve, at least in part, increased ovarian sympathetic nerve activity.
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Ciclo Estral , Lipopolissacarídeos/toxicidade , Ovário/efeitos dos fármacos , Receptor de Fator de Crescimento Neural/metabolismo , Sistema Nervoso Simpático/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Técnicas Imunoenzimáticas , Ovário/inervação , Ovário/metabolismo , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Psychosocial stress disrupts reproduction and interferes with pulsatile LH secretion. The posterodorsal medial amygdala (MePD) is an upstream modulator of the reproductive axis and stress. Corticotropin-releasing factor type 2 receptors (CRFR2s) are activated in the presence of psychosocial stress together with increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in the MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress on LH pulsatility. First, we administered Ucn3 into the MePD and monitored the effect on LH pulses in ovariectomized mice. Next, we delivered Astressin2B, a selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected Ucn3-cre-tdTomato mice with inhibitory designer receptor exclusively activated by designer drugs (DREADDs) targeting MePD Ucn3 neurons while exposing mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone release. Administration of Ucn3 into the MePD dose-dependently inhibited LH pulses and administration of Astressin2B blocked the suppressive effect of TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses and corticosterone release. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and corticosterone secretion. Ucn3 signalling in the MePD plays a role in modulating the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal center in the interaction between the reproductive and stress axes.
Assuntos
Tonsila do Cerebelo/metabolismo , Hormônio Luteinizante/metabolismo , Estresse Psicológico/metabolismo , Urocortinas/fisiologia , Animais , Feminino , Sistema Hipotálamo-Hipofisário/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Sistema Hipófise-Suprarrenal/metabolismo , Urocortinas/genéticaRESUMO
BACKGROUND: Chronic subdural hematoma (cSDH) is a common pathology, and recurrence is a common complication, which may be predicted by certain patient and radiologic factors. Empiric radiologic surveillance has been shown to convey no benefit. METHODS: A retrospective review of a prospectively collated database was performed. Preoperative and postoperative noncontrast computed tomography scans were reviewed. Radiologic appearance, preoperative hematoma volume, patient age, presence of bilateral hematomas, maximal hematoma thickness, and therapeutic coagulopathy were assessed as predictors. Receiver operating characteristic curve analysis, logistic regression, and LASSO regression were used to select potential predictors. A multivariate model was then fitted, and a score was derived. RESULTS: A total of 142 patients were included. Maximal hematoma thickness >12 mm (P = 0.02) and age >65 years (P = 0.01) were found to correlate with the likelihood of recurrence. Bilateral hematomas and a hyperdense or mixed density appearance were also identified on LASSO regression. Bilateral hematomas (P = 0.19), hyperdense or mixed density (P = 0.66), maximum thickness >12 mm (P = 0.01), and age >65 years (P = 0.02) were included in the multivariate model. A 6-point score was derived. A score of >3 had a sensitivity of 89% (95% confidence interval [CI] 78%-97%) and specificity of 26% (95% CI, 17%-34%) for predicting recurrence, with recurrence significantly more likely in patients with a score of 4-6 versus those with a score of 0-3 (P = 0.02). CONCLUSIONS: Certain radiologic findings may predict the recurrence of cSDH following evacuation. The score derived may be useful in identifying patients who might benefit from routine postoperative surveillance imaging.
Assuntos
Hematoma Subdural Crônico/diagnóstico por imagem , Hematoma Subdural Crônico/cirurgia , Recidiva Local de Neoplasia/diagnóstico por imagem , Idoso , Feminino , Humanos , Masculino , Estudos Retrospectivos , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios XRESUMO
Pulsatile GnRH release is essential for normal reproductive function. Kisspeptin secreting neurons found in the arcuate nucleus, known as KNDy neurons for co-expressing neurokinin B, and dynorphin, drive pulsatile GnRH release. Furthermore, gonadal steroids regulate GnRH pulsatile dynamics across the ovarian cycle by altering KNDy neurons' signalling properties. However, the precise mechanism of regulation remains mostly unknown. To better understand these mechanisms, we start by perturbing the KNDy system at different stages of the estrous cycle using optogenetics. We find that optogenetic stimulation of KNDy neurons stimulates pulsatile GnRH/LH secretion in estrous mice but inhibits it in diestrous mice. These in vivo results in combination with mathematical modelling suggest that the transition between estrus and diestrus is underpinned by well-orchestrated changes in neuropeptide signalling and in the excitability of the KNDy population controlled via glutamate signalling. Guided by model predictions, we show that blocking glutamate signalling in diestrous animals inhibits LH pulses, and that optic stimulation of the KNDy population mitigates this inhibition. In estrous mice, disruption of glutamate signalling inhibits pulses generated via sustained low-frequency optic stimulation of the KNDy population, supporting the idea that the level of network excitability is critical for pulse generation. Our results reconcile previous puzzling findings regarding the estradiol-dependent effect that several neuromodulators have on the GnRH pulse generator dynamics. Therefore, we anticipate our model to be a cornerstone for a more quantitative understanding of the pathways via which gonadal steroids regulate GnRH pulse generator dynamics. Finally, our results could inform useful repurposing of drugs targeting the glutamate system in reproductive therapy.