RESUMEN
Astrocytes play active roles at synapses and can monitor, respond, and adapt to local synaptic activity. While there is abundant evidence that astrocytes modulate excitatory transmission in the hippocampus, evidence for astrocytic modulation of hippocampal synaptic inhibition remains more limited. Furthermore, to better investigate roles for astrocytes in modulating synaptic transmission, more tools that can selectively activate native G protein signaling pathways in astrocytes with both spatial and temporal precision are needed. Here, we utilized AAV8-GFAP-Optoα1AR-eYFP (Optoα1AR), a viral vector that enables activation of Gq signaling in astrocytes via light-sensitive α1-adrenergic receptors. To determine if stimulating astrocytic Optoα1AR modulates hippocampal synaptic transmission, recordings were made in CA1 pyramidal cells with surrounding astrocytes expressing Optoα1AR, channelrhodopsin (ChR2), or GFP. Both high-frequency (20 Hz, 45-ms light pulses, 5 mW, 5 min) and low-frequency (0.5 Hz, 1-s pulses at increasing 1, 5, and 10 mW intensities, 90 s per intensity) blue light stimulation were tested. 20 Hz Optoα1AR stimulation increased both inhibitory and excitatory postsynaptic current (IPSC and EPSC) frequency, and the effect on miniature IPSCs (mIPSCs) was largely reversible within 20 min. However, low-frequency stimulation of Optoα1AR did not modulate either IPSCs or EPSCs, suggesting that astrocytic Gq -dependent modulation of basal synaptic transmission in the hippocampus is stimulation-dependent. By contrast, low-frequency stimulation of astrocytic ChR2 was effective in increasing both synaptic excitation and inhibition. Together, these data demonstrate that Optoα1AR activation in astrocytes changes basal GABAergic and glutamatergic transmission, but only following high-frequency stimulation, highlighting the importance of temporal dynamics when using optical tools to manipulate astrocyte function.
Asunto(s)
Astrocitos , Transmisión Sináptica , Astrocitos/fisiología , Transmisión Sináptica/fisiología , Hipocampo , Células Piramidales/fisiología , Sinapsis/fisiologíaRESUMEN
Central output of gonadotropin-releasing hormone (GnRH) neurons controls fertility and is sculpted by sex-steroid feedback. A switch of estradiol action from negative to positive feedback initiates a surge of GnRH release, culminating in ovulation. In ovariectomized mice bearing constant-release estradiol implants (OVX+E), GnRH neuron firing is suppressed in the morning (AM) by negative feedback and activated in the afternoon (PM) by positive feedback; no time-of-day-dependent changes occur in OVX mice. In this daily surge model, GnRH neuron intrinsic properties are shifted to favor increased firing during positive feedback. It is unclear whether this shift and the observed concomitant increase in GABAergic transmission, which typically excites GnRH neurons, are independently sufficient for increasing GnRH neuron firing rate during positive feedback or whether both are needed. To test this, we used dynamic clamp to inject selected previously recorded trains of GABAergic postsynaptic conductances (PSgs) collected during the different feedback states of the daily surge model into GnRH neurons from OVX, OVX+E AM, and OVX+E PM mice. PSg trains mimicking positive feedback initiated more action potentials in cells from OVX+E PM mice than negative feedback or OVX (open feedback loop) trains in all three animal models, but the positive-feedback train was most effective when applied to cells during positive feedback. In silico studies of model GnRH neurons in which >1000 PSg trains were tested exhibited the same results. These observations support the hypothesis that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during positive feedback.SIGNIFICANCE STATEMENT Infertility affects 15%-20% of couples; failure to ovulate is a common cause. Understanding how the brain controls ovulation is critical for new developments in both infertility treatment and contraception. Ovarian estradiol alters both the intrinsic properties of gonadotropin-releasing hormone (GnRH) neurons and synaptic inputs to these cells coincident with production of sustained GnRH release that ultimately triggers ovulation. We demonstrate here using dynamic clamp and mathematical modeling that estradiol-induced shifts in synaptic transmission alone can increase firing output, but that the intrinsic properties of GnRH neurons during positive feedback further poise these cells for increased response to higher frequency synaptic transmission. These data suggest that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during the preovulatory GnRH surge.
Asunto(s)
Encéfalo/fisiología , Estradiol/fisiología , Retroalimentación Fisiológica , Hormona Liberadora de Gonadotropina/fisiología , Neuronas/fisiología , Ovulación/fisiología , Transmisión Sináptica , Potenciales de Acción , Animales , Femenino , Ratones Transgénicos , Modelos Neurológicos , Ovariectomía , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Synaptic and intrinsic properties interact to sculpt neuronal output. Kisspeptin neurons in the hypothalamic arcuate nucleus help convey homeostatic estradiol feedback to central systems controlling fertility. Estradiol increases membrane depolarization induced by GABAA receptor activation in these neurons. We hypothesized that the mechanisms underlying estradiol-induced alterations in postsynaptic response to GABA, and also AMPA, receptor activation include regulation of voltage-gated potassium currents. Whole-cell recordings of arcuate kisspeptin neurons in brain slices from ovariectomized (OVX) and OVX+estradiol (OVX+E) female mice during estradiol negative feedback revealed that estradiol reduced capacitance, reduced transient and sustained potassium currents, and altered voltage dependence and kinetics of transient currents. Consistent with these observations, estradiol reduced rheobase and action potential latency. To study more directly interactions between synaptic and active intrinsic estradiol feedback targets, dynamic clamp was used to simulate GABA and AMPA conductances. Both GABA and AMPA dynamic clamp-induced postsynaptic potentials (PSPs) were smaller in neurons from OVX than OVX+E mice; blocking transient potassium currents eliminated this difference. To interrogate the role of the estradiol-induced changes in passive intrinsic properties, different Markov model structures based on the properties of the transient potassium current in cells from OVX or OVX+E mice were combined in silico with passive properties reflecting these two endocrine conditions. Some of tested models reproduced the effect on PSPs in silico, revealing that AMPA PSPs were more sensitive to changes in capacitance. These observations support the hypothesis that PSPs in arcuate kisspeptin neurons are regulated by estradiol-sensitive mechanisms including potassium conductances and membrane properties.SIGNIFICANCE STATEMENT Kisspeptin neurons relay estradiol feedback to gonadotropin-releasing hormone neurons, which regulate the reproductive system. The fast synaptic neurotransmitters GABA and glutamate rapidly depolarize arcuate kisspeptin neurons and estradiol increases this depolarization. Estradiol reduced both potassium current in the membrane potential range typically achieved during response to fast synaptic inputs and membrane capacitance. Using simulated GABA and glutamate synaptic inputs, we showed changes in both the passive and active intrinsic properties induced by in vivo estradiol treatment affect the response to synaptic inputs, with capacitance having a greater effect on response to glutamate. The suppression of both passive and active intrinsic properties by estradiol feedback thus renders arcuate kisspeptin neurons more sensitive to fast synaptic inputs.
Asunto(s)
Estradiol/metabolismo , Kisspeptinas/metabolismo , Neuronas/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Núcleo Arqueado del Hipotálamo/metabolismo , Estradiol/farmacología , Femenino , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Pulsatile gonadotropin-releasing hormone (GnRH) release is critical for reproduction. Disruptions to GnRH secretion patterns may contribute to polycystic ovary syndrome (PCOS). Prenatally androgenized (PNA) female mice recapitulate many neuroendocrine abnormalities observed in PCOS patients. PNA and development induce changes in spontaneous GnRH neuron firing rate, response to synaptic input, and the afterhyperpolarization potential of the action potential. We hypothesized potassium currents are altered by PNA treatment and/or development. Whole-cell patch-clamp recordings were made of transient and residual potassium currents of GnRH neurons in brain slices from 3-week-old and adult control and PNA females. At 3 weeks of age, PNA treatment increased transient current density versus controls. Development and PNA altered voltage-dependent activation and inactivation of the transient current. In controls, transient current activation and inactivation were depolarized at 3 weeks of age versus in adulthood. In GnRH neurons from 3-week-old mice, transient current activation and inactivation were more depolarized in control than PNA mice. Development and PNA treatment interacted to shift the time-dependence of inactivation and recovery from inactivation. Notably, in cells from adult PNA females, recovery was prolonged compared to all other groups. Activation of the residual current occurred at more depolarized membrane potentials in 3-week-old than adult controls. PNA depolarized activation of the residual current in adults. These findings demonstrate the properties of GnRH neuron potassium currents change during typical development, potentially contributing to puberty, and further suggest PNA treatment may both alter some typical developmental changes and induce additional modifications, which together may underlie aspects of the PNA phenotype. There was not any clinical trial involved in this work.
Asunto(s)
Síndrome del Ovario Poliquístico , Efectos Tardíos de la Exposición Prenatal , Animales , Femenino , Humanos , Ratones , Embarazo , Andrógenos/farmacología , Hormona Liberadora de Gonadotropina/fisiología , Ratones Transgénicos , Neuronas/fisiología , VirilismoRESUMEN
Estradiol has both negative and positive feedback actions upon gonadotropin-releasing hormone (GnRH) release; the latter actions trigger the preovulatory GnRH surge. Although neurobiological mechanisms of the transitions between feedback modes are becoming better understood, the roles of voltage-gated potassium currents, major contributors to neuronal excitability, are unknown. Estradiol alters two components of potassium currents in these cells: a transient current, I(A), and a sustained current, I(K). Kisspeptin is a potential mediator between estradiol and GnRH neurons and can act directly on GnRH neurons. We examined how estradiol, time of day, and kisspeptin interact to regulate these conductances in a mouse model exhibiting daily switches between estradiol negative (morning) and positive feedback (evening). Whole-cell voltage clamp recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice and from OVX mice treated with estradiol (OVX+E). There were no diurnal changes in either I(A) or I(K) in GnRH neurons from OVX mice. In contrast, in GnRH neurons from OVX+E mice, I(A) and I(K) were greater during the morning when GnRH neuron activity is low and smaller in the evening when GnRH neuron activity is high. Estradiol increased I(A) in the morning and decreased it in the evening, relative to that in cells from OVX mice. Exogenously applied kisspeptin reduced I(A) regardless of time of day or estradiol status. Estradiol, interacting with time of day, and kisspeptin both depolarized I(A) activation. These findings extend our understanding of both the neurobiological mechanisms of estradiol negative vs. positive regulation of GnRH neurons and of kisspeptin action on these cells.
Asunto(s)
Ritmo Circadiano/fisiología , Estradiol/fisiología , Retroalimentación Fisiológica/fisiología , Hormona Liberadora de Gonadotropina/fisiología , Kisspeptinas/fisiología , Neuronas/fisiología , Canales de Potasio con Entrada de Voltaje/fisiología , Animales , Encéfalo/citología , Estradiol/farmacología , Femenino , Proteínas Fluorescentes Verdes/genética , Kisspeptinas/farmacología , Ratones , Ratones Transgénicos , Modelos Animales , Neuronas/citología , Ovariectomía , Técnicas de Placa-Clamp , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Gonadotropin-releasing hormone (GnRH) drives pituitary secretion of luteinizing hormone and follicle-stimulating hormone, which in turn regulate gonadal functions including steroidogenesis. The pattern of GnRH release and thus fertility depend on gonadal steroid feedback. Under homeostatic (negative) feedback conditions, removal of the gonads from either females or males increases the amplitude and frequency of GnRH release and alters the long-term firing pattern of these neurons in brain slices. The neurobiological mechanisms intrinsic to GnRH neurons that are altered by homeostatic feedback are not well studied and have not been compared between sexes. During estradiol-positive feedback, which is unique to females, there are correlated changes in voltage-gated potassium currents and neuronal excitability. We thus hypothesized that these same mechanisms would be engaged in homeostatic negative feedback. Voltage-gated potassium channels play a direct role in setting excitability and action potential properties. Whole-cell patch-clamp recordings of GFP-identified GnRH neurons in brain slices from sham-operated and castrated adult female and male mice were made to assess fast and slow inactivating potassium currents as well as action potential properties. Surprisingly, no changes were observed among groups in most potassium current properties, input resistance, or capacitance, and this was reflected in a lack of differences in excitability and specific action potential properties. These results support the concept that, in contrast to positive feedback, steroid-negative feedback regulation of GnRH neurons in both sexes is likely conveyed to GnRH neurons via mechanisms that do not induce major changes in the biophysical properties of these cells.
Asunto(s)
Retroalimentación Fisiológica , Hormona Liberadora de Gonadotropina , Potasio , Animales , Estradiol , Femenino , Masculino , Ratones , Neuronas , OvariectomíaRESUMEN
Kisspeptin-expressing neurons in the anteroventral-periventricular nucleus (AVPV) are part of a neural circuit generating the gonadotropin-releasing hormone (GnRH) surge. This process is estradiol-dependent and occurs on the afternoon of proestrus in female mice. On proestrus, AVPV kisspeptin neurons express more kisspeptin and exhibit higher frequency action potentials and burst firing compared with diestrus, which is characterized by a pulsatile rather than a prolonged surge of GnRH secretion. We hypothesized changes in voltage-gated potassium conductances shape activity profiles of these cells in a cycle-dependent manner. Whole-cell voltage-clamp recordings of GFP-identified AVPV kisspeptin neurons in brain slices from diestrous and proestrous mice revealed three subcomponents of the voltage-sensitive K+ current: fast-transient slow-transient, and residual. During proestrus, the V50 of inactivation of the fast-transient current was depolarized and the amplitude of the slow-transient component was reduced compared with diestrus; the residual component was consistent across both stages. Computational models were fit to experimental data, including published estrous-cycle effects on other voltage-gated currents. Computer simulations suggest proestrus-typical K+ currents are suppressive compared with diestrus. Interestingly, larger T-type, persistent-sodium, and hyperpolarization-activated currents during proestrus compensate for this suppressive effect while also enabling postinhibitory rebound bursting. These findings suggest modulation of voltage-gated K+ and multiple subthreshold depolarizing currents across the negative to positive feedback transition maintain AVPV kisspeptin neuron excitability in response to depolarizing stimuli. These changes also enable firing in response to hyperpolarization, providing a net increase in neuronal excitability, which may contribute to activation of this population leading up to the preovulatory GnRH surge.
Asunto(s)
Kisspeptinas , Potasio , Animales , Estradiol/farmacología , Ciclo Estral , Femenino , Hormona Liberadora de Gonadotropina , Hipotálamo Anterior/metabolismo , Kisspeptinas/metabolismo , Ratones , Neuronas/metabolismoRESUMEN
GnRH neurons are the final central neural output regulating fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (KNDy neurons) are considered the main regulator of GnRH output. GnRH and KNDy neurons are surrounded by astrocytes, which can modulate neuronal activity and communicate over distances. Prostaglandin E2 (PGE2), synthesized primarily by astrocytes, increases GnRH neuron activity and downstream pituitary release of luteinizing hormone (LH). We hypothesized that glial fibrillary acidic protein (GFAP)-expressing astrocytes play a role in regulating GnRH and/or KNDy neuron activity and LH release. We used adeno-associated viruses to target designer receptors exclusively activated by designer drugs (DREADDs) to GFAP-expressing cells to activate Gq- or Gi-mediated signaling. Activating Gq signaling in the preoptic area, near GnRH neurons, but not in the arcuate, increases LH release in vivo and GnRH firing in vitro via a mechanism in part dependent upon PGE2. These data suggest that astrocytes can activate GnRH/LH release in a manner independent of KNDy neurons.
Asunto(s)
Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Hormona Liberadora de Gonadotropina/farmacología , Hormona Luteinizante/efectos de los fármacos , Neuronas/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/citología , Núcleo Arqueado del Hipotálamo/metabolismo , Kisspeptinas/metabolismo , Hormona Luteinizante/metabolismo , Masculino , Ratones , Ratones Transgénicos , Hipófisis/metabolismo , Área Preóptica/metabolismoRESUMEN
Gonadotropin-releasing hormone (GnRH) neurons control anterior pituitary, and thereby gonadal, function. GnRH neurons are active before outward indicators of puberty appear. Prenatal androgen (PNA) exposure mimics reproductive dysfunction of the common fertility disorder polycystic ovary syndrome (PCOS) and reduces prepubertal GnRH neuron activity. Early neuron activity can play a critical role in establishing circuitry and adult function. We tested the hypothesis that changing prepubertal GnRH neuron activity programs adult GnRH neuron activity and reproduction independent of androgen exposure in female mice. Activating (3Dq) or inhibitory (4Di) designer receptors exclusively activated by designer drugs (DREADDs) were targeted to GnRH neurons using Cre-lox technology. In control studies, the DREADD ligand clozapine n-oxide (CNO) produced the expected changes in GnRH neuron activity in vitro and luteinizing hormone (LH) release in vivo CNO was administered to control or PNA mice between two and three weeks of age, when GnRH neuron firing rate is reduced in PNA mice. In controls, reducing prepubertal GnRH neuron activity with 4Di increased adult GnRH neuron firing rate and days in diestrus but did not change puberty onset or GABA transmission to these cells. In contrast, activating GnRH neurons had no effect on reproductive parameters or firing rate and did not rescue reproductive phenotypes in PNA mice. These studies support the hypothesis that prepubertal neuronal activity sculpts elements of the adult reproductive neuroendocrine axis and cyclicity but indicate that other PNA-induced programming actions are required for full reproductive phenotypes and/or that compensatory mechanisms overcome activity-mediated changes to mitigate reproductive changes in adults.
Asunto(s)
Hormona Liberadora de Gonadotropina , Efectos Tardíos de la Exposición Prenatal , Animales , Femenino , Ratones , Neuronas , Embarazo , Reproducción , Maduración SexualRESUMEN
Gonadotropin-releasing hormone (GnRH) neurons form the final pathway for the central neuronal control of fertility. GnRH is released in pulses that vary in frequency in females, helping drive hormonal changes of the reproductive cycle. In the common fertility disorder polycystic ovary syndrome (PCOS), persistent high-frequency hormone release is associated with disrupted cycles. We investigated long- and short-term action potential patterns of GnRH neurons in brain slices before and after puberty in female control and prenatally androgenized (PNA) mice, which mimic aspects of PCOS. A Monte Carlo (MC) approach was used to randomize action potential interval order. Dataset distributions were analysed to assess (i) if organization persists in GnRH neuron activity in vitro, and (ii) to determine if any organization changes with development and/or PNA treatment. GnRH neurons in adult control, but not PNA, mice produce long-term patterns different from MC distributions. Short-term patterns differ from MC distributions before puberty but become absorbed into the distributions with maturation, and the distributions narrow. These maturational changes are blunted by PNA treatment. Firing patterns of GnRH neurons in brain slices thus maintain organization dictated at least in part by the biologic status of the source and are disrupted in models of disease.
RESUMEN
In the brain, ischemic preconditioning (IPC) diminishes mitochondrial dysfunction after ischemia and confers neuroprotection. Activation of epsilon protein kinase C (epsilonPKC) has been proposed to be a key neuroprotective pathway during IPC. We tested the hypothesis that IPC increases the levels of epsilonPKC in synaptosomes from rat hippocampus, resulting in improved synaptic mitochondrial respiration. Preconditioning significantly increased the level of hippocampal synaptosomal epsilonPKC to 152% of sham-operated animals at 2 d of reperfusion, the time of peak neuroprotection. We tested the effect of epsilonPKC activation on hippocampal synaptic mitochondrial respiration 2 d after preconditioning. Treatment with the specific epsilonPKC activating peptide, tat-psiepsilonRACK (tat-psiepsilon-receptor for activated C kinase), increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV to 157, 153, and 131% of control (tat peptide alone). In parallel, we found that epsilonPKC activation in synaptosomes from preconditioned animals resulted in altered levels of phosphorylated mitochondrial respiratory chain proteins: increased serine and tyrosine phosphorylation of 18 kDa subunit of complex I, decreased serine phosphorylation of FeS protein in complex III, increased threonine phosphorylation of COX IV (cytochrome oxidase IV), increased mitochondrial membrane potential, and decreased H2O2 production. In brief, ischemic preconditioning promoted significant increases in the level of synaptosomal epsilonPKC. Activation of epsilonPKC increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins. We propose that, at 48 h of reperfusion after ischemic preconditioning, epsilonPKC is poised at synaptic mitochondria to respond to ischemia either by direct phosphorylation or activation of the epsilonPKC signaling pathway.
Asunto(s)
Isquemia Encefálica/enzimología , Precondicionamiento Isquémico/métodos , Mitocondrias/enzimología , Proteína Quinasa C-epsilon/fisiología , Sinaptosomas/enzimología , Animales , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Respiración de la Célula/fisiología , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Proteína Quinasa C-epsilon/genética , Ratas , Ratas Sprague-Dawley , Sinaptosomas/metabolismoRESUMEN
Cardiovascular pathologies in the French are not prevalent despite high dietary saturated fat consumption. This is commonly referred to as the "French Paradox" attributing its anti-lipidemic effects to moderate consumption of red wine. Resveratrol, a phytoalexin found in red wine, is currently the focus of intense research both in the cardiovascular system and the brain. Current research suggests resveratrol may enhance prognosis of neurological disorders such as, Parkinson's, Huntington's, Alzheimer's diseases and stroke. The beneficial effects of resveratrol include: antioxidation, free radical scavenger, and modulation of neuronal energy homeostasis and glutamatergic receptors/ion channels. Resveratrol directly increases sirtuin 1 (SIRT1) activity, a NAD(+) (oxidized form of nicotinamide adenine dinucleotide)-dependent histone deacetylase related to increased lifespan in various species similar to calorie restriction. We recently demonstrated that brief resveratrol pretreatment conferred neuroprotection against cerebral ischemia via SIRT1 activation. This neuroprotective effect produced by resveratrol was similar to ischemic preconditioning-induced neuroprotection, which protects against lethal ischemic insults in the brain and other organ systems. Inhibition of SIRT1 abolished ischemic preconditioning-induced neuroprotection in CA1 region of the hippocampus. Since resveratrol and ischemic preconditioning-induced neuroprotection require activation of SIRT1, this common signaling pathway may provide targeted therapeutic treatment modalities as it relates to stroke and other brain pathologies. In this review, we will examine common signaling pathways, cellular targets of resveratrol, and ischemic preconditioning-induced neuroprotection as it relates to the brain.
Asunto(s)
Isquemia Encefálica/metabolismo , Isquemia Encefálica/prevención & control , Precondicionamiento Isquémico , Fármacos Neuroprotectores/farmacología , Estilbenos/farmacología , Animales , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Humanos , Precondicionamiento Isquémico/métodos , Fármacos Neuroprotectores/uso terapéutico , Resveratrol , Transducción de Señal/efectos de los fármacos , Estilbenos/uso terapéutico , Sinapsis/efectos de los fármacos , Sinapsis/metabolismoRESUMEN
Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (epsilonPKC). A major goal in our laboratory is to define the cellular targets by which epsilonPKC confers protection. We tested the hypothesis that epsilonPKC targets the mitochondrial K(+)(ATP) channel (mtK(+)(ATP)) after IPC. Our results demonstrated a rapid translocation of epsilonPKC to rat hippocampal mitochondria after IPC. Because in other tissues epsilonPKC targets mtK(+)(ATP) channels, but its presence in brain mitochondria is controversial, we determined the presence of the K(+)(ATP) channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether mtK(+)(ATP) channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the K(+)(ATP) channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or epsilonPKC agonist-induced preconditioning. Blockade of mtK(+)(ATP) channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or epsilonPKC preconditioning. Both ischemic and epsilonPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK(+)(ATP) channel subunit Kir6.2. After IPC, selective inhibition of epsilonPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of epsilonPKC or its downstream effectors. Our results support the hypothesis that the brain mtK(+)(ATP) channel is an important target of IPC and the signal transduction pathways initiated by epsilonPKC.
Asunto(s)
Hipocampo/citología , Precondicionamiento Isquémico/métodos , Canales KATP/metabolismo , Mitocondrias/metabolismo , Neuronas/ultraestructura , Proteína Quinasa C-epsilon/fisiología , Animales , Animales Recién Nacidos , Antihipertensivos/farmacología , Diazóxido/farmacología , Inhibidores Enzimáticos/farmacología , Hipocampo/fisiología , Inmunoprecipitación , Técnicas In Vitro , Isquemia/metabolismo , Masculino , Membranas Mitocondriales/efectos de los fármacos , Modelos Biológicos , Neuronas/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Factores de TiempoAsunto(s)
Isquemia Encefálica/fisiopatología , Encéfalo/irrigación sanguínea , Circulación Cerebrovascular , Proteína Quinasa C-delta/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Daño por Reperfusión/fisiopatología , Animales , Isquemia Encefálica/patología , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Masculino , Microvasos/patología , Oligopéptidos/farmacología , Oxígeno/metabolismo , Proteína Quinasa C-delta/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
During the female reproductive cycle, estradiol exerts negative and positive feedback at both the central level to alter gonadotropin-releasing hormone (GnRH) release and at the pituitary to affect response to GnRH. Many studies of the neurobiologic mechanisms underlying estradiol feedback have been done on ovariectomized, estradiol-replaced (OVX+E) mice. In this model, GnRH neuron activity depends on estradiol and time of day, increasing in estradiol-treated mice in the late afternoon, coincident with a daily luteinizing hormone (LH) surge. Amplitude of this surge appears lower than in proestrous mice, perhaps because other ovarian factors are not replaced. We hypothesized GnRH neuron activity is greater during the proestrous-preovulatory surge than the estradiol-induced surge. GnRH neuron activity was monitored by extracellular recordings from fluorescently tagged GnRH neurons in brain slices in the late afternoon from diestrous, proestrous, and OVX+E mice. Mean GnRH neuron firing rate was low on diestrus; firing rate was similarly increased in proestrous and OVX+E mice. Bursts of action potentials have been associated with hormone release in neuroendocrine systems. Examination of the patterning of action potentials revealed a shift toward longer burst duration in proestrous mice, whereas intervals between spikes were shorter in OVX+E mice. LH response to an early afternoon injection of GnRH was greater in proestrous than diestrous or OVX+E mice. These observations suggest the lower LH surge amplitude observed in the OVX+E model is likely not attributable to altered mean GnRH neuron activity, but because of reduced pituitary sensitivity, subtle shifts in action potential pattern, and/or excitation-secretion coupling in GnRH neurons.
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Estradiol/fisiología , Hormona Liberadora de Gonadotropina/fisiología , Hormona Luteinizante/sangre , Hipófisis/fisiología , Proestro/fisiología , Potenciales de Acción , Animales , Femenino , Ratones Endogámicos C57BL , Neuronas/metabolismoRESUMEN
Pulsatile release of gonadotropin-releasing hormone (GnRH) is key to fertility. Pulse frequency is modulated by gonadal steroids and likely arises subsequent to coordination of GnRH neuron firing activity. The source of rhythm generation and the site of steroid feedback remain critical unanswered questions. Arcuate neurons that synthesize kisspeptin, neurokinin B, and dynorphin (KNDy) may be involved in both of these processes. We tested the hypotheses that action potential firing in KNDy neurons is episodic and that gonadal steroids regulate this pattern. Targeted extracellular recordings were made of green fluorescent protein-identified KNDy neurons in brain slices from adult male mice that were intact, castrated, or castrated and treated with estradiol or dihydrotestosterone (DHT). KNDy neurons exhibited marked peaks and nadirs in action potential firing activity during recordings lasting 1 to 3.5 hours. Peaks, identified by Cluster analysis, occurred more frequently in castrated than intact mice, and either estradiol or DHT in vivo or blocking neurokinin type 3 receptor in vitro restored peak frequency to intact levels. The frequency of peaks in firing rate and estradiol regulation of this frequency is similar to that observed for GnRH neurons, whereas DHT suppressed firing in KNDy but not GnRH neurons. We further examined the patterning of action potentials to identify bursts that may be associated with increased neuromodulator release. Burst frequency and duration are increased in castrated compared with intact and steroid-treated mice. The observation that KNDy neurons fire in an episodic manner that is regulated by steroid feedback is consistent with a role for these neurons in GnRH pulse generation and regulation.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Andrógenos/farmacología , Núcleo Arqueado del Hipotálamo/citología , Dihidrotestosterona/farmacología , Estradiol/farmacología , Estrógenos/farmacología , Neuronas/efectos de los fármacos , Orquiectomía , Potenciales de Acción/fisiología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiología , Análisis por Conglomerados , Dinorfinas/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Proteínas Fluorescentes Verdes/genética , Kisspeptinas/genética , Kisspeptinas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuroquinina B/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-Clamp , Receptores de Neuroquinina-3/antagonistas & inhibidoresRESUMEN
Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction and are inhibited by negative energy balance. In normal adults, these neurons maintain elevated intracellular chloride so that GABA(A) receptor activation is excitatory. We hypothesized that fasting alters homeostatic mechanisms to eliminate excitatory responses to GABA but rejected this hypothesis when brief, local GABA application elicited action currents in GnRH neurons from fed and fasted mice. This response was specific to GABA(A) receptors, because glycine elicited no response. We next found that fasting reduced the frequency of spontaneous GABAergic postsynaptic currents (PSCs) and that this was reversed by in vivo treatment with leptin during the fast. In the presence of tetrodotoxin to minimize presynaptic actions, leptin also potentiated the postsynaptic response of these cells to GABA(A) receptor activation. Postsynaptic effects of leptin on GABAergic miniature PSCs were eliminated by inhibiting JAK2/3 (Janus kinase), the tyrosine kinase through which leptin receptors signal. In all experiments, elimination of PSCs at ECl or by treatment with the GABAA receptor antagonist bicuculline confirmed that PSCs were specifically mediated by GABA(A) receptor chloride channels. These data demonstrate that fasting and leptin act presynaptically and postsynaptically to alter GABAergic drive to GnRH neurons, providing evidence for GABAergic communication of metabolic cues to GnRH neurons, and suggest the possibility for functional leptin receptors on GnRH neurons. They further demonstrate cytokine modulation of the postsynaptic response to GABA in mammals, which may be important to central neural regulation in both healthy and diseased states.
Asunto(s)
Fertilidad/fisiología , Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/fisiología , Terminales Presinápticos/fisiología , Transmisión Sináptica/fisiología , Animales , Glucemia , Cloruros/metabolismo , Metabolismo Energético/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Ayuno/fisiología , Femenino , Proteínas Fluorescentes Verdes , Hipotálamo/citología , Hipotálamo/fisiología , Técnicas In Vitro , Leptina/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estado Nutricional/fisiología , Técnicas de Placa-Clamp , Área Preóptica/citología , Área Preóptica/fisiología , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Leptina , Transducción de Señal/fisiología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
gamma-Aminobutyric acid (GABA) provides a major synaptic input to GnRH neurons. GnRH neurons maintain high intracellular chloride levels and respond to exogenous GABA with depolarization and action potential firing. We examined the role of synaptic GABA type A receptor (GABA(A)R) activation on the firing activity of GnRH neurons. Targeted extracellular recordings were used to detect firing activity of GnRH neurons in brain slices from adult female mice. Because the brain slice preparation preserves both glutamatergic and GABAergic neuronal networks, the effects of GABA(A)Rs on GnRH neurons were isolated by blocking ionotropic glutamatergic receptors (iGluR). With iGluR blocked, many GnRH neurons remained spontaneously active. Consistent with an excitatory role for GABA, subsequent blockade of GABA(A)Rs suppressed the firing rate in active cells from diestrous females by approximately 40% (P < 0.05; n = 10). GABA(A)R blockade did not affect inactive cells (n = 7), indicating that GABA(A)R-mediated inhibition was not responsible for the lack of firing. In prenatally androgenized females, GnRH neurons exhibit larger, more frequent GABAergic postsynaptic currents than control females. Most cells from prenatally androgenized animals fired spontaneously, and the firing rate was suppressed approximately 80% after GABA(A)R blockade (P < 0.01; n = 8). Blocking GABA(A)R without blocking iGluRs increased the firing rate in GnRH neurons from diestrous females (P < 0.05; n = 6), perhaps attributable to hyperexcitability within the slice network. Our results indicate that GABAergic inputs help generate a portion of action potentials in GnRH neurons; this fraction depends on the level of GABA transmission and postsynaptic responsiveness. The complexities of the GnRH neuron response to GABA make this a potentially critical integration point for central regulation of fertility.
Asunto(s)
Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/fisiología , Ácido gamma-Aminobutírico/fisiología , Potenciales de Acción , Andrógenos/farmacología , Animales , Diestro , Dihidrotestosterona/farmacología , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Antagonistas del GABA/farmacología , Antagonistas de Receptores de GABA-A , Ácido Glutámico/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiología , Técnicas In Vitro , Ratones , Ratones Transgénicos , Red Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal , Receptores de Glutamato/efectos de los fármacos , Transmisión Sináptica , Ácido gamma-Aminobutírico/metabolismoRESUMEN
GnRH neurons are regulated by estradiol feedback through unknown mechanisms. Voltage-gated potassium channels determine the pattern of activity and response to synaptic inputs in many neurons. We used whole-cell patch-clamp to test whether estradiol feedback altered potassium currents in GnRH neurons. Adult mice were ovariectomized and some treated with estradiol implants to suppress reproductive neuroendocrine function; 1 wk later, brain slices were prepared for recording. Estradiol affected the amplitude, decay time, and the voltage dependence of both inactivation and activation of A-type potassium currents in these cells. Estradiol also altered a slowly inactivating current, I(K.) The estradiol-induced changes in I(A) contributed to marked changes in action potential properties. Estradiol increased excitability in GnRH neurons, decreasing both threshold and latency for action potential generation. To test whether estradiol altered phosphorylation of the channels or associated proteins, the broad-spectrum kinase inhibitor H7 was included in the recording pipette. H7 acutely reversed some but not all effects of estradiol on potassium currents. Estradiol did not affect I(A) or I(K) in paraventricular neurosecretory neurons, demonstrating a degree of specificity in these effects. Potassium channels are thus one target for estradiol regulation of GnRH neurons; this regulation involves changes in phosphorylation of potassium channel components.
Asunto(s)
Encéfalo/fisiología , Estradiol/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/metabolismo , Canales de Potasio/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Cloruro de Cadmio/farmacología , Electrofisiología/métodos , Inhibidores Enzimáticos/farmacología , Estradiol/farmacología , Retroalimentación Fisiológica , Femenino , Hormona Liberadora de Gonadotropina/genética , Proteínas Fluorescentes Verdes , Técnicas In Vitro , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Ovariectomía , Núcleo Hipotalámico Paraventricular/metabolismo , Fosforilación , Canales de Potasio/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/metabolismo , Regiones Promotoras GenéticasRESUMEN
Gamma-aminobutyric acid (GABA), acting through GABA(A) receptors (GABA(A)R), is hypothesized to suppress reproduction by inhibiting GnRH secretion, but GABA actions directly on GnRH neurons are not well established. In green fluorescent protein-identified adult mouse GnRH neurons in brain slices, gramicidin-perforated-patch-clamp experiments revealed the reversal potential (E(GABA)) for current through GABA(A)Rs was depolarized relative to the resting potential. Furthermore, rapid GABA application elicited action potentials in GnRH neurons but not controls. The consequence of GABA(A)R activation depends on intracellular chloride levels, which are maintained by homeostatic mechanisms. Membrane proteins that typically extrude chloride (KCC-2 cotransporter, CLC-2 channel) were absent from the GT1-7 immortalized GnRH cell line and GnRH neurons in situ or were not localized to the proper cell compartment for function. In contrast, GT1-7 cells and some GnRH neurons expressed the chloride-accumulating cotransporter, NKCC-1. Patch-clamp experiments showed that blockade of NKCC hyperpolarized E(GABA) by lowering intracellular chloride. Regardless of reproductive state, rapid GABA application excited GnRH neurons. In contrast, bath application of the GABA(A)R agonist muscimol transiently increased then suppressed firing; suppression persisted 4-15 min. Rapid activation of GABA(A)R thus excites GnRH neurons whereas prolonged activation reduces excitability, suggesting the physiological consequence of synaptic activation of GABA(A)R in GnRH neurons is excitation.