scholarly journals Estradiol Negative and Positive Feedback in a Prenatal Androgen-Induced Mouse Model of Polycystic Ovarian Syndrome

Endocrinology ◽  
2012 ◽  
Vol 154 (2) ◽  
pp. 796-806 ◽  
Author(s):  
Aleisha M. Moore ◽  
Melanie Prescott ◽  
Rebecca E. Campbell
Keyword(s):  
Positive Feedback ◽  
Polycystic Ovarian Syndrome ◽  
Fluorescent Protein ◽  
Spine Density ◽  
Gnrh Neuron ◽  
Feedback Effects ◽  
Gnrh Neurons ◽  
Green Fluorescent ◽  
Prenatal Androgen ◽  
Ovarian Syndrome

Gonadal steroid hormone feedback is impaired in polycystic ovarian syndrome (PCOS), a common endocrine disorder characterized by hyperandrogenism and an associated increase in LH pulse frequency. Using a prenatal androgen (PNA)-treated mouse model of PCOS, we aimed to investigate negative and positive feedback effects of estrogens on the hypothalamic-pituitary axis regulation of LH. PNA-treated mice exhibited severely disrupted estrous cycles, hyperandrogenism, significantly reduced fertility, and altered ovarian morphology. To assess the negative feedback effects of estrogens, LH was measured before and after ovariectomy and after estradiol (E2) administration. Compared with controls, PNA-treated mice exhibited a blunted postcastration rise in LH (P < .001) and an absence of LH suppression after E2 administration. To assess E2-positive feedback, control and PNA-treated GnRH-green fluorescent protein transgenic mice were subjected to a standard ovariectomy with E2-replacement regimen, and both plasma and perfusion-fixed brains were collected at the time of the expected GnRH/LH surge. Immunocytochemistry and confocal imaging of cFos and green fluorescent protein were used to assess GnRH neuron activation and spine density. In the surged group, both control and PNA-treated mice had significantly increased LH and cFos activation in GnRH neurons (P < .05) compared with nonsurged animals. Spine density was quantified in cFos-positive and -negative GnRH neurons to examine whether there was an increase in spine density in cFos-expressing GnRH neurons of surged mice as expected. A significant increase in spine density in cFos-expressing GnRH neurons was evident in control animals; however, no significant increase was observed in the PNA-treated mice because spine density was elevated across all GnRH neurons. These data support that PNA treatment results in a PCOS-like phenotype that includes impaired E2-negative feedback. Additionally, although E2-positive feedback capability is retained in PNA mice, elevated GnRH neuron spine density may reflect altered synaptic regulation.

scholarly journals Estradiol-Sensitive Afferents Modulate Long-Term Episodic Firing Patterns of GnRH Neurons

Endocrinology ◽  
2002 ◽  
Vol 143 (6) ◽  
pp. 2284-2292 ◽  
Author(s):  
Craig S. Nunemaker ◽  
R. Anthony DeFazio ◽  
Suzanne M. Moenter
Keyword(s):  
Green Fluorescent Protein Expression ◽  
Fluorescent Protein ◽  
Gnrh Neuron ◽  
Action Current ◽  
Neuron Firing ◽  
Firing Patterns ◽  
Gnrh Neurons ◽  
Episode Frequency ◽  
Green Fluorescent

Abstract GnRH neurons comprise the final common pathway of an estrogen-sensitive pattern generator controlling fertility. To determine estradiol effects on GnRH neuron firing patterns, adult transgenic mice were ovariectomized (OVX), and half were treated with estradiol (OVX+E). One week later targeted single-unit extracellular recordings were made from GnRH neurons identified by green fluorescent protein expression. Estradiol markedly affected GnRH neuron firing patterns, increasing the percentage and duration of time these cells were quiescent (≤1 action current/min). Estradiol increased the interval between episodes of increased firing rate determined by Cluster analysis of recordings more than 45 min (OVX+E 38.8 ± 7.2 min, OVX 16.7 ± 2.1 min, n = 6 each). Possible mechanisms of estradiol modulation were examined by simultaneously blocking ionotropic secretion of γ-aminobutyric acid and glutamatergic receptors. This treatment had no effect on cells from OVX mice (n = 10), indicating episodic firing of GnRH neurons is not driven by activation of these receptors. Receptor blockade eliminated estradiol effects on GnRH neurons in the midventral preoptic area (n = 7) but not elsewhere (n = 7). Individual GnRH neurons thus display episodic firing patterns at intervals previously reported for secretory pulses. Estradiol modulates episode frequency to exert feedback control; in a substantial subset of GnRH neurons, estradiol feedback is enforced via GABAergic and/or glutamatergic afferents.

scholarly journals Postnatal Remodeling of Dendritic Structure and Spine Density in Gonadotropin-Releasing Hormone Neurons

Endocrinology ◽  
2006 ◽  
Vol 147 (8) ◽  
pp. 3652-3661 ◽  
Author(s):  
Elizabeth C. Cottrell ◽  
Rebecca E. Campbell ◽  
Seong-Kyu Han ◽  
Allan E. Herbison
Keyword(s):  
Green Fluorescent Protein ◽  
Adult Male ◽  
Fluorescent Protein ◽  
Dendritic Tree ◽  
Spine Density ◽  
Male Mice ◽  
Fixed Tissue ◽  
Gnrh Neurons ◽  
Green Fluorescent

The GnRH neurons represent the output cells of the neuronal network controlling gonadal function. Their activation initiates the onset of puberty, but the underlying mechanisms remain unclear. Using a GnRH-green fluorescent protein mouse model, we have been able to fill individual GnRH neurons with biocytin in the acute brain slice preparation to examine their morphological characteristics across puberty. GnRH neurons in prepubertal male mice [postnatal d 10–15 (PND10–15)] exhibited half as many dendritic and somal spines as adult male mice (>PND60; P < 0.05) but, surprisingly, a much more complex dendritic tree with 5-fold greater branch points (P < 0.05). Experiments examining somal and proximal dendritic spine numbers in vivo, in perfusion-fixed tissue from GnRH-green fluorescent protein mice, revealed the same pattern of approximately twice as many spines on adult GnRH neurons compared with PND10 male mice (P < 0.01). In contrast to the spine density alterations, reflecting changing excitatory input, confocal immunofluorescence studies revealed no differences in the numbers of vesicular γ-aminobutyric acid transporter-immunoreactive elements adjacent to GnRH soma or proximal dendrites in prepubertal and adult male mice. Experiments evaluating dendritic tree structure in vivo (PND3, -10, and -35 and adult) revealed that GnRH neurons located in the rostral preoptic area, but not the medial septum, exhibited a more complex branching pattern at PND10, but that this was adult-like by PND35. These studies demonstrate unexpected dendritic tree remodeling in the GnRH neurons and provide evidence for an increase in direct excitatory inputs to GnRH neurons across the time of puberty.

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

Endocrinology ◽  
2015 ◽  
Vol 156 (7) ◽  
pp. 2582-2594 ◽  
Author(s):  
Siew Hoong Yip ◽  
Ulrich Boehm ◽  
Allan E. Herbison ◽  
Rebecca E. Campbell
Keyword(s):  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Close Contact ◽  
Third Ventricle ◽  
Gnrh Neuron ◽  
Tract Tracing ◽  
Neuron Populations ◽  
Gnrh Neurons ◽  
A Cell ◽  
Green Fluorescent

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

scholarly journals Enhancement of a robust arcuate GABAergic input to gonadotropin-releasing hormone neurons in a model of polycystic ovarian syndrome

2014 ◽  
Vol 112 (2) ◽  
pp. 596-601 ◽  
Author(s):  
Aleisha M. Moore ◽  
Mel Prescott ◽  
Christopher J. Marshall ◽  
Siew Hoong Yip ◽  
Rebecca E. Campbell
Keyword(s):  
Progesterone Receptor ◽  
Polycystic Ovarian Syndrome ◽  
Steroid Hormone ◽  
Pulse Frequency ◽  
Gonadotropin Releasing Hormone ◽  
Receptor Expression ◽  
Spine Density ◽  
Releasing Hormone ◽  
Gnrh Neurons ◽  
Ovarian Syndrome

Polycystic ovarian syndrome (PCOS), the leading cause of female infertility, is associated with an increase in luteinizing hormone (LH) pulse frequency, implicating abnormal steroid hormone feedback to gonadotropin-releasing hormone (GnRH) neurons. This study investigated whether modifications in the synaptically connected neuronal network of GnRH neurons could account for this pathology. The PCOS phenotype was induced in mice following prenatal androgen (PNA) exposure. Serial blood sampling confirmed that PNA elicits increased LH pulse frequency and impaired progesterone negative feedback in adult females, mimicking the neuroendocrine abnormalities of the clinical syndrome. Imaging of GnRH neurons revealed greater dendritic spine density that correlated with increased putative GABAergic but not glutamatergic inputs in PNA mice. Mapping of steroid hormone receptor expression revealed that PNA mice had 59% fewer progesterone receptor-expressing cells in the arcuate nucleus of the hypothalamus (ARN). To address whether increased GABA innervation to GnRH neurons originates in the ARN, a viral-mediated Cre-lox approach was taken to trace the projections of ARN GABA neurons in vivo. Remarkably, projections from ARN GABAergic neurons heavily contacted and even bundled with GnRH neuron dendrites, and the density of fibers apposing GnRH neurons was even greater in PNA mice (56%). Additionally, this ARN GABA population showed significantly less colocalization with progesterone receptor in PNA animals compared with controls. Together, these data describe a robust GABAergic circuit originating in the ARN that is enhanced in a model of PCOS and may underpin the neuroendocrine pathophysiology of the syndrome.

scholarly journals Retrograde Endocannabinoid Signaling Reduces GABAergic Synaptic Transmission to Gonadotropin-Releasing Hormone Neurons

Endocrinology ◽  
2010 ◽  
Vol 151 (12) ◽  
pp. 5818-5829 ◽  
Author(s):  
Imre Farkas ◽  
Imre Kalló ◽  
Levente Deli ◽  
Barbara Vida ◽  
Erik Hrabovszky ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cannabinoid Receptor ◽  
Gaba Release ◽  
Vesicle Fusion ◽  
Gnrh Neuron ◽  
Neuron Firing ◽  
Gabaergic Neurotransmission ◽  
Functional Studies ◽  
Gnrh Neurons ◽  
Green Fluorescent

Cannabinoids suppress fertility via reducing hypothalamic GnRH output. γ-Aminobutyric acid (GABA)A receptor (GABAA-R)-mediated transmission is a major input to GnRH cells that can be excitatory. We hypothesized that cannabinoids act via inhibiting GABAergic input. We performed loose-patch electrophysiological studies of acute slices from adult male GnRH-green fluorescent protein transgenic mice. Bath application of type 1 cannabinoid receptor (CB1) agonist WIN55,212 decreased GnRH neuron firing rate. This action was detectable in presence of the glutamate receptor antagonist kynurenic acid but disappeared when bicuculline was also present, indicating GABAA-R involvement. In immunocytochemical experiments, CB1-immunoreactive axons formed contacts with GnRH neurons and a subset established symmetric synapses characteristic of GABAergic neurotransmission. Functional studies were continued with whole-cell patch-clamp electrophysiology in presence of tetrodotoxin. WIN55,212 decreased the frequency of GABAA-R-mediated miniature postsynaptic currents (mPSCs) (reflecting spontaneous vesicle fusion), which was prevented with the CB1 antagonist AM251, indicating collectively that activation of presynaptic CB1 inhibits GABA release. AM251 alone increased mPSC frequency, providing evidence that endocannabinoids tonically inhibit GABAA-R drive onto GnRH neurons. Increased mPSC frequency was absent when diacylglycerol lipase was blocked intracellularly with tetrahydrolipstatin, showing that tonic inhibition is caused by 2-arachidonoylglycerol production of GnRH neurons. CdCl2 in extracellular solution can maintain both action potentials and spontaneous vesicle fusion. Under these conditions, when endocannabinoid-mediated blockade of spontaneous vesicle fusion was blocked with AM251, GnRH neuron firing increased, revealing an endogenous endocannabinoid brake on GnRH neuron firing. Retrograde endocannabinoid signaling may represent an important mechanism under physiological and pathological conditions whereby GnRH neurons regulate their excitatory GABAergic inputs.

scholarly journals Suppression of Basal Spontaneous Gonadotropin-Releasing Hormone Neuronal Activity during Lactation: Role of Inhibitory Effects of Neuropeptide Y

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 333-340 ◽  
Author(s):  
Jing Xu ◽  
Melissa A. Kirigiti ◽  
Michael A. Cowley ◽  
Kevin L. Grove ◽  
M. Susan Smith
Keyword(s):  
Neuropeptide Y ◽  
Neuronal Activity ◽  
Fluorescent Protein ◽  
Resting Membrane Potential ◽  
Inhibitory Effects ◽  
Gnrh Neurons ◽  
Cell Current ◽  
Green Fluorescent ◽  
Hypothalamic Slices

Increased neuropeptide Y (NPY) activity drives the chronic hyperphagia of lactation and may contribute to the suppression of GnRH activity. The majority of GnRH neurons are contacted by NPY fibers, and GnRH cells express NPY Y5 receptor (Y5R). Therefore, NPY provides a neurocircuitry for information about food intake/energy balance to be directly transmitted to GnRH neurons. To investigate the effects of lactation on GnRH neuronal activity, hypothalamic slices were prepared from green fluorescent protein-GnRH transgenic rats. Extracellular loose-patch recordings determined basal GnRH neuronal activity from slices of ovariectomized control and lactating rats. Compared with controls, hypothalamic slices from lactating rats had double the number of quiescent GnRH neurons (14.51 ± 2.86 vs. 7.04 ± 2.84%) and significantly lower firing rates of active GnRH neurons (0.25 ± 0.02 vs. 0.37 ± 0.03 Hz). To study the NPY-postsynaptic Y5R system, whole-cell current-clamp recordings were performed in hypothalamic slices from control rats to examine NPY/Y5R antagonist effects on GnRH neuronal resting membrane potential. Under tetrodotoxin treatment, NPY hyperpolarized GnRH neurons from −56.7 ± 1.94 to −62.1 ± 1.83 mV; NPY’s effects were blocked by Y5R antagonist. To determine whether increased endogenous NPY tone contributes to GnRH neuronal suppression during lactation, hypothalamic slices were treated with Y5R antagonist. A significantly greater percentage of GnRH cells were activated in slices from lactating rats (52%) compared with controls (28%). These results suggest that: 1) basal GnRH neuronal activity is suppressed during lactation; 2) NPY can hyperpolarize GnRH neurons via postsynaptic Y5R; and 3) increased inhibitory NPY tone during lactation is a component of the mechanisms responsible for suppression of GnRH neuronal activity. Neuropeptide Y (NPY) directly hyperpolarizes GnRH neurons via postsynaptic NPY Y5 receptor. Increased inhibitory NPY tone during lactation is an important component of the suppression of GnRH neuronal activity.

scholarly journals Small-Conductance Calcium-Activated Potassium Channels Control Excitability and Firing Dynamics in Gonadotropin-Releasing Hormone (GnRH) Neurons

Endocrinology ◽  
2008 ◽  
Vol 149 (7) ◽  
pp. 3598-3604 ◽  
Author(s):  
Xinhuai Liu ◽  
Allan E. Herbison
Keyword(s):  
Fluorescent Protein ◽  
Current Injection ◽  
Sk Channels ◽  
Cellular Mechanisms ◽  
Burst Frequency ◽  
Single Action ◽  
Sk Channel ◽  
Gnrh Neurons ◽  
Green Fluorescent ◽  
Small Conductance

The cellular mechanisms determining the firing patterns of GnRH neurons are presently under intense investigation. In this study, we used GnRH-green fluorescent protein transgenic mice and perforated-patch electrophysiology to examine the role of small conductance calcium-activated potassium (SK) channels in determining the electrical excitability and burst-firing characteristics of adult GnRH neurons. After establishing an appropriate protocol for examining the afterhyperpolarization potential (AHP) currents in GnRH neurons, the highly selective SK channel blocker apamin was used to demonstrate that all GnRH neurons express functional SK channels (35.7 ± 2.7 pA, mean decay time constant = 2167 msec, apamin IC50 = 9.6 nm) and that this channel underlies approximately 90% of the AHP in these cells. Current-clamp experiments showed that apamin-sensitive SK channels were tonically active in the majority (74%) of GnRH neurons, with apamin (100 nm) administration resulting in a mean 6.9 ± 0.5 mV membrane depolarization. Apamin also elevated the firing rate of GnRH neurons, including increased burst frequency and duration in spontaneously bursting cells as well as the ability of GnRH neurons to fire action potentials in response to current injection. In GnRH neurons activated by current injection, apamin significantly enhanced the amplitude of the afterdepolarization potential after a single action potential and eliminated spike frequency adaptation. Together, these studies show that apamin-sensitive SK channels play a key role in restraining GnRH neuron excitability. Through direct modulation of the AHP and indirect actions on the afterdepolarization potential, the SK channel exerts a powerful tonic influence upon the firing dynamics of GnRH neurons.

scholarly journals Dose-Dependent Switch in Response of Gonadotropin-Releasing Hormone (GnRH) Neurons to GnRH Mediated through the Type I GnRH Receptor

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 728-735 ◽  
Author(s):  
Chun Xu ◽  
Xu-Zhi Xu ◽  
Craig S. Nunemaker ◽  
Suzanne M. Moenter
Keyword(s):  
Firing Rate ◽  
Fluorescent Protein ◽  
Brain Slices ◽  
Dose Dependence ◽  
Gnrh Receptor ◽  
Neuron Activity ◽  
Type I ◽  
Pulsatile Release ◽  
Gnrh Neurons ◽  
Green Fluorescent

Abstract Pulsatile release of GnRH provides central control of reproduction. GnRH neuron activity is likely synchronized to produce hormone pulses, but the mechanisms are largely unknown. One candidate for communication among these neurons is GnRH itself. Cultured embryonic and immortalized GnRH neurons express GnRH receptor type I (GnRHR-1), but expression has not been shown in adult GnRH neurons. Using mice that express green fluorescent protein (GFP) in GnRH neurons, we tested whether adult GnRH neurons express GnRHR-1. GFP-positive (n = 42) and -negative neurons (n = 22) were harvested from brain slices, and single-cell RT-PCR was performed with cell contents. Fifty-two percent of the GnRH neurons tested expressed GnRHR-1, but only 9% of non-GnRH hypothalamic neurons expressed GnRHR-1; no false harvest controls (n = 13) were positive. GnRHR-1 expression within GnRH neurons suggested a physiological ultrashort loop feedback role for GnRH. Thus, we examined the effect of GnRH on the firing rate of GnRH neurons. Low-dose GnRH (20 nm) significantly decreased firing rate in 12 of 22 neurons (by 42 ± 4%, P < 0.05), whereas higher doses increased firing rate (200 nm, five of 10 neurons, 72 ± 26%; 2000 nm, nine of 13 neurons, 53 ± 8%). Interestingly, the fraction of GnRH neurons responding was similar to the fraction in which GnRHR-1 was detected. Together, these data demonstrate that a subpopulation of GnRH neurons express GnRHR-1 and respond to GnRH with altered firing. The dose dependence suggests that this autocrine control of GnRH neurons may be not only a mechanism for generating and modulating pulsatile release, but it may also be involved in the switch between pulse and surge modes of release.

scholarly journals Knockdown of GABAA Receptor Signaling in GnRH Neurons Has Minimal Effects upon Fertility

Endocrinology ◽  
2010 ◽  
Vol 151 (9) ◽  
pp. 4428-4436 ◽  
Author(s):  
Kiho Lee ◽  
Robert Porteous ◽  
Rebecca E. Campbell ◽  
Bernhard Lüscher ◽  
Allan E. Herbison
Keyword(s):  
Gabaa Receptor ◽  
Fluorescent Protein ◽  
Feedback Mechanism ◽  
Postnatal Life ◽  
Negative Feedback Mechanism ◽  
Postsynaptic Currents ◽  
Electrophysiological Recordings ◽  
Gnrh Neurons ◽  
Positive Feedback Mechanism ◽  
Green Fluorescent

The amino acid γ-aminobutyric acid (GABA) is thought to play a key role in shaping the activity of the GnRH neurons throughout embryonic and postnatal life. However, the physiological roles of direct GABA inputs to GnRH neurons remain unknown. Using a Cre-LoxP strategy, we generated a targeted mouse line, in which all (98 ± 1%) GnRH neurons had the γ2-subunit of the GABAA receptor deleted. Electrophysiological recordings of GABAA-mediated postsynaptic currents from green fluorescent protein-tagged GnRH neurons with the γ2-subunit knocked out (GnRH γ2 KO) showed that the amplitude and frequency of GABAA postsynaptic currents were reduced by 70% (P < 0.01) and 77% (P < 0.05), respectively, and that the response to exogenous GABA was reduced by 90% (P < 0.01). Evaluation of male and female GnRH γ2 KO mice revealed completely normal fecundity, estrous cycles, and puberty onset. Further investigation with gonadectomy and different steroid replacement regimens showed normal basal levels of LH in both sexes, and a normal estradiol-evoked positive feedback mechanism in females. However, the increment in LH after gonadectomy in GnRH γ2 KO female mice was double that of controls (P < 0.05) and also more potently suppressed by 17-β-estradiol (P < 0.05). A similar but nonsignificant trend was observed in GnRH γ2 KO male mice. Together, these findings show that 70–90% reductions in the normal levels of GABAA receptor activity at the GnRH neuron appear to impact upon the estrogen negative feedback mechanism but are, nevertheless, compatible with normal fertility in mice.

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