Effect of Steroid Milieu on Gonadotropin-Releasing Hormone-1 Neuron Firing Pattern and Luteinizing Hormone Levels in Male Mice

Department of Internal Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.
Biology of Reproduction (Impact Factor: 3.32). 06/2006; 74(5):931-7. DOI: 10.1095/biolreprod.105.049619
Source: PubMed


GnRH neuronal function is regulated by gonadal hormone feedback. In males, testosterone can act directly or be converted to either dihydrotestosterone (DHT) or estradiol (E2). We examined central steroid feedback by recording firing of green fluorescent protein (GFP)-identified GnRH neurons in brain slices from male mice that were intact, castrated, or castrated and treated with implants containing DHT, E2, or E2 + DHT. Castration increased LH levels. DHT or E2 alone partially suppressed LH, whereas E2 + DHT reduced LH to intact levels. Despite the inhibitory actions on LH, the combination of E2 + DHT increased GnRH neuron activity relative to other treatments, reflected in mean firing rate, amplitude of peaks in firing rate, and area under the curve of firing rate vs. time. Cluster8 was used to identify peaks in firing activity that may be correlated with hormone release. Castration increased the frequency of peaks in firing rate. Treatment with DHT failed to reduce frequency of these peaks. In contrast, treatment with E2 reduced peak frequency to intact levels. The frequency of peaks in firing rate was intermediate in animals treated with E2 + DHT, perhaps suggesting the activating effects of this combination partially counteracts the inhibitory actions of E2. These data indicate that E2 mediates central negative feedback in males primarily by affecting the pattern of GnRH neuron activity, and that androgens combined with estrogens have a central activating effect on GnRH neurons. The negative feedback induced by E2 + DHT to restore LH to intact levels may mask an excitatory central effect of this combination.

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    • "We next recorded from GFP-identified GnRH neurons in brain slices (Suter et al., 2000). Female OVX+E+P mice were used as this treatment produces low endogenous GnRH neuron activity (Pielecka et al., 2006). Because FSCV detects changes in concentration rather than absolute concentration, a low baseline was desired for initial tests. "
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    ABSTRACT: Pulsatile gonadotropin-releasing hormone (GnRH) release is critical for the central regulation of fertility. There is no method allowing real-time GnRH detection in brain slices. We developed fast-scan cyclic voltammetry (FSCV) using carbon-fiber microelectrodes (CFME) to detect GnRH release and validated it using a biologically relevant system. FSCV parameters (holding potential, switching potential, and scan rate) were determined for stable GnRH detection in vitro, then optimized for GnRH detection in mouse brain slices. Placement of CFMEs in the median eminence (ME) near GnRH terminals allowed detection of both KCl-evoked and spontaneous GnRH release. GnRH release was also detected from GnRH fibers passing near GnRH soma and near fiber-fiber appositions in the preoptic area. No GnRH signal was detected from CFMEs in the ME of hpg mice, which lack GnRH, or in regions not containing GnRH neurons in wild-type mice; application of exogenous GnRH produced a signal similar to that observed for spontaneous/evoked endogenous GnRH release. Using an established mouse model that produces diurnal variations in GnRH neuron activity, we demonstrated corresponding changes in spontaneous GnRH release in the median eminence. These results validate FSCV to detect GnRH in brain slices and provide new information on the sites and amounts of GnRH release, providing insight into its neuromodulatory functions.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2012; 32(42):14664-9. DOI:10.1523/JNEUROSCI.1303-12.2012 · 6.34 Impact Factor
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    • "In the present study castration, which increases GnRH release, markedly increased I h in GnRH neurons. In males there is substantial conversion within the brain of circulating testosterone from the gonads to estradiol (Woolley, 2007), which provides the primary negative feedback signal to reduce the GnRH-dependent secretion of gonadotropins from the pituitary, as well as the activity of GnRH neurons (Roselli and Resko, 1990; Scott et al., 1997; Fisher et al., 1998; Pielecka and Moenter, 2006). Treatment of castrated male mice with estradiol restored I h to levels observed in gonadal intact mice suggesting that estradiol-induced changes in I h are a component of this negative feedback mechanism. "
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    ABSTRACT: Pulsatile release of gonadotropin-releasing hormone (GnRH) is required for fertility and is regulated by steroid feedback. Hyperpolarization-activated currents (I(h)) play a critical role in many rhythmic neurons. We examined the contribution of I(h) to the membrane and firing properties of GnRH neurons and the modulation of this current by steroid milieu. Whole-cell voltage- and current-clamp recordings were made of GFP-identified GnRH neurons in brain slices from male mice that were gonad-intact, castrated, or castrated and treated with estradiol implants. APV, CNQX, and bicuculline were included to block fast synaptic transmission. GnRH neurons (47%) expressed a hyperpolarization-activated current with pharmacological and biophysical characteristics of I(h). The I(h)-specific blocker ZD7288 reduced hyperpolarization-induced sag and rebound potential, decreased GnRH neuron excitability and action potential firing, and hyperpolarized membrane potential in some cells. ZD7288 also altered the pattern of burst firing and reduced the slope of recovery from the after-hyperpolarization potential. Activation of I(h) by hyperpolarization increased spike frequency, whereas inactivation of I(h) by depolarization reduced spike frequency. Castration increased I(h) compared with that in gonad-intact males. This effect was reversed by in vivo estradiol replacement. Together, these data indicate I(h) provides an excitatory drive in GnRH neurons that contributes to action potential burst firing and that estradiol regulates I(h) in these cells. As estradiol is the primary central negative feedback hormone on GnRH neuron firing in males, this provides insight into the mechanisms by which steroid hormones potentially alter the intrinsic properties of GnRH neurons to change their activity.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2010; 30(40):13373-83. DOI:10.1523/JNEUROSCI.1687-10.2010 · 6.34 Impact Factor
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    • "Thus, GABA A receptor-mediated conductance changes, while depolarizing, may nonetheless shunt more distally generated active responses and result in diminished firing frequency recorded at the soma. While further studies are required to determine whether GABA depolarizes or hyperpolarizes GnRH neurons in AAS-treated subjects, the results presented here contrast with prior studies in gonadectomized male mice indicating coordinate enhancement of GABA A receptor-mediated PSC frequency (Chen and Moenter, 2009) and AP firing (Pielecka and Moenter, 2006) in GnRH neurons. These data further underscore that actions of supraphysiological levels of synthetic steroids may significantly diverge from effects imposed by castration or by replacement in castrates with physiological androgens or estrogens. "
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    ABSTRACT: Gonadotropin-releasing hormone (GnRH) neurons are the central regulators of reproduction. GABAergic transmission plays a critical role in pubertal activation of pulsatile GnRH secretion. Self-administration of excessive doses of anabolic androgenic steroids (AAS) disrupts reproductive function and may have critical repercussions for pubertal onset in adolescent users. Here, we demonstrate that chronic treatment of adolescent male mice with the AAS 17alpha-methyltestosterone significantly decreased action potential frequency in GnRH neurons, reduced the serum gonadotropin levels, and decreased testes mass. AAS treatment did not induce significant changes in GABAA receptor subunit mRNA levels or alter the amplitude or decay kinetics of GABAA receptor-mediated spontaneous postsynaptic currents (sPSCs) or tonic currents in GnRH neurons. However, AAS treatment significantly increased action potential frequency in neighboring medial preoptic area (mPOA) neurons and GABAA receptor-mediated sPSC frequency in GnRH neurons. In addition, physical isolation of the more lateral aspects of the mPOA from the medially localized GnRH neurons abrogated the AAS-induced increase in GABAA receptor-mediated sPSC frequency and the decrease in action potential firing in the GnRH cells. Our results indicate that AAS act predominantly on steroid-sensitive presynaptic neurons within the mPOA to impart significant increases in GABAA receptor-mediated inhibitory tone onto downstream GnRH neurons, resulting in diminished activity of these pivotal mediators of reproductive function. These AAS-induced changes in central GABAergic circuits of the forebrain may significantly contribute to the disruptive actions of these drugs on pubertal maturation and the development of reproductive competence in male steroid abusers.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 05/2010; 30(19):6497-506. DOI:10.1523/JNEUROSCI.5383-09.2010 · 6.34 Impact Factor
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