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Abstract
Background
The gonadotropin releasing hormone (GnRH) neurons play a pivotal role in the central regulation of fertility. Kisspeptin binds to the G-protein coupled receptor 54 (GPR54) and GPR54 has been shown to be essential for puberty and subsequent fertility in humans. The recent in vivo studies have proved that kisspeptin is an extremely potent activator of GnRH neurons. However, the precise mechanism of action of kisspeptin on the GnRH neurons and the age-dependent kisspeptin effects are not yet fully understood. In this study, we investigated the effects of kisspeptin on the GnRH neurons over the developmental stages in male and female mice.
Methods
Young (< P30) and adult (> P35) GnRH-GFP transgenic mice expressing green fluorescent protein were used in this study. Acute coronal brain slices containing the preoptic area were prepared, and the identified GnRH neurons were recorded using the gramicidin perforated-patch clamp technique.
Results
In young mice, GnRH neurons were excited by bath application of kisspeptin in 36% (13/36) in male, 17% (4/23) in female tested neurons. In adult mice, GnRH neurons were excited in the majority (30/40, 75%) in male, (21/31, 68%) in female neurons tested. However, there was no significant difference between the effects of kisspeptin in male and female mice. In addition, we tested kisspeptin effects in diestrus, proestrus and estrus animals. There were no significant differences of kisspeptin effects over the estrous cycle. Kisspeptin failed to induce excitatory effects on GnRH neurons (6/7, 86%) neurons) by pretreatment of U73122, a protein lipase C (PLC) inhibitor and kisspeptin-induced excitatory effects were decreased by U73122 application (n = 2).
Figures and Tables
Fig. 1
Minority of young GnRH neurons get affected by kisspeptin. Gramicidin perforated patch recording of a young male GnRH neuron showing no response (A), depolarization with increase of spontaneous firing (B) and hyperpolarization (C) by 30 nM kisspeptin. D, Spontaneously active young female GnRH neuron responding to kisspeptin with transient depolarization accompanied by action potential firing. Bars represent the duration of kisspeptin application
Fig. 2
Majority of adult GnRH neurons are activated by kisspeptin. A, An adult male GnRH neuron depolarized by kisspeptin for > 40 minutes, exhibiting frequent burst of action potential. B, Representative voltage trace of an adult male GnRH neuron showing kisspeptin-induced potent increase in action potential firing. without membrane depolarization. C, A trace from adult female (proestrus) GnRH neuron exhibiting a membrane depolarization accompanied by intense firing of action potential in response to kisspeptin. Bars represent the duration of kisspeptin application.
Fig. 3
Effect of kisspeptin over postnatal development. Scattered plots showing the individual membrane potential changes from all GnRH neurons recorded. Male (open circles), female (filled circles).
Fig. 4
Bar graph showing developmental differences in the ability of kisspeptin to elicit GnRH neuronal excitability. Comparison of the stimulatory response to kisspeptin between young (< P30) and adult (> P35) male and female mice. NS: not significant, ** represents P < 0.01 (χ2-test).
Fig. 5
Comparison of the stimulatory effects of kisspeptin on GnRH neurons over estrous cycle. A, Percentage responses to kisspeptin among adult male (M) and adult female over diestrus (D), proestrus (P) and estrus (E). B, Comparison of kisspeptin-evoked mean membrane potential changes among adult male and female over estrous cyclicity. Error bars represent SEM.
Fig. 6
Intracellular signaling cascade involved in mediating kisspeptin-induced stimulatory effects. A, An adult male GnRH neuron depolarized by kisspeptin for > 40 minutes, exhibiting frequent burst of action potential. B, Representative voltage trace from an adult male GnRH neuron showing no response to 30 nM kisspeptin following pretreatment with 30 µM U73122 (phospholipase C inhibitor). C, A bar graph showing mean (± SEM) response of GnRH neurons to applied kisspeptin versus kisspeptin in the presence of U73122. D, Voltage trace of male representing inhibition of kisspeptin-induced action potential firing with slight hyperpolarization effect by U73122 on prolonged depolarization induced by kisspeptin.
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