mRNA expression of ion channels in GnRH neurons: Subtype-specific regulation by 17β-estradiol

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Abstract

Burst firing of neurons optimizes neurotransmitter release. GnRH neurons exhibit burst firing activity and T-type calcium channels, which are vital for burst firing activity, are regulated by 17β-estradiol (E2) in GnRH neurons. To further elucidate ion channel expression and E2 regulation during positive and negative feedback on GnRH neurosecretion, we used single cell RT-PCR and real-time qPCR to quantify channel mRNA expression in GnRH neurons. GFP-GnRH neurons expressed numerous ion channels important for burst firing activity. E2-treatment sufficient to induce an LH surge increased mRNA expression of HCN1 channels, which underlie the pacemaker current, the calcium-permeable CaV1.3, CaV2.2, CaV2.3 channels, and TRPC4 channels, which mediate the kisspeptin excitatory response. E2 also decreased mRNA expression of SK3 channels underlying the medium AHP current. Therefore, E2 exerts fundamental changes in ion channel expression in GnRH neurons, to prime them to respond to incoming stimuli with increased excitability at the time of the surge.

Highlights

► Single-cell quantitative PCR of channel transcripts in GnRH neurons was validated and used. ► E2 increased or decreased the mRNA expression of ion channels in GnRH neurons. ► Altered channel mRNA expression leads to changes in the excitability of GnRH neurons.

Introduction

As demonstrated in a number of species including the rat, sheep and rhesus monkey, the preovulatory luteinizing hormone (LH) surge is accompanied by a surge in gonadotropin-releasing hormone (GnRH) (Caraty et al., 1989, Chappell and Levine, 2000, Levine and Ramirez, 1982, Pau et al., 1993), suggesting increased activity of GnRH neurons at the time of the GnRH surge. This increased activity is primarily due to increased estradiol levels, because treatment with 17β-estradiol (E2) in ovariectomized (OVX) females can mimic the positive feedback regulation of GnRH and LH secretion (Caraty et al., 1989, Chappell and Levine, 2000). Cell-attached single-unit extracellular recording to evaluate GnRH neuronal firing activity during negative as compared to positive feedback has revealed that the GnRH neuronal firing rate is low during the morning negative feedback period and significantly higher during the evening positive feedback period (Christian et al., 2005). Collectively, these findings would indicate that there are fundamental changes in GnRH neuronal firing activity during the different stages of the ovulatory cycle and during E2-induced negative and positive feedback. The timing of the GnRH (LH) surge and thus the increased activity of GnRH neurons at the time of the surge is E2-dependent, but it is also entrained to a circadian input from the suprachiasmatic nucleus (SCN) at least in some rodent species (Chappell et al., 2009, Christian et al., 2005, Christian and Moenter, 2008, Legan et al., 1975). The mechanism by which the SCN affects GnRH neuronal activity is not known, but circadian cues may involve vasopressin input to kisspeptin neurons and vasoactive intestinal peptide input to GnRH neurons (Christian and Moenter, 2008, Vida et al., 2010, Ward et al., 2009).

In addition to GnRH neurons, kisspeptin neurons are essential for reproductive development and reproductive competence (Oakley et al., 2009). These hypothalamic neurons express ERα, are affected by E2 feedback, and are strongly excitatory to GnRH neurons (Oakley et al., 2009). Kisspeptin excites GnRH neurons by actions on the G protein-coupled receptor 54 (GPR54), also called kisspeptin receptor (Zhang et al., 2008). Evidence from GT1–7 GnRH neuronal cells suggests that GPR54 exhibits an E2-dependent diurnal variation in mRNA expression (Tonsfeldt et al., 2011). These findings, however, have not been confirmed in native GnRH neurons.

Based on a model similar to that described and validated for thalamocortical relay neurons and hypothalamic neurosecretory neurons (Chemin et al., 2002, Erickson et al., 1993b, Kelly and Rønnekleiv, 1994, Kim et al., 2001), we had predicted that T-type calcium channels together with the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) are essential for induction of burst firing in GnRH neurons, and that the calcium-dependent, small-conductance calcium-activated potassium channels (SK) -type channels, which underlie afterhyperpolarization (AHP) are crucial for allowing repetitive cycles of burst firing (Kelly and Rønnekleiv, 1994, Kelly and Wagner, 2002). All of these channels are active in GnRH neurons and contribute significantly to their signaling pattern (Bosch et al., 2002, Chu et al., 2009, Chu et al., 2010, Kato et al., 2006, Lee et al., 2010, Liu and Herbison, 2008, Spergel, 2007, Zhang et al., 2007, Zhang et al., 2009). Additional channels important for GnRH neuronal firing include canonical transient receptor potential (TRPC) channels, which are activated by kisspeptin, and high voltage activated (HVA) calcium channels, which are important for calcium homeostasis and peptide release (Sun et al., 2010, Zhang et al., 2008).

While previous studies have demonstrated that E2 regulates the expression and/or function of a number of channels in GnRH neurons including T-type and L-type calcium channels (Sun et al., 2010, Zhang et al., 2009), little is known about the channel subtype expression and the E2 and diurnal regulation of the majority of ion channels in GnRH neurons. To begin to understand the E2-induced changes in GnRH neurons, we have explored the mRNA expression of HCN, TRPC, SK, and HVA calcium channels in the morning (negative feedback) and the expression in the evening (positive feedback) in oil- and E2-treated females. Indeed, we have found an E2-induced increased mRNA expression of HCN1, TRPC4, CaV1.3 (L), CaV2.2 (N) and CaV2.3 (R)-type calcium channels in GnRH neurons. In contrast, SK3 mRNA was decreased in GnRH neurons, whereas GPR54 mRNA was not altered at any time-points. These findings indicate that the rising E2-levels exert specific fundamental changes in ion channels expression in GnRH neurons, to prime these neurons for altered responsiveness to incoming stimuli leading to changes in excitability in an E2-dependent manner.

Section snippets

Animals

Adult female CBB6 mice (GnRH-GFP) (Suter et al., 2000) were maintained under constant temperature and lights. Two different lighting cycles were used, where lights were on between 0600 h (zeitgeber time (ZT) 0) and 1800 h (ZT 12) or where lights were on between 0200 h (ZT 0) and 1400 h (ZT 12) local time. The breeders and most of the research animals were kept permanently under reversed lighting schedule, and these animals were used for the majority of evening experiments with some exceptions as

E2-induced LH release

OVX animals were injected with a low priming dose followed by a LH surge-inducing dose of E2. With this procedure, we consistently induced a LH surge in CBB6 GnRH mice (Fig. 1). The timing of the surge was similar to that reported previously in mice kept under 12 h light and 12 h dark conditions (Gee et al., 1984, Wintermantel et al., 2006). All animals had low serum LH levels in the morning at ZT 4–5 (n = 6) following E2-treatment. The mean serum LH levels were slightly, but significantly elevated

Discussion

Presently we evaluated the mRNA expression and dominant subunits of numerous ion channels important for burst firing and transmitter (neuropeptide) release, including HCN, HVA, TRPC and SK channels, as well as their expression during E2 negative and positive feedback regulation of LH secretion. The salient findings were the following: (1) both HCN1 and HCN2 channel mRNAs were highly expressed in mouse GnRH neurons, but only the mRNA expression of HCN1 was regulated by E2. (2) The majority of

Acknowledgements

Research reported in this publication was supported by National Institutes of Health Grant NS 43330. K.J.T. was supported by the Reproductive Biology Training Grant NIH T32HD007133 and Steinberg Funds from OHSU Department of Physiology and Pharmacology. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

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