Classically, 17beta-estradiol (E2) is thought to control homeostatic functions such as reproduction, stress responses, feeding, sleep cycles, temperature regulation, and motivated behaviors through transcriptional events. Although it is increasingly evident that E2 can also rapidly activate kinase pathways to have multiple downstream actions in CNS neurons, the receptor(s) and the signal transduction pathways involved have not been identified. We discovered that E2 can alter mu-opioid and GABA neurotransmission rapidly through nontranscriptional events in hypothalamic GABA, proopiomelanocortin (POMC), and dopamine neurons. Therefore, we examined the effects of E2 in these neurons using whole-cell recording techniques in ovariectomized female guinea pigs. E2 reduced rapidly the potency of the GABAB receptor agonist baclofen to activate G-protein-coupled, inwardly rectifying K+ channels in hypothalamic neurons. These effects were mimicked by the membrane impermeant E2-BSA and selective estrogen receptor modulators, including a new diphenylacrylamide compound, STX, that does not bind to intracellular estrogen receptors alpha or beta, suggesting that E2 acts through a unique membrane receptor. We characterized the coupling of this estrogen receptor to a Galpha(q)-mediated activation of phospholipase C, leading to the upregulation of protein kinase Cdelta and protein kinase A activity in these neurons. Moreover, using single-cell reverse transcription-PCR, we identified the critical transcripts, PKCdelta and its downstream target adenylyl cyclase VII, for rapid, novel signaling of E2 in GABA, POMC, and dopamine neurons. Therefore, this unique Gq-coupled estrogen receptor may be involved in rapid signaling in hypothalamic neurons that are critical for normal homeostatic functions.

The timing of puberty is controlled by many genes. The elements coordinating this process have not, however, been identified. Here we show that an epigenetic mechanism of transcriptional repression times the initiation of female puberty in rats. We identify silencers of the Polycomb group (PcG) as major contributors to this mechanism, and show that PcG proteins repress Kiss1, a puberty-activating gene. Hypothalamic expression of two key PcG genes, Eed and Cbx7, decreases and methylation of their promoters increases preceding puberty. Inhibiting DNA methylation blocks both events and results in pubertal failure. The pubertal increase in Kiss1 is accompanied by EED loss from the Kiss1 promoter and enrichment of histone H3 modifications associated with gene activation. Preventing the eviction of EED from the Kiss1 promoter disrupts pulsatile GnRH release, delays puberty, and compromises fecundity. Our results identify epigenetic silencing as a novel mechanism underlying the neuroendocrine control of female puberty.

Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

Hypothalamic proopiomelanocortin (POMC) neurons are critical for controlling homeostatic functions in the mammal. We used a transgenic mouse model in which the POMC neurons were labeled with enhanced green fluorescent protein to perform visualized, whole-cell patch recordings from prepubertal female hypothalamic slices. The mouse POMC-enhanced green fluorescent protein neurons expressed the same endogenous conductances (a transient outward K(+) current and a hyperpolarization-activated, cation current) that have been described for guinea pig POMC neurons. In addition, the selective micro -opioid receptor agonist DAMGO induced an outward current (maximum of 12.8 +/- 1.2 pA), which reversed at K(+) equilibrium potential (E(K+)), in the majority (85%) of POMC neurons with an EC(50) of 102 nM. This response was blocked by the opioid receptor antagonist naloxone with an inhibition constant of 3.1 nM. In addition, the gamma-aminobutyric acid(B) receptor agonist baclofen (40 micro M) caused an outward current (21.6 +/- 4.0 pA) that reversed at E(K+) in these same neurons. The ATP-sensitive potassium channel opener diazoxide also induced an outward K(+) current (maximum of 18.7 +/- 2.2 pA) in the majority (92%) of POMC neurons with an EC(50) of 61 micro M. The response to diazoxide was blocked by the sulfonylurea tolbutamide, indicating that the POMC neurons express both Kir6.2 and sulfonylurea receptor 1 channel subunits, which was verified using single cell RT-PCR. This pharmacological and molecular profile suggested that POMC neurons might be sensitive to metabolic inhibition, and indeed, we found that their firing rate varied with changes in glucose concentrations. Therefore, it appears that POMC neurons may function as an integrator of metabolic cues and synaptic input for controlling homeostasis in the mammal.

Leptin can exert its potent appetite-suppressing effects via activation of hypothalamic proopiomelanocortin (POMC) neurons. It depolarizes POMC neurons via activation of a yet unidentified nonselective cation current. Therefore, we sought to identify the conductance activated by leptin using whole-cell recording in EGFP-POMC neurons from transgenic mice. The TRPC channel blockers SKF96365 (1-[␤-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), flufenamic acid, and 2-APB (2-aminoethyl diphenylborinate) potently inhibited the leptin-induced current. Also, lanthanum (La 3ϩ ) and intracellular Ca 2ϩ potentiated the effects of leptin. Moreover, the diacylglycerol-permeable analog OAG (2-acetyl-1-oleoyl-sn-glycerol) failed to activate any TRPC current. Using a Cs ϩ -gluconate-based internal solution, the leptin-activated current reversed near Ϫ20 mV. After replacement of external Na ϩ and K ϩ with Cs ϩ , the reversal shifted to near 0 mV, and the I/V curve exhibited a negative slope conductance at voltages more negative than Ϫ40 mV. Based on scRT-PCR, TRPC1 and TRPC4 -7 mRNA were expressed in POMC neurons, with TRPC5 being the most prevalent. The leptin-induced current was blocked by the Jak2 inhibitor AG490, the PI3 kinase inhibitor wortmannin, and the phospholipase C inhibitors, U73122 and ET-18-OCH3. Notably, we identified PLC␥1 transcripts in the majority of POMC neurons. Therefore, leptin through a Jak2-PI3 kinase-PLC␥ pathway activates TRPC channels, and TRPC1, 4, and 5 appear to be the key channels mediating the depolarizing effects of leptin in POMC neurons.

Kisspeptin and its cognate receptor, GPR54, are critical for reproductive development and for the regulation of gonadotropin-releasing hormone (GnRH) secretion. Although kisspeptin has been found to depolarize GnRH neurons, the underlying ionic mechanism has not been elucidated. Presently, we found that kisspeptin depolarized GnRH neurons in a concentration-dependent manner with a maximum depolarization of 22.6 Ϯ 0.6 mV and EC 50 of 2.8 Ϯ 0.2 nM. Under voltage-clamp conditions, kisspeptin induced an inward current of 18.2 Ϯ 1.6 pA (V hold ϭ Ϫ60 mV) that reversed near Ϫ115 mV in GnRH neurons. The more negative reversal potential than E K ϩ (Ϫ90 mV) was caused by the concurrent inhibition of barium-sensitive, inwardly rectifying (Kir) potassium channels and activation of sodiumdependent, nonselective cationic channels (NSCCs). Indeed, reducing extracellular Na ϩ (to 5 mM) essentially eliminated the kisspeptininduced inward current. The current-voltage relationships of the kisspeptin-activated NSCC currents exhibited double rectification with negative slope conductance below Ϫ40 mV in the majority of the cells. Pharmacological examination showed that the kisspeptin-induced inward currents were blocked by TRPC (canonical transient receptor potential) channel blockers 2-APB (2-aminoethyl diphenylborinate), flufenamic acid, SKF96365 (1-[␤-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), and Cd 2ϩ, but not by lanthanum (100 M). Furthermore, single-cell reverse transcription-PCR analysis revealed that TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 subunits were expressed in GnRH neurons. Therefore, it appears that kisspeptin depolarizes GnRH neurons through activating TRPC-like channels and, to a lesser extent, inhibition of Kir channels. These actions of kisspeptin contribute to the pronounced excitation of GnRH neurons that is critical for mammalian reproduction.

Kisspeptin (Kiss1) and neurokinin B (NKB) neurocircuits are essential for pubertal development and fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (Kiss1ARH) co-express Kiss1, NKB, dynorphin and glutamate and are postulated to provide an episodic, excitatory drive to gonadotropin-releasing hormone 1 (GnRH) neurons, the synaptic mechanisms of which are unknown. We characterized the cellular basis for synchronized Kiss1ARH neuronal activity using optogenetics, whole-cell electrophysiology, molecular pharmacology and single cell RT-PCR in mice. High-frequency photostimulation of Kiss1ARH neurons evoked local release of excitatory (NKB) and inhibitory (dynorphin) neuropeptides, which were found to synchronize the Kiss1ARH neuronal firing. The light-evoked synchronous activity caused robust excitation of GnRH neurons by a synaptic mechanism that also involved glutamatergic input to preoptic Kiss1 neurons from Kiss1ARH neurons. We propose that Kiss1ARH neurons play a dual role of driving episodic secretion of GnRH through the differential release of peptide and amino acid neurotransmitters to coordinate reproductive function.DOI: http://dx.doi.org/10.7554/eLife.16246.001

SUMMARY Proopiomelanocortin (POMC) neurons within the hypothalamic arcuate nucleus are vital anorexigenic neurons. Although both the leptin receptor and insulin receptor are coupled to activation of phosphatidylinositide3-kinase (PI3K) in POMC neurons, they are thought to have disparate actions on POMC excitability. Using whole-cell recording and selective pharmacological tools, we have found that similar to leptin, purified insulin depolarized POMC, and adjacent kisspeptin neurons via activation of TRPC5 channels, which are highly expressed in these neurons. In contrast, insulin hyperpolarized and inhibited NPY/AgRP neurons via activation of KATP channels. Moreover, Zn2+, which is found in insulin formulations at nanomolar concentrations, inhibited POMC neurons via activation of KATP channels. Finally as predicted, insulin given intracerebroventrically robustly inhibited food intake and activated c-fos expression in arcuate POMC neurons. Our results show that purified insulin excites POMC neurons in the arcuate nucleus, which we propose is a major mechanism by which insulin regulates energy homeostasis.