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Starving animals are less likely to defend their home territory and more likely to engage in risky foraging behaviors. This work describes a circuit involving hypothalamic AgRP neurons projecting to neurons in the medial nucleus of the amygdala and their projections to the bed nucleus of the stria terminalis, which, when activated, mimics these behaviors in mice that are well fed. In the face of starvation, animals will engage in high-risk behaviors that would normally be considered maladaptive. Starving rodents, for example, will forage in areas that are more susceptible to predators and will also modulate aggressive behavior within a territory of limited or depleted nutrients. The neural basis of these adaptive behaviors likely involves circuits that link innate feeding, aggression and fear. Hypothalamic agouti-related peptide (AgRP)-expressing neurons are critically important for driving feeding and project axons to brain regions implicated in aggression and fear. Using circuit-mapping techniques in mice, we define a disynaptic network originating from a subset of AgRP neurons that project to the medial nucleus of the amygdala and then to the principal bed nucleus of the stria terminalis, which suppresses territorial aggression and reduces contextual fear. We propose that AgRP neurons serve as a master switch capable of coordinating behavioral decisions relative to internal state and environmental cues.

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. Puberty and fertility are necessary for survival of the species. An evolutionarily ancient region of the brain called the hypothalamus regulates these processes. The hypothalamus releases a chemical messenger called gonadotropin-releasing hormone (or GnRH for short), which is then transported from the brain to the pituitary gland. GnRH activates the pituitary gland, which in turn releases reproductive hormones that control ovulation in females and sperm production in males. For this process to work correctly in females, the hypothalamus must release GnRH in appropriately timed pulses and produce one massive release, or “surge”, of GnRH to trigger ovulation. Two populations of neurons within the hypothalamus produce a peptide molecule called Kisspeptin and drive the activity and subsequent release of GnRH. One population resides in an area called the arcuate nucleus and the other in the preoptic nucleus. Recent findings suggest that the arcuate nucleus is the “pulse generator” responsible for triggering the rhythmic release of GnRH by the hypothalamus, whereas the preoptic nucleus induces the surge of GnRH. However, how these brain regions do this remains unclear. Using a technique called optogenetics, Qiu, Nestor et al. explored whether kisspeptin-producing neurons in the arcuate nucleus are able to communicate with each other to drive pulses of GnRH release. The idea was to selectively activate a subset of kisspeptin neurons in mice and determine whether this would activate the remaining neurons at the same time. Qiu, Nestor et al. introduced a light-sensitive protein into the kisspeptin-producing neurons on one side of the arcuate nucleus, and then used light to activate those neurons. As predicted, this caused kisspeptin neurons throughout the arcuate nucleus to coordinate their activity. In addition to their namesake peptide, kisspeptin-producing neurons also make the neurotransmitter glutamate, the excitatory peptide neurokinin B, and the inhibitory peptide dynorphin. Light-induced stimulation of the arcuate nucleus caused its kisspeptin neurons to also release neurokinin B and dynorphin, which synchronized the firing of the kisspeptin neurons. The hypothalamus then translates this coordinated activity into pulses of GnRH release. The light-induced stimulation also triggered the release of glutamate, which caused kisspeptin neurons within the preoptic nucleus to fire in bursts. This in turn robustly excited the GnRH neurons, giving rise to the surge of GnRH. These findings show that peptide and classical neurotransmitters collaborate to control GnRH neuron activity and, consequently, fertility. The results obtained by Qiu, Nestor et al. can be used to further explore kisspeptin-GnRH neuronal circuits, and to obtain insights into the role of neuronal peptide signaling in healthy as well as diseased states.

Anal squamous cell carcinoma (SCC) will be diagnosed in an estimated 9,080 adults in the United States this year, and rates have been rising over the last several decades. Most people that develop anal SCC have associated human papillomavirus (HPV) infection (~85–95%), with approximately 5–15% of anal SCC cases occurring in HPV-negative patients from unknown etiology. This study identified and characterized the Kras -driven, female sex hormone-dependent development of anal squamous cell carcinoma (SCC) in the LSL-Kras G12D ; Pdx1-Cre (KC) mouse model that is not dependent on papillomavirus infection. One hundred percent of female KC mice develop anal SCC, while no male KC mice develop tumors. Both male and female KC anal tissue express Pdx1 and Cre-recombinase mRNA, and the activated mutant Kras G12D gene. Although the driver gene mutation Kras G12D is present in anus of both sexes, only female KC mice develop Kras -mutant induced anal SCC. To understand the sex-dependent differences, KC male mice were castrated and KC female mice were ovariectomized. Castrated KC males displayed an unchanged phenotype with no anal tumor formation. In contrast, ovariectomized KC females demonstrated a marked reduction in anal SCC development, with only 15% developing anal SCC. Finally, exogenous administration of estrogen rescued the tumor development in ovariectomized KC female mice and induced tumor development in castrated KC males. These results confirm that the anal SCC is estrogen mediated. The delineation of the role of female sex hormones in mediating mutant Kras to drive anal SCC pathogenesis highlights a subtype of anal SCC that is independent of papillomavirus infection. These findings may have clinical applicability for the papillomavirus-negative subset of anal SCC patients that typically respond poorly to standard of care chemoradiation.

The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty in association with the decrease in MKRN3 expression in the medial basal hypothalamus of mice before the initiation of reproductive maturation suggests that MKRN3 is acting as a brake on gonadotropin-releasing hormone (GnRH) secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, decreases as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus and that MKRN3 repressed promoter activity of human KISS1 and TAC3, 2 key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve an MKRN3-directed ubiquitination-mediated mechanism.

Mammalian reproductive function depends upon a neuroendocrine circuit that evokes the pulsatile release of gonadotropin hormones (luteinizing hormone and follicle-stimulating hormone) from the pituitary. This reproductive circuit is sensitive to metabolic perturbations. When challenged with starvation, insufficient energy reserves attenuate gonadotropin release, leading to infertility. The reproductive neuroendocrine circuit is well established, composed of two populations of kisspeptin-expressing neurons (located in the anteroventral periventricular hypothalamus, Kiss1AVPV, and arcuate hypothalamus, Kiss1ARH), which drive the pulsatile activity of gonadotropin-releasing hormone (GnRH) neurons. The reproductive axis is primarily regulated by gonadal steroid and circadian cues, but the starvation-sensitive input that inhibits this circuit during negative energy balance remains controversial. Agouti-related peptide (AgRP)-expressing neurons are activated during starvation and have been implicated in leptin-associated infertility. To test whether these neurons relay information to the reproductive circuit, we used AgRP-neuron ablation and optogenetics to explore connectivity in acute slice preparations. Stimulation of AgRP fibers revealed direct, inhibitory synaptic connections with Kiss1ARH and Kiss1AVPV neurons. In agreement with this finding, Kiss1ARH neurons received less presynaptic inhibition in the absence of AgRP neurons (neonatal toxin-induced ablation). To determine whether enhancing the activity of AgRP neurons is sufficient to attenuate fertility in vivo, we artificially activated them over a sustained period and monitored fertility. Chemogenetic activation with clozapine N-oxide resulted in delayed estrous cycles and decreased fertility. These findings are consistent with the idea that, during metabolic deficiency, AgRP signaling contributes to infertility by inhibiting Kiss1 neurons.

In this study, the authors show that an epigenetic program, operating in the hypothalamus, can regulate the timing of female puberty. They find that increased promoter methylation of two Polycomb group family members reduces their expression at the onset of puberty, allowing expression of Kiss1. 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 principal 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 , decreased and methylation of their promoters increased before puberty. Inhibiting DNA methylation blocked both events and resulted in pubertal failure. The pubertal increase in Kiss1 expression was 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 disrupted pulsatile gonadotropin-releasing hormone release, delayed puberty and compromised fecundity. Our results identify epigenetic silencing as a mechanism underlying the neuroendocrine control of female puberty.

The neuropeptides tachykinin2 (Tac2) and kisspeptin (Kiss1) in hypothalamic arcuate nucleus Kiss1 (Kiss1ARH) neurons are essential for pulsatile release of GnRH and reproduction. Since 17β-estradiol (E2) decreases Kiss1 and Tac2 mRNA expression in Kiss1ARH neurons, the role of Kiss1ARH neurons during E2-driven anorexigenic states and their coordination of POMC and NPY/AgRP feeding circuits have been largely ignored. Presently, we show that E2 augmented the excitability of Kiss1ARH neurons by amplifying Cacna1g, Hcn1 and Hcn2 mRNA expression and T-type calcium and h-currents. E2 increased Slc17a6 mRNA expression and glutamatergic synaptic input to arcuate neurons, which excited POMC and inhibited NPY/AgRP neurons via metabotropic receptors. Deleting Slc17a6 in Kiss1 neurons eliminated glutamate release and led to conditioned place preference for sucrose in E2-treated KO female mice. Therefore, the E2-driven increase in Kiss1 neuronal excitability and glutamate neurotransmission may play a key role in governing the motivational drive for palatable food in females.

Inactivating mutations in the melanocortin 4 receptor ( MC4R ) gene cause monogenic obesity. Interestingly, female patients also display various degrees of reproductive disorders, in line with the subfertile phenotype of Mc4r KO female mice. However, the cellular mechanisms by which MC4R regulates reproduction are unknown. Kiss1 neurons directly stimulate gonadotropin-releasing hormone (GnRH) release through two distinct populations: the Kiss1 ARH neurons, controlling GnRH pulses, and the sexually dimorphic Kiss1 AVPV/PeN neurons controlling the preovulatory luteinizing hormone (LH) surge. Here, we show that Mc4r expressed in Kiss1 neurons regulates fertility in females. In vivo, deletion of Mc4r from Kiss1 neurons in female mice replicates the reproductive impairments of Mc4r KO mice without inducing obesity. Conversely, re-insertion of Mc4r in Kiss1 neurons of Mc4r null mice restores estrous cyclicity and LH pulsatility without reducing their obese phenotype. In vitro, we dissect the specific action of Mc4r on Kiss1 ARH versus Kiss1 AVPV/PeN neurons and show that Mc4r activation excites Kiss1 ARH neurons through direct synaptic actions. In contrast, Kiss1 AVPV/PeN neurons are normally inhibited by MC4R activation except under elevated estradiol levels, thus facilitating the activation of Kiss1 AVPV/PeN neurons to induce the LH surge driving ovulation in females. Our findings demonstrate that POMC ARH neurons acting through MC4R directly regulate reproductive function in females by stimulating the ‘pulse generator’ activity of Kiss1 ARH neurons and restricting the activation of Kiss1 AVPV/PeN neurons to the time of the estradiol-dependent LH surge, and thus unveil a novel pathway of the metabolic regulation of fertility by the melanocortin system.

Hypothalamic kisspeptin (Kiss1) neurons are vital for pubertal development and reproduction. Arcuate nucleus Kiss1 (Kiss1 ARH ) neurons are responsible for the pulsatile release of gonadotropin-releasing hormone (GnRH). In females, the behavior of Kiss1 ARH neurons, expressing Kiss1, neurokinin B (NKB), and dynorphin (Dyn), varies throughout the ovarian cycle. Studies indicate that 17β-estradiol (E2) reduces peptide expression but increases Slc17a6 ( Vglut2 ) mRNA and glutamate neurotransmission in these neurons, suggesting a shift from peptidergic to glutamatergic signaling. To investigate this shift, we combined transcriptomics, electrophysiology, and mathematical modeling. Our results demonstrate that E2 treatment upregulates the mRNA expression of voltage-activated calcium channels, elevating the whole-cell calcium current that contributes to high-frequency burst firing. Additionally, E2 treatment decreased the mRNA levels of canonical transient receptor potential (TPRC) 5 and G protein-coupled K + (GIRK) channels. When Trpc5 channels in Kiss1 ARH neurons were deleted using CRISPR/SaCas9, the slow excitatory postsynaptic potential was eliminated. Our data enabled us to formulate a biophysically realistic mathematical model of Kiss1 ARH neurons, suggesting that E2 modifies ionic conductances in these neurons, enabling the transition from high-frequency synchronous firing through NKB-driven activation of TRPC5 channels to a short bursting mode facilitating glutamate release. In a low E2 milieu, synchronous firing of Kiss1 ARH neurons drives pulsatile release of GnRH, while the transition to burst firing with high, preovulatory levels of E2 would facilitate the GnRH surge through its glutamatergic synaptic connection to preoptic Kiss1 neurons.

Kisspeptin is a neuropeptide that signals via a Gαq-coupled receptor, GPR54, in gonadotropin-releasing hormone (GnRH) neurons and is essential for pubertal maturation and fertility. Kisspeptin depolarizes and excites GnRH neurons primarily through the activation of canonical transient receptor potential (TRPC) channels and the inhibition of K + channels. The gonadal steroid 17β-estradiol (E 2 ) upregulates not only kisspeptin (Kiss1) mRNA but also increases the excitability of the rostral forebrain Kiss1 neurons. In addition, a primary postsynaptic action of E 2 on GnRH neurons is to upregulate the expression of channel transcripts that orchestrate the downstream signaling of kisspeptin in GnRH neurons. These include not only TRPC4 channels but also low-voltage-activated T-type calcium channels and high-voltage-activated L-, N- and R-type calcium channel transcripts. Moreover, E 2 has direct membrane-initiated actions to alter the excitability of GnRH neurons by enhancing ATP-sensitive potassium channel activity, which is critical for maintaining GnRH neurons in a hyperpolarized state for the recruitment of T-type calcium channels that are important for burst firing. Therefore, E 2 modulates the excitability of GnRH neurons as well as of Kiss1 neurons by altering the expression and/or function of ion channels; moreover, kisspeptin provides critical excitatory input to GnRH neurons to facilitate burst firing activity and peptide release.