POMC neurons control fertility through differential signaling of MC4R in Kisspeptin neurons.

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 MC4RKO 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 Kiss1ARH neurons, controlling GnRH pulses, and the sexually dimorphic Kiss1AVPV/PeN neurons controlling the preovulatory LH surge. Here, we show that Mc4r expressed in Kiss1 neurons is required for fertility in females. In vivo, deletion of Mc4r from Kiss1 neurons in female mice replicates the reproductive impairments of MC4RKO mice without inducing obesity. Conversely, reinsertion 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 Kiss1ARH vs Kiss1AVPV/PeN neurons and show that MC4R activation excites Kiss1ARH neurons through direct synaptic actions. In contrast, Kiss1AVPV/PeN neurons are normally inhibited by MC4R activation except under elevated estradiol levels, thus facilitating the activation of Kiss1AVPV/PeN neurons to induce the LH surge driving ovulation in females. Our findings demonstrate that POMCARH neurons acting through MC4R, directly regulate reproductive function in females by stimulating the "pulse generator" activity of Kiss1ARH neurons and restricting the activation of Kiss1AVPV/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.

Estradiol elicits distinct firing patterns in arcuate nucleus kisspeptin neurons of females through altering ion channel conductances.

Hypothalamic kisspeptin (Kiss1) neurons are vital for pubertal development and reproduction. Arcuate nucleus Kiss1 (Kiss1ARH) neurons are responsible for the pulsatile release of Gonadotropin-releasing Hormone (GnRH). In females, the behavior of Kiss1ARH neurons, expressing Kiss1, Neurokinin B (NKB), and Dynorphin (Dyn), varies throughout the ovarian cycle. Studies indicate that 17ß-estradiol (E2) reduces peptide expression but increases 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 and that contribute 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 Kiss1ARH neurons were deleted using CRISPR, the slow excitatory postsynaptic potential (sEPSP) was eliminated. Our data enabled us to formulate a biophysically realistic mathematical model of the Kiss1ARH neuron, suggesting that E2 modifies ionic conductances in Kiss1ARH 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 Kiss1ARH 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.

CRISPR/SaCas9 mutagenesis of stromal interaction molecule 1 in proopiomelanocortin neurons increases glutamatergic excitability and protects against diet-induced obesity.

OBJECTIVE: Proopiomelanocortin (POMC) neurons are the key anorexigenic hypothalamic neuron for integrating metabolic cues to generate the appropriate output for maintaining energy homeostasis and express the requisite channels as a perfect synaptic integrator in this role. Similar to the metabolic hormones leptin and insulin, glutamate also excites POMC neurons via group I metabotropic glutamate receptors (mGluR1 and 5, mGluR1/5) that activate Transient Receptor Potential Canonical (TRPC 5) Channels to cause depolarization. A key modulator of TRPC 5 channel activity is stromal interaction molecule 1 (STIM1), which is involved in recruitment of TRPC 5 channels from receptor-operated to store-operated calcium entry following depletion of calcium from the endoplasmic reticulum. METHODS: We used a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas) and a single guide RNA (sgRNA) to mutate Stim1 in POMCCre neurons in male mice, verified by qPCR of Stim1 mRNA expression in single POMC neurons. Whole-cell patch clamp experiments were conducted to validate the effects of Stim1 mutagenesis. Body weight and food intake were measured in male mice to assess disruptions in energy balance. RESULTS: Reduced Stim1 expression augmented the efficacy of the mGluR1/5 agonist 3, 5-Dihydroxyphenylglycine (DHPG) to depolarize POMC neurons via a Gαq-coupled signaling pathway, which is an essential part of excitatory glutamatergic input in regulating energy homeostasis. The TRPC 5 channel blockers HC070 and Pico145 antagonized the excitatory effects of DHPG. As proof of principle, mutagenesis of Stim1 in POMC neurons reduced food intake, attenuated weight gain, reduced body fat and fat pad mass in mice fed a high fat diet. CONCLUSIONS: Using CRISPR technology we have uncovered a critical role of STIM1 in modulating glutamatergic activation of TRPC 5 channels in POMC neurons, which ultimately is important for maintaining energy balance.

Hypothalamic Kisspeptin Neurons and the Control of Homeostasis.

Hypothalamic kisspeptin (Kiss1) neurons provide indispensable excitatory transmission to gonadotropin-releasing hormone (GnRH) neurons for the coordinated release of gonadotropins, estrous cyclicity, and ovulation. But maintaining reproductive functions is metabolically demanding so there must be a coordination with multiple homeostatic functions, and it is apparent that Kiss1 neurons play that role. There are 2 distinct populations of hypothalamic Kiss1 neurons, namely arcuate nucleus (Kiss1ARH) neurons and anteroventral periventricular and periventricular nucleus (Kiss1AVPV/PeN) neurons in rodents, both of which excite GnRH neurons via kisspeptin release but are differentially regulated by ovarian steroids. Estradiol (E2) increases the expression of kisspeptin in Kiss1AVPV/PeN neurons but decreases its expression in Kiss1ARH neurons. Also, Kiss1ARH neurons coexpress glutamate and Kiss1AVPV/PeN neurons coexpress gamma aminobutyric acid (GABA), both of which are upregulated by E2 in females. Also, Kiss1ARH neurons express critical metabolic hormone receptors, and these neurons are excited by insulin and leptin during the fed state. Moreover, Kiss1ARH neurons project to and excite the anorexigenic proopiomelanocortin neurons but inhibit the orexigenic neuropeptide Y/Agouti-related peptide neurons, highlighting their role in regulating feeding behavior. Kiss1ARH and Kiss1AVPV/PeN neurons also project to the preautonomic paraventricular nucleus (satiety) neurons and the dorsomedial nucleus (energy expenditure) neurons to differentially regulate their function via glutamate and GABA release, respectively. Therefore, this review will address not only how Kiss1 neurons govern GnRH release, but how they control other homeostatic functions through their peptidergic, glutamatergic and GABAergic synaptic connections, providing further evidence that Kiss1 neurons are the key neurons coordinating energy states with reproduction.

Sex-dependent development of Kras-induced anal squamous cell carcinoma in mice.

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-KrasG12D; 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 KrasG12D gene. Although the driver gene mutation KrasG12D 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.

Deletion of Stim1 in Hypothalamic Arcuate Nucleus Kiss1 Neurons Potentiates Synchronous GCaMP Activity and Protects against Diet-Induced Obesity.

Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. High-frequency firing of hypothalamic arcuate Kiss1 (Kiss1ARH) neurons releases kisspeptin into the median eminence, and neurokinin B (NKB) and dynorphin onto neighboring Kiss1ARH neurons to generate a slow EPSP mediated by TRPC5 channels that entrains intermittent, synchronous firing of Kiss1ARH neurons. High-frequency optogenetic stimulation of Kiss1ARH neurons also releases glutamate to excite the anorexigenic proopiomelanocortin (POMC) neurons and inhibit the orexigenic neuropeptide Y/agouti-related peptide (AgRP) neurons via metabotropic glutamate receptors. At the molecular level, the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1) is critically involved in the regulation of neuronal Ca2+ signaling and neuronal excitability through its interaction with plasma membrane (PM) calcium (e.g., TRPC) channels. Therefore, we hypothesized that deletion of Stim1 in Kiss1ARH neurons would increase neuronal excitability and their synchronous firing, which ultimately would affect energy homeostasis. Using optogenetics in combination with whole-cell recording and GCaMP6 imaging in slices, we discovered that deletion of Stim1 in Kiss1 neurons significantly increased the amplitude and duration of the slow EPSP and augmented synchronous [Ca2+]i oscillations in Kiss1ARH neurons. Deletion of Stim1 in Kiss1ARH neurons amplified the actions of NKB and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD). Therefore, STIM1 appears to play a critical role in regulating synchronous firing of Kiss1ARH neurons, which ultimately affects the coordination between energy homeostasis and reproduction.SIGNIFICANCE STATEMENT Hypothalamic arcuate kisspeptin (Kiss1ARH) neurons are essential for stimulating the pulsatile release of gonadotropin-releasing hormone (GnRH) and maintaining fertility. However, Kiss1ARH neurons appear to be a key player in coordinating energy balance with reproduction. The regulation of calcium channels and hence calcium signaling is critically dependent on the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1), which interacts with the plasma membrane (PM) calcium channels. We have conditionally deleted Stim1 in Kiss1ARH neurons and found that it significantly increased the excitability of Kiss1ARH neurons and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD).

Arcuate and Preoptic Kisspeptin Neurons Exhibit Differential Projections to Hypothalamic Nuclei and Exert Opposite Postsynaptic Effects on Hypothalamic Paraventricular and Dorsomedial Nuclei in the Female Mouse.

Kisspeptin (Kiss1) neurons provide indispensable excitatory input to gonadotropin-releasing hormone (GnRH) neurons, which is important for the coordinated release of gonadotropins, estrous cyclicity and ovulation. However, Kiss1 neurons also send projections to many other brain regions within and outside the hypothalamus. Two different populations of Kiss1 neurons, one in the arcuate nucleus (Kiss1ARH) and another in the anteroventral periventricular nucleus (AVPV) and periventricular nucleus (PeN; Kiss1AVPV/PeN) of the hypothalamus are differentially regulated by ovarian steroids, and are believed to form direct contacts with GnRH neurons as well as other neurons. To investigate the projection fields from Kiss1AVPV/PeN and Kiss1ARH neurons in female mice, we used anterograde projection analysis, and channelrhodopsin-assisted circuit mapping (CRACM) to explore their functional input to select target neurons within the paraventricular (PVH) and dorsomedial (DMH) hypothalamus, key preautonomic nuclei. Cre-dependent viral (AAV1-DIO-ChR2 mCherry) vectors were injected into the brain to label the two Kiss1 neuronal populations. Immunocytochemistry (ICC) for mCherry and neuropeptides combined with confocal microscopy was used to determine the projection-fields of both Kiss1 neuronal groups. Whole-cell electrophysiology and optogenetics were used to elucidate the functional input to the PVH and DMH. Our analysis revealed many common but also several clearly separate projection fields between the two different populations of Kiss1 neurons. In addition, optogenetic stimulation of Kiss1 projections to PVH prodynorphin, Vglut2 and DMH CART-expressing neurons, revealed excitatory glutamatergic input from Kiss1ARH neurons and inhibitory GABAergic input from Kiss1AVPV/PeN neurons. Therefore, these steroid-sensitive Kiss1 neuronal groups can differentially control the excitability of target neurons to coordinate autonomic functions with reproduction.

CRISPR knockdown of Kcnq3 attenuates the M-current and increases excitability of NPY/AgRP neurons to alter energy balance.

OBJECTIVE: Arcuate nucleus neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior. The M-current, a subthreshold non-inactivating potassium current, plays a critical role in regulating NPY/AgRP neuronal excitability. Fasting decreases while 17ß-estradiol increases the M-current by regulating the mRNA expression of Kcnq2, 3, and 5 (Kv7.2, 3, and 5) channel subunits. Incorporating KCNQ3 into heteromeric channels has been considered essential to generate a robust M-current. Therefore, we investigated the behavioral and physiological effects of selective Kcnq3 deletion from NPY/AgRP neurons. METHODS: We used a single adeno-associated viral vector containing a recombinase-dependent Staphylococcus aureus Cas9 with a single-guide RNA to selectively delete Kcnq3 in NPY/AgRP neurons. Single-cell quantitative measurements of mRNA expression and whole-cell patch clamp experiments were conducted to validate the selective knockdown. Body weight, food intake, and locomotor activity were measured in male mice to assess disruptions in energy balance. RESULTS: The virus reduced the expression of Kcnq3 mRNA without affecting Kcnq2 or Kcnq5. The M-current was attenuated, causing NPY/AgRP neurons to be more depolarized, exhibit a higher input resistance, and require less depolarizing current to fire action potentials, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it significantly reduced the locomotor activity as measured by open-field testing. Control mice on a high-fat diet exhibited an enhanced M-current and increased Kcnq2 and Kcnq3 expression, but the M-current remained significantly attenuated in KCNQ3 knockdown animals. CONCLUSIONS: The M-current plays a critical role in modulating the intrinsic excitability of NPY/AgRP neurons that is essential for maintaining energy homeostasis.

Membrane and nuclear initiated estrogenic regulation of homeostasis.

Reproduction and energy balance are inextricably linked in order to optimize the evolutionary fitness of an organism. With insufficient or excessive energy stores a female is liable to suffer complications during pregnancy and produce unhealthy or obesity-prone offspring. The quintessential function of the hypothalamus is to act as a bridge between the endocrine and nervous systems, coordinating fertility and autonomic functions. Across the female reproductive cycle various motivations wax and wane, following levels of ovarian hormones. Estrogens, more specifically 17ß-estradiol (E2), coordinate a triumvirate of hypothalamic neurons within the arcuate nucleus (ARH) that govern the physiological underpinnings of these behavioral dynamics. Arising from a common progenitor pool of cells, this triumvirate is composed of the kisspeptin (Kiss1ARH), proopiomelanocortin (POMC), and neuropeptide Y/agouti-related peptide (AgRP) neurons. Although the excitability of these neuronal subpopulations is subject to genomic and rapid estrogenic regulation, kisspeptin neurons are the most sensitive, reflecting their integral function in female fertility. Based on the premise that E2 coordinates autonomic functions around reproduction, we will review the recent findings on the synaptic interactions between Kiss1, AgRP and POMC neurons and how the rapid membrane-initiated and intracellular signaling cascades activated by E2 in these neurons are critical for control of homeostatic functions supporting reproduction.

MKRN3 inhibits the reproductive axis through actions in kisspeptin-expressing neurons.

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.

Estradiol Protects Neuropeptide Y/Agouti-Related Peptide Neurons against Insulin Resistance in Females.

When it comes to obesity, men exhibit a higher incidence of metabolic syndrome than women in early adult life, but this sex advantage wanes in postmenopausal women. A key diagnostic of the metabolic syndrome is insulin resistance in both peripheral tissues and brain, especially in the hypothalamus. Since the anorexigenic hormone 17ß-estradiol (E2) regulates food intake in part by inhibiting the excitability of the hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons, we hypothesized that E2 would protect against insulin resistance in NPY/AgRP neurons with diet-induced obesity (DIO). Therefore, we did whole-cell recordings and single cell quantitative polymerase chain reaction in arcuate NPYGFP neurons from both female and male mice to test the efficacy of insulin with DIO. The resting membrane potential and input resistance of NPY/AgRP neurons were significantly increased in DIO versus control-diet fed males. Most notably, the efficacy of insulin to activate KATP channels in NPY/AgRP neurons was significantly attenuated, although the KATP channel opener diazoxide was fully effective in NPY/AgRP neurons from DIO males, indicating that the KATP channels were expressed and functional. In contrast, insulin was fully efficacious to activate KATP channels in DIO females, and the response was reversed by the KATP channel blocker tolbutamide. However, the ability of insulin to activate KATP channels was abrogated with ovariectomy but fully restored with E2 replacement. Insulin resistance in obese males was likely mediated by an increase in suppressor of cytokine signaling-3 (SOCS-3), protein tyrosine phosphatase B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP) activity, since the expression of all 3 mRNAs were upregulated in the obese males but not in females. As proof of principle, pre-incubation of hypothalamic slices from DIO males with the PTP1B/TCPTP inhibitor CX08005 completely rescued the effects of insulin. Therefore, E2 protects NPY/AgRP neurons in females against insulin resistance through, at least in part, attenuating phosphatase activity. The neuroprotective effects of E2 may explain sex differences in the expression of metabolic syndrome that disappears with the loss of E2 in aging.

Arcuate Kisspeptin Neurons Coordinate Reproductive Activities with Metabolism.

Hypothalamic control of fertility is the quintessential homeostatic function. However, fertility is metabolically demanding; so, there must be coordination between energy states and reproductive functions. Because gonadotropin-releasing hormone (GnRH) neurons are devoid of many of the critical metabolic hormone receptors for sensing nutrient levels, it has long been recognized that the sensing of energy stores had to be done by neurons presynaptic to GnRH neurons. Some of the obvious players have been the anorexigenic proopiomelanocortin (POMC) and orexigenic neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons, both of which are in close apposition to the median eminence, a circumventricular organ. Indeed, POMC and NPY/AgRP neurons are inversely regulated by glucose and metabolic hormones including insulin and leptin. However, their synaptic connections with GnRH neurons are sparse and/or GnRH neurons are lacking the postsynaptic receptors to mediate the appropriate physiological response. Kisspeptin neurons were discovered in the early part of this century and subsequently shown to project to and control GnRH neuronal excitability. In fact, more recently the arcuate kisspeptin neurons have been identified as the command neurons driving pulsatile release of GnRH. Subsequently, it was shown that arcuate kisspeptin neurons express not only steroid hormone receptors but also metabolic hormone receptors such that similar to POMC neurons, they are excited by insulin and leptin. Therefore, based on the premise that arcuate kisspeptin neurons are the key neurons coordinating energy states with reproduction, we will review not only how these vital neurons control pulsatile GnRH release but how they control energy homeostasis through their synaptic connections with POMC and NPY/AgRP neurons and ultimately how E2 can regulate their excitability.

Estradiol Drives the Anorexigenic Activity of Proopiomelanocortin Neurons in Female Mice.

Energy balance is regulated by anorexigenic proopiomelanocortin (POMC) and orexigenic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons of the hypothalamic arcuate nucleus. POMC neurons make extensive projections and are thought to release both amino acid and peptide neurotransmitters. However, whether they communicate directly with NPY/AgRP neurons is debated. Initially, using single-cell RT-PCR, we determined that mouse POMCeGFP neurons express Slc17a6 (Vglut2) and Slc18a2 (Vmat2), but not Slc31a1 (Vgat) mRNA, suggesting glutamate and non-canonical GABA release. Quantitative (q)RT-PCR of POMCeGFP cells revealed that Vglut2 and Vmat2 expression was significantly increased in E2- versus oil-treated, ovariectomized (OVX) female mice. Since 17ß-estradiol (E2) is anorexigenic, we hypothesized that an underlying mechanism is enhancement of POMC signaling. Therefore, we optogenetically stimulated POMC neurons in hypothalamic slices to examine evoked release of neurotransmitters onto NPY/AgRP neurons. Using brief light pulses, we primarily observed glutamatergic currents and, based on the paired pulse ratio (PPR), determined that release probability was higher in E2- versus oil-treated, OVX female, congruent with increased Vlgut2 expression. Moreover, bath perfusion of the Gq-coupled membrane estrogen receptor (ER) agonist STX recapitulated the effects of E2 treatment. In addition, high-frequency (20 Hz) stimulation generated a slow outward current that reversed near Ek+ and was antagonized by naloxone, indicative of ß-endorphin release. Furthermore, individual NPY/AgRP neurons were found to express Oprm1, the transcript for µ-opioid receptor, and DAMGO, a selective agonist, elicited an outward current. Therefore, POMC excitability and neurotransmission are enhanced by E2, which would facilitate decreased food consumption through marked inhibition of NPY/AgRP neurons.

Estrogenic-dependent glutamatergic neurotransmission from kisspeptin neurons governs feeding circuits in females.

The neuropeptides tachykinin2 (Tac2) and kisspeptin (Kiss1) in hypothalamic arcuate nucleus Kiss1 (Kiss1ARH) neurons are essential for pulsatile release of GnRH and reproduction. Since 17ß-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.

TRPCing around the hypothalamus.

All of the canonical transient receptor potential channels (TRPC) with the exception of TRPC 2 are expressed in hypothalamic neurons and are involved in multiple homeostatic functions. Although the metabotropic glutamate receptors have been shown to be coupled to TRPC channel activation in cortical and sub-cortical brain regions, in the hypothalamus multiple amine and peptidergic G protein-coupled receptors (GPCRs) and growth factor/cytokine receptors are linked to activation of TRPC channels that are vital for reproduction, temperature regulation, arousal and energy homeostasis. In addition to the neurotransmitters, circulating hormones like insulin and leptin through their cognate receptors activate TRPC channels in POMC neurons. Many of the post-synaptic effects of the neurotransmitters and hormones are regulated in different physiological states by expression of TRPC channels in the post-synaptic neurons. Therefore, TRPC channels are key targets not only for neurotransmitters but circulating hormones in their vital role to control multiple hypothalamic functions, which is the focus of this review.

Diverse actions of estradiol on anorexigenic and orexigenic hypothalamic arcuate neurons.

Contribution to Special Issue on Fast effects of steroids. There is now compelling evidence for membrane-associated estrogen receptors in hypothalamic neurons that are critical for the hypothalamic control of homeostatic functions. It has been known for some time that estradiol (E2) can rapidly alter hypothalamic neuronal activity within seconds, indicating that some cellular effects can occur via membrane initiated events. However, our understanding of how E2 signals via membrane-associated receptors and how these signals impact physiological functions is only just emerging. Thus, E2 can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell excitability and even gene transcription in hypothalamic neurons. One population of hypothalamic neurons, the anorexigenic proopiomelanocortin (POMC) neurons, has long been considered to be a target of E2's actions based on gene (Pomc) expression studies. However, we now know that E2 can rapidly alter POMC neuronal activity within seconds and activate several intracellular signaling cascades that ultimately affect gene expression, actions which are critical for maintaining sensitivity to insulin in metabolically stressed states. E2 also affects the orexigenic Neuropeptide Y/Agouti-related Peptide (NPY/AgRP) neurons in similarly rapid but antagonistic manner. Therefore, this review will summarize our current state of knowledge of how E2 signals via rapid membrane-initiated and intracellular signaling cascades in POMC and NPY/AgRP neurons to regulate energy homeostasis.

Estradiol Protects Proopiomelanocortin Neurons Against Insulin Resistance.

Insulin resistance is at the core of the metabolic syndrome, and men exhibit a higher incidence of metabolic syndrome than women in early adult life, but this sex advantage diminishes sharply when women reach the postmenopausal state. Because 17ß-estradiol (E2) augments the excitability of the anorexigenic proopiomelanocortin (POMC) neurons, we investigated the neuroprotective effects of E2 against insulin resistance in POMC neurons from diet-induced obese (DIO) female and male mice. The efficacy of insulin to activate canonical transient receptor potential 5 (TRPC5) channels and depolarize POMC neurons was significantly reduced in DIO male mice but not in DIO female mice. However, the insulin response in POMC neurons was abrogated in ovariectomized DIO females but restored with E2 replacement. E2 increased T-type calcium channel Cav3.1 messenger RNA (mRNA) expression and whole-cell currents but downregulated stromal-interaction molecule 1 mRNA, which rendered POMC neurons more excitable and responsive to insulin-mediated TRPC5 channel activation. Moreover, E2 prevented the increase in suppressor of cytokine signaling-3 mRNA expression with DIO as seen in DIO males. As proof of principle, insulin [intracerebroventricular injection into the third ventricle (ICV)] decreased food intake and increased metabolism in female but not male guinea pigs fed a high-fat diet. The uncoupling of the insulin receptor from its downstream effector system was corroborated by the reduced expression of phosphorylated protein kinase B in the arcuate nucleus of male but not female guinea pigs following insulin. Therefore, E2 protects female POMC neurons from insulin resistance by enhancing POMC neuronal excitability and the coupling of insulin receptor to TRPC5 channel activation.

GLP-1R Signaling Directly Activates Arcuate Nucleus Kisspeptin Action in Brain Slices but Does not Rescue Luteinizing Hormone Inhibition in Ovariectomized Mice During Negative Energy Balance.

Kisspeptin (Kiss1) neurons in the hypothalamic arcuate nucleus (ARC) are key components of the hypothalamic-pituitary-gonadal axis, as they regulate the basal pulsatile release of gonadotropin releasing hormone (GnRH). ARC Kiss1 action is dependent on energy status, and unmasking metabolic factors responsible for modulating ARC Kiss1 neurons is of great importance. One possible factor is glucagon-like peptide 1 (GLP-1), an anorexigenic neuropeptide produced by brainstem preproglucagon neurons. Because GLP fiber projections and the GLP-1 receptor (GLP-1R) are abundant in the ARC, we hypothesized that GLP-1R signaling could modulate ARC Kiss1 action. Using ovariectomized mice, we found that GLP-producing fibers come in close apposition with ARC Kiss1 neurons; these neurons also contain Glp1r mRNA. Electrophysiological recordings revealed that liraglutide (a long-acting GLP-1R agonist) increased action potential firing and caused a direct membrane depolarization of ARC Kiss1 cells in brain slices. We determined that brainstem preproglucagon mRNA is decreased after a 48-h fast in mice, a negative energy state in which ARC Kiss1 expression and downstream GnRH/luteinizing hormone (LH) release are potently suppressed. However, activation of GLP-1R signaling in fasted mice with liraglutide was not sufficient to prevent LH inhibition. Furthermore, chronic central infusions of the GLP-1R antagonist, exendin(9-39), in ad libitum-fed mice did not alter ARC Kiss1 mRNA or plasma LH. As a whole, these data identify a novel interaction of the GLP-1 system with ARC Kiss1 neurons but indicate that CNS GLP-1R signaling alone is not critical for the maintenance of LH during fasting or normal feeding.

AgRP to Kiss1 neuron signaling links nutritional state and fertility.

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.

High-frequency stimulation-induced peptide release synchronizes arcuate kisspeptin neurons and excites GnRH neurons.

Kisspeptin (Kiss1) and neurokinin B (NKB) neurocircuits are essential for pubertal development and fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (Kiss1(ARH)) 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 Kiss1(ARH) neuronal activity using optogenetics, whole-cell electrophysiology, molecular pharmacology and single cell RT-PCR in mice. High-frequency photostimulation of Kiss1(ARH) neurons evoked local release of excitatory (NKB) and inhibitory (dynorphin) neuropeptides, which were found to synchronize the Kiss1(ARH) 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 Kiss1(ARH) neurons. We propose that Kiss1(ARH) 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.