Canonical transient receptor potential channels and hypothalamic control of homeostatic functions.

Recent molecular biological and electrophysiological studies have identified multiple transient receptor potential (TRP) channels in hypothalamic neurons as critical modulators of homeostatic functions. In particular, the canonical transient receptor potential channels (TRPCs) are expressed in hypothalamic neurons that are vital for the control of fertility and energy homeostasis. Classical neurotransmitters such as serotonin and glutamate and peptide neurotransmitters such as kisspeptin, neurokinin B and pituitary adenylyl cyclase-activating polypeptide signal through their cognate G protein-coupled receptors to activate TPRC 4, 5 channels, which are essentially ligand-gated calcium channels. In addition to neurotransmitters, circulating hormones like insulin and leptin signal through insulin receptor (InsR) and leptin receptor (LRb), respectively, to activate TRPC 5 channels in hypothalamic arcuate nucleus pro-opiomelanocortin (POMC) and kisspeptin (arcuate Kiss1 [Kiss1ARH]) neurons to have profound physiological (excitatory) effects. Besides its overt depolarizing effects, TRPC channels conduct calcium ions into the cytoplasm, which has a plethora of downstream effects. Moreover, not only the expression of Trpc5 mRNA but also the coupling of receptors to TRPC 5 channel opening are regulated in different physiological states. In particular, the mRNA expression of Trpc5 is highly regulated in kisspeptin neurons by circulating estrogens, which ultimately dictates the firing pattern of kisspeptin neurons. In obesity states, InsRs are "uncoupled" from opening TRPC 5 channels in POMC neurons, rendering them less excitable. Therefore, in this review, we will focus on the critical role of TRPC 5 channels in regulating the excitability of Kiss1ARH and POMC neurons in different physiological and pathological states.

Functional Outcomes of Congenital Scoliosis at a Mean 35-Year Follow-up Post In Situ Fusion. Revisiting Patients From the 2002 Goldberg et al Study.

BACKGROUND: The management of congenital scoliosis poses a significant challenge for treating surgeons. The aim of our study was to provide insight into the long-term clinical results of spinal fusion in congenital scoliosis. METHODS: We performed a retrospective review of the scoliosis database in our institution for the period 1976 until 2002 identifying 43 patients with congenital scoliosis who underwent spinal fusion. Patient demographics, diagnosis, levels fused, and radiographs were evaluated. Patients were evaluated for unplanned return to the operating room (UPROR) via SRS 22, EQ5D-5L, and Oswestry Disability Index (ODI). RESULTS: Of the 43 patients who fulfilled the inclusion criteria, 22 patients agreed to participate, 3 patients were known to be deceased and 18 patients were lost to follow-up or declined to participate and were excluded. The mean age of the respondents was 40.7 years (range, 30 to 47 y) with a mean follow-up from index surgery of 35 years (range, 20 to 44 y). At most recent follow-up, 12 patients (54%) underwent UPROR. The mean age at diagnosis was 3.4 years (range, birth to 11.5 y), and the mean age for first surgery was 5.8 years (range, 1 to 13 y). As regards radiologic follow-up; the mean number of levels fused was 5.2 (range, 2 to 12). Thoracic fusion was performed in 17 patients (77%). The mean T1 to T12 height at index surgery and maturity was 166 mm (range, 130 to 240 mm) and 202 mm (range, 125 to 270 mm), respectively. The mean functional scores at follow-up were SRS 22: 4.5 (range, 2.4 to 5), cumulative EQ5D-5L score 7.2 (range, 5 to 15), and ODI: 8% (range, 2 to 30%). All respondents completed high school, 10 patients (45%) completed university, and 2 patients were awarded doctorates. Currently, 17 patients (77%) are in paid employment. CONCLUSIONS: This report constitutes the largest series of patients treated by spinal arthrodesis for congenital scoliosis followed into maturity. We demonstrate the thorax continues to grow after index fusion, patient-reported outcomes were satisfactory with superior educational and employment rates and unplanned return to theatre is rare in adult life. LEVEL OF EVIDENCE: Therapeutic Level IV.

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.

The novel estrogen receptor modulator STX attenuates Amyloid-ß neurotoxicity in the 5XFAD mouse model of Alzheimer's disease.

Based on previous evidence that the non-steroidal estrogen receptor modulator STX mitigates the effects of neurotoxic Amyloid-ß (Aß) in vitro, we have evaluated its neuroprotective benefits in a mouse model of Alzheimer's disease. Cohorts of 5XFAD mice, which begin to accumulate cerebral Aß at two months of age, were treated with orally-administered STX starting at 6 months of age for two months. After behavioral testing to evaluate cognitive function, biochemical and immunohistochemical assays were used to analyze key markers of mitochondrial function and synaptic integrity. Oral STX treatment attenuated Aß-associated mitochondrial toxicity and synaptic toxicity in the brain, as previously documented in cultured neurons. STX also moderately improved spatial memory in 5XFAD mice. In addition, STX reduced markers for reactive astrocytosis and microgliosis surrounding amyloid plaques, and also unexpectedly reduced overall levels of cerebral Aß in the brain. The neuroprotective effects of STX were more robust in females than in males. These results suggest that STX may have therapeutic potential in Alzheimer's Disease.

Estrogenic regulation of reproduction and energy homeostasis by a triumvirate of hypothalamic arcuate neurons.

Pregnancy is energetically demanding and therefore, by necessity, reproduction and energy balance are inextricably linked. With insufficient or excessive energy stores a female is liable to suffer complications during pregnancy or produce unhealthy offspring. Gonadotropin-releasing hormone neurons are responsible for initiating both the pulsatile and subsequent surge release of luteinizing hormone to control ovulation. Meticulous work has identified two hypothalamic populations of kisspeptin (Kiss1) neurons that are critical for this pattern of release. The involvement of the hypothalamus is unsurprising because its quintessential function is to couple the endocrine and nervous systems, coordinating energy balance and reproduction. Estrogens, more specifically 17ß-estradiol (E2 ), orchestrate the activity of 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, these cells differentiate into ARH kisspeptin, pro-opiomelanocortin (POMC), and agouti related peptide/neuropeptide Y (AgRP) neurons. Although the excitability of all these subpopulations is subject to genomic and rapid estrogenic regulation, Kiss1 neurons are the most sensitive, reflecting their integral function in female fertility. Based on the premise that E2 coordinates autonomic functions around reproduction, we review recent findings on how Kiss1 neurons interact with gonadotropin-releasing hormone, AgRP and POMC neurons, as well as how the rapid membrane-initiated and intracellular signaling cascades activated by E2 in these neurons are critical for control of homeostatic functions supporting reproduction. In particular, we highlight how Kiss1 and POMC neurons conspire to inhibit AgRP neurons and diminish food motivation in service of reproductive success.

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.

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.

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.

Photorelease of 2-Arachidonoylglycerol in Live Cells.

2-Arachidonoylglycerol (2-AG) is acting as a full agonist of cannabinoid receptor 1 and 2. Direct manipulation of 2-AG levels is a challenging task. The amphiphilic properties and the instability of 2-AG in aqueous media complicate its use as a drug-like molecule. Additionally, inhibition of the protein machinery that regulates 2-AG levels may also affect other monoacylglycerols. Therefore, we developed a novel method to elevate 2-AG levels with a flash of light. The resulting tool is a photoactivatable "caged" 2-arachidonoylglycerol (cg2-AG) allowing for the rapid photorelease of the signaling lipid in live cells. We characterized the mechanism of uncaging and the effect of 2-AG on the regulation of the ß-cell signaling network. After uncaging of 2-AG, we monitored calcium levels, CB1-GIRK channel coupling, and CB1-mediated inhibition of adenylate cyclase and protein kinase A activity.

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.

The 3rd World Conference on Kisspeptin, "Kisspeptin 2017: Brain and Beyond":Unresolved questions, challenges and future directions for the field.

The 3rd World Conference on Kisspeptin, "Kisspeptin 2017: Brain and Beyond" was held March 30-31 at the Rosen Centre Hotel in Orlando, Florida, providing an international forum for multidisciplinary scientists to meet and share cutting-edge research on kisspeptin biology and its relevance to human health and disease. The meeting built upon previous world conferences focused on the role of kisspeptin and associated peptides in the control of gonadotropin-releasing hormone (GnRH) secretion and reproduction. Based on recent discoveries, the scope of this meeting was expanded to include functions of kisspeptin and related peptides in other physiological systems including energy homeostasis, pregnancy, ovarian and uterine function, and thermoregulation. In addition, discussions addressed the translation of basic knowledge of kisspeptin biology to the treatment of disease, with the goal of seeking consensus about the best approaches to improve human health. The two-day meeting featured a non-traditional structure, with each day starting with poster sessions followed by lunch discussions and facilitated large-group sessions with short presentations to maximize the exchange of new, unpublished data. Topics were identified by a survey prior to the meeting, and focused on major unresolved questions, important controversies, and future directions in the field. Finally, career development activities provided mentoring for trainees and junior investigators, and networking opportunities for those individuals with established researchers in the field. Overall, the meeting was rated as a success by attendees and covered a wide range of lively and provocative discussion topics on the changing nature of the field of "kisspeptinology" and its future. This article is protected by copyright. All rights reserved.

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.

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.