Multi-unit recordings first done in female rhesus macaques identified synchronous volleys of action potentials 16 in the hypothalamic arcuate nucleus that correlated with pulsatile luteinizing hormone (LH) release (Knobil, 1981;  17 Wilson et al., 1984) . It was later proposed that hypothalamic arcuate kisspeptin (Kiss1 ARH ) neurons were 18 responsible for the "pulse-generator" activity that stimulates pulsatile secretion of gonadotropin releasing 3 releases NKB and dynorphin (Qiu J. et al., 2016) . NKB binds to tachykinin 3 receptor (TacR3) in neighboring 27 Kiss1 ARH neurons to activate canonical transient receptor potential 5 (TRPC5) channels to cause a robust 28 depolarization (slow EPSP), whereas co-released dynorphin feeds back to bind to presynaptic κ-opioid receptors 29 to limit the release of NKB to discrete bursts of activity (Qiu J. et al., 2016) . The co-release of the two peptide 
A key molecule modulating the activity of TRPC channels is stromal-interaction molecule 1 (STIM1), which 67 is localized to the endoplasmic reticulum (ER) membrane of cells, and its N-terminal domain contains an EF-68 hand that protrudes into the lumen of the ER to sense changes in ER Ca 2+ 
Previous results show that insulin receptor (Insr) and Trpc5 mRNA are expressed and involved in sculpting 04 the excitability in Kiss1 ARH neurons (Qiu J. et al., 2011; Qiu X. et al., 2013) , and insulin excites Kiss1 ARH neurons 05 through activation of TPRC5 channels (Qiu J. et al., 2014) . To see if Insr and Trpc5 mRNAs are co-localized in 06 Kiss1 ARH neurons, we harvested 64 Kiss1 ARH neurons from 3 females and did scRT-PCR for Insr and Trpc5 07 6 ( Figure 1C) . The single-cell analysis revealed that Insr transcript was detected in 53% of Kiss1 ARH neurons and 08 GCaMP6s and imaged these neurons using spinning disk confocal microscopy (Figure 2-video supplement 1) .
ER Ca 2+ stores were released by treatment with 2 M thapsigargin (Tg), a blocker of the SERCA 21 (sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase) pump. As expected, Tg treatment of neurons bathed in Ca 2+ -22 free aCSF generated an initial wave of cytoplasmic Ca 2+ release ([Ca 2+ ]i ) as measured by an increase in 23 GCaMP6s activity both in control and Stim1-deleted neurons ( Figure 1D , E and F). As long as neurons were 24 kept in Ca 2+ -free aCSF, the ER stores remained empty, a situation that was presumably sensed by the Ca 2+ 25 sensor STIMs. Upon switching to a normal aCSF containing 2 mM Ca 2+ , an immediate SOCE response was 26 observed as a second wave of cytoplasmic Ca 2+ rise. Consistent with a role for STIM1 regulation, we observed 27 an attenuation of SOCE in Stim1 kko neurons ( Figure 1D , E F and G: ΔF/F0*100 =1274.5 ± 49.4, n = 4, Kiss1 ARH 28 group versus 389.0 ± 86.1, n = 4, Stim1 kko group, which was measured from the 15 minute time point to the peak, 29 unpaired t-test, t(6) = 8.921, p = 0.0001, ***p < 0.005), indicating that STIM1 plays a major role in SOCE after Tg- To test the hypothesis that insulin potentiates the slow EPSP in Stim1 kko neurons to a greater extent than in 88 9 Kiss1 ARH neurons, we pretreated ChR2-mCherry expressing arcuate slices from Stim1 kko mice with insulin.
Indeed, we found that insulin augmented the slow EPSP by 60% beyond the effects of Stim1 deletion on the (Figure 6A, B) . Moreover, the 05 average fat mass of Stim1 kko mice was significantly less that of Kiss1 Cre controls by week 6 (Stim1 kko versus 06 Kiss1 Cre mice: 7.6 ± 0.9 g, n=10 versus 11.4 ± 1.1 g, n=10 ) ( Figure 6C ). The lean mass of Stim1 kko mice was 07 significantly less versus the Kiss1 Cre mice (Stim1 kko versus the Kiss1 Cre mice: 16.9 ± 0.4 g, n=10 versus 18.9 ± 08 0.4 g, n=10) ( Figure 6D ). After 6 weeks, both Stim1 kko and Kiss1 Cre controls were assessed for glucose tolerance 09 using an i.p. glucose tolerance test (see Methods). Both Stim1 kko and Kiss1 Cre females started at relatively the 10 same blood glucose levels after an overnight fast ( Figure 6E , time 0), suggesting similar whole-body homeostatic 11 conditions after fasting. However, Stim1 kko female mice had significantly lower glucose levels after i.p. glucose 12 compared to Kiss1 Cre females, indicating that Stim1 kko females were more glucose tolerant compared to Kiss1 Cre 13 controls. Moreover, Stim1 kko females had a significantly different glucose clearance rate than controls based on 14 the integrated area under the curve (Stim1 kko versus the Kiss1 Cre controls AUC: 20,232 ± 868 mg/dL × min, n = 15 10 6 vs 22,622 ± 624 mg/dL × min, n = 6). Finally, when both groups were euthanized after eight weeks on HFD 16 and tissues harvested for mRNA measurements, both the intrascapular brown adipose tissue (iBAT) and 
The rapid insulin effects were abrogated by PI3K kinase inhibitor wortmannin. Importantly, the enhanced TacR3 50 signaling in Stim1 kko female mice afforded protection against diet-induced obesity and glucose intolerance. 51 52 We first discovered that TRPC5 channels are coupled to metabolic hormone signaling in both POMC and Software) was used to design primers that cross at least one intron-exon boundary. In order to confirm that 08 STIM1 was knocked out, STIM1 primers were designed to include part of exon 2 (see Table 1 ). Single cell PCR 09 conditions were optimized for primer concentration, magnesium concentration and annealing temperature.
Standard curves were generated using hypothalamic cDNA with dilutions from 1:50 to 1:12,800 for primers used 11 for qPCR to determine the efficiency (E = 10 (-1/m) -1; table 1). Primer pairs with efficiencies of 90-100% permit 12 21 the use of the comparative ΔΔCT method for analysis (Livak and Schmittgen, 2001; Pfaffl, 2001 on high fat diet (unpaired, two-tailed t-test for B, t(18) = 3.222, **p = 0.0047; unpaired two-tailed t test for C, t(18) = 19 2.662, *p = 0.0159; unpaired, two-tailed t test for D, t(18) = 3.489, *p = 0.0026). E, six weeks after high fat diet, 20 there was a significant difference in GTTs between the two groups (two-way ANOVA: main effect of treatment 21 (F(1, 9) = 6.282, p = 0.0335), main effect of time (F(4, 36) = 88.01, p < 0.0001) and interaction (F(4, 36) = 3.527, p = 22 0.0158); Kiss1 Cre , n = 6, Stim1 kko , n = 5; post hoc Bonferroni test, **p < 0.01, *p < 0.05). F and G, both 23 interscapular brown adipose tissue (iBAT) and perigonadal adipose tissue (GAT) mass of Stim1 kko were lighter 24 than that of Kiss1 Cre mice on a fat diet after eight weeks (unpaired, two-tailed t test for iBAT, t(18) = 2.127, *p = 25 0.0475; unpaired two-tailed t-test for GAT, t(18) = 2.711, *p = 0.0143). 58 channels thereby engaging these Ca 2+ channels as store-operated channels, which are activated with