E₂ increases the whole-cell T-type calcium current and the mRNA expression of Cav3.1, but decreases the function of Stim1 in POMC neurons. (A) The current amplitude was normalized to the cell capacitance to calculate current density. Bar graphs summarize the density of T-type calcium current in POMC neurons from oil- and E₂-treated animals. (B) Cav 3.1 mRNA expression was determined using the Taqman real-time PCR method in POMC neuronal pools (five cells in each pool). (C) Stim1 was measured in the arcuate nucleus in oil- and E₂-treated females using SYBR Green real-time PCR (see “Materials and Methods”). (D) Based on single-cell RT-PCR analysis (65 cells from three female mice) >75% of POMC neurons express Stim1 mRNA. A representative gel illustrating that single POMC-EGFP neurons express mRNA for Pomc and Stim1, but not AgRP. –RT indicates that single cell reacted without RT; + indicates positive tissue control (with RT); – indicates negative tissue control (without RT). (E) In OVX females, insulin (Ins; 20 µM/4 µL “puff” application into bath) generated an inward current (4 pA) that washed out after several minutes, during which time the store-operated Ca²⁺ channel inhibitor GSK 7975A (10 µM) was applied. A second application of insulin (puff application) generated 8 pA current. V_hold = –60 mV. (F) GSK augmented the insulin-induced inward current by 2.9 ± 0.9-fold (n = 8). Data points represent the mean ± SEM. Unpaired two-tailed Student t test: t₍₁₅₎ = 3.986, P = 0.0012 (for T-type current); t₍₈₎ = 4.818, P = 0.0013 (for Cav3.1 mRNA); t₍₁₀₎ = 2.764, P = 0.020 (for Stim1 mRNA); t₍₂₅₎ = 2.184, P = 0.0386 (for GSK augmentation of insulin-induced inward current). *P < 0.05; **P < 0.01. EB, estradiol benzoate; MM, molecular marker.