Brain slice recordings of EGFP (i.e., Nts) neurons in the LHA of Nts-EGFP (Control) and Nts-LepRbKO-EGFP (Nts-LepRbKO) mice treated with vehicle (V) or leptin (100 nM, L). Representative traces (A, B, C; bottom panel, on the expanded time scale) and aggregate responses (D, E) from leptin-depolarized (A, D-grey bars) and leptin-hyperpolarized neurons (B, D- white bars) in Control mice, and (C, E) all recorded neurons in Nts-LepRbKO mice. All the recordings were performed in the presence of GABA_A, glycine and glutamate receptor antagonists. Data represent mean ± SEM, * = p<0.01 by paired t test.

12–14 week old male Nts-LepRbKO (KO) and Control mice (KO n=20, control n = 16) were subjected to CLAMS analysis to determine (A) VO₂ and (B) ambulatory locomotor activity. Data are shown for dark cycle (Dark), light cycle (Light) and averaged over 24 hours (Total). Graphed data represent average values ± SEM. ANOVA: *p < 0.05, **p < 0.01 and ***p < 0.001 for Control vs. KO; # p<0.05 for light vs. dark.

Adult (12–15 wk) male Nts-LepRbKO (KO) and Control mice were ad libitum fed (A, C) or fasted for 26h (B, D) and analyzed by immunohistochemistry for cFos (green nuclei, pseudocolored) and immunofluoresence of OX neurons (red). (E) Percentage of OX neurons containing cFos in fed and fasted Control and KO mice. Control fed n= 4, control fasted n=5, KO fed n=6, KO fasted n=7. (F, G) Adult male Control and KO mice were treated with PBS or leptin (IP, 5 mg/kg) every 12h for 26 h total for the assessment of body weight and Ox expression. Control + PBS n=12, control + leptin n=10, KO + PBS n=12, KO + PBS n=15. (F) Weight change over 26h of treatment in control and KO mice. (G) Ox expression in the LHA of control and KO mice. Data are plotted as mean ± SEM; * p<0.05 via one-way ANOVA with Bonferroni post test.

(A) Immunofluorescent detection of GFP (green) and Nts (red) in colchicine-treated LepRb^EGFP animals. (B) Enlargement of (A). (C) Schematic diagram depicting the generation of Nts-LepRbKO mice lacking functional LepRb only in Nts neurons. (D–I) Nts^cre mice were bred onto the Rosa26-EGFP reporter line so that cre induces the expression of EGFP to identify cre-containing (Nts) neurons. Nts-EGFP (Control) and Nts-LepRbKO-EGFP (KO) mice (n=4 each) were treated with leptin (IP, 5 mg/kg, 2h) and analyzed via immunohistochemistry for pSTAT3 (red nuclei, pseudocolored) and immunofluoresence for EGFP. (D) LHA of a leptin-treated control mouse. (E) LHA of a leptin-treated KO mouse. (F, G) Leptin-induced pSTAT3 is similar in the ARC of control (F) and KO mice (G). (H, I) Leptin-induced pSTAT3 is similar in the DMH of control (H) and KO mice (I). Insets: higher magnification view of labeled LHA neurons. Scale bar = 100 μm. 3V= third cerebral ventricle; F = fornix.

(A) Body weight, (B) gonadal fat pad weight, (C) percentage fat mass, and (D) percentage lean mass of male Control and Nts-LepRbKO (KO) mice (12–14 wk old, n=23–25 per genotype). (E) ELISA analysis of terminal serum for leptin (13–15 wk old, KO n=15, control n = 16). (F) Weekly food intake from 4–5 wk of age. (n=25 per genotype). Graphed data represent average values ± SEM. *p<0.05 by Student's t test.

(A) Schematic diagram showing cre-mediated expression of EGFPf and WGA in infected Nts-expressing neurons following stereotaxic injection of Ad-iZ/EGFPf or Ad-IN/WED, respectively, into the LHA of Nts^cre mice. (B) Immunofluorescent detection of EGFPf in the LHA of Nts^cre mice following intra-LHA injection of Ad-iZ/EGFPf identifies cell bodies and fibers (projections) in the LHA. (C) Immunofluorescent detection of EGFPf in the VTA of Nts^cre mice after intra-LHA injection of Ad-iZ/EGFPf. (D, E) Enlargements of B and D, respectively. (F–I) LHA images from leptin-treated (2h, IP, 5 mg/kg) Nts^EGFP mice injected with the retrograde tracer FG in the VTA. (F) GFP-IR, (G) FG-IR, (H) pSTAT3-IR, and (I) merged image. Yellow arrows indicate neurons positive for pSTAT3, FG and GFP. White arrows identify GFP+FG neurons that do not contain pSTAT3. (J–L) LHA from an Nts^cre mouse injected with Ad-IN/WED. (J) Immunofluoresence for WGA (green), (K) OX (red), and (L) merged. Yellow arrows: neurons that contain OX + WGA. Scale bars in (B) = 100 μm. F= fornix; 3V = third cerebral ventricle; VTA = ventral tegmental area; SN = substantia nigra (pars compacta); IP= interpeduncular tubercle; * = injection site.

(A) Adult male Nts-LepRbKO (KO) and littermate Control mice were analyzed for AMPH-induced locomotor activity. Mice were placed in the activity monitoring chamber at time 0, given an IP saline injection at 60 min, AMPH (4 mg/kg, IP) at 120 minutes, and were followed for an additional 90 minutes. Graphed data represent the average total distance (cm) ambulated ± SEM, control n=15, KO n=20. (B) Relative Th expression in the VTA and (C) DA in the NAc of control and KO mice. Data are plotted as the average ± SEM, control n=12, Lepr^NtsKO n=15. (D, E) Mean evoked dopamine spike amplitude (D) and t1/2 (E) in the NAc shell in KO and Control mice. Amplitude: KO, n=36 stimulations from 8 slices in 5 animals; Control, n=42 stimulations from 9 slices in 5 animals. t1/2: KO, n=36 stimulations from 8 slices in 5 animals; Control n=42 stimulations from 9 slices in 5 animals. Data are plotted as the average ± SEM, *p < 0.05 by ANOVA.