Figure 3. DMH^LepR neuron inactivation disrupts diurnal patterns of food intake, LMA, heat production, and substrate utilization.

Two-hour binned continuous measures (left panels) and mean values across the light (L) and dark (D) periods (right panels) 30 days following microinjection of GFP:TeTx (TeTx; n=7) or GFP control (control; n=7) to the dorsomedial hypothalamic nucleus (DMH) of LepR-Cre+ male mice. Shaded areas indicate dark cycle (ZT14 – ZT24). (A) Food intake. Two-way ANOVA: F_(1,12)=12; p=0.0047 (main effect of TeTx). F_(87,1044)=2.354; p<0.0001 (time x TeTx interaction). (B) Mean food intake from (A) during L, D, and 24-hr periods. Two-way ANOVA: F_(1,12)=9.567; p=0.0093 (main effect of TeTx). (C) Locomotor activity (LMA). Two-way ANOVA: F_(1,12)=93.22; p<0.0001 (main effect of TeTx). (D) Mean LMA from (C) during L, D, and 24-hr periods. Two-way ANOVA: F_(1,12)=110.4; p<0.0001 (main effect of TeTx). (E) Heat production. Two-way ANOVA: F_(1,12)=1.006; p=0.3357 (main effect of TeTx). (F) Mean heat production from (E) during L and D periods. Two-way ANOVA: F_(1,12)=1.209; p=0.2930 (main effect of TeTx). (G) Respiratory exchange ratio (RER). Two-way ANOVA: F_(1,12)=2.789; p=0.1208 (main effect of TeTx). (H) Mean RER from (G) during L and D periods. Two-way ANOVA: F_(1,12)=2.04; p=0.1788 (main effect of TeTx). Data are mean ± SEM. For repeated measures, post hoc, Sidak’s test at each time point is indicated on the graph. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

Figure 3—figure supplement 1. Silencing DMH^LepR neurons rapidly and robustly disrupts diurnal rhythms in food intake, peripheral substrate utilization, and LMA.

(A) Representative viral expression in DMH^LepR from LepR-Cre+ male mice injected bilaterally with an AAV encoding Cre-dependent GFP (left) as a control (control; n=7) or Cre-dependent GFP:TeTx (right; TeTx; n=7). (B) Daily body weight expressed as %day 0 value. Two-way ANOVA: F_(1,12)=37.82; p<0.0001 (main effect of TeTx); F_(7,84)=25.17; p<0.0001 (time x TeTx interaction). Dashed boxes indicate periods wherein mice were housed in calorimetry 2 days after microinjection (this figure) and 30 days after microinjection (Figure 3). Shaded areas indicate dark cycle (ZT14–ZT24). (C) Food intake (2 hr binned). (D) Photoperiod-averaged food intake from (C). Two-way ANOVA: F_(1,12)=3.151; p=0.1012 (main effect of TeTx); F_(1,12)=5.725; p=0.034 (time x TeTx interaction). (E) Respiratory exchange ratio (RER; 2 hr binned). (F) Photoperiod-averaged RER from (E). Two-way ANOVA: F_(1,12)=25.16; p=0.0003 (main effect of TeTx); F_{( 1,12)}=3.747; p=0.0768 (time x TeTx interaction). (G) Locomotor activity (LMA; 2 hr binned). (H) Photoperiod-averaged LMA from (G). Two-way ANOVA: F_(1,12)=4.673; p=0.0516 (main effect of TeTx); F_(1,12)=17.64; p=0.0012 (time x TeTx interaction).

Figure 3—figure supplement 2. Silencing DMH^LepR neurons in female mice recapitulates the effect in males to disrupt diurnal rhythms.

Two-hour binned continuous measures (left panels) and photoperiod-averaged values (right panels) 30 days following bilateral microinjection of Cre-dependent GFP:TeTx (TeTx; n=9) or GFP control (control; n=5) to the dorsomedial hypothalamic nucleus (DMH) of LepR-Cre+ female mice. Shaded areas indicate dark cycle (ZT14–ZT24). (A) Food intake. Two-way ANOVA: F_(1,12)=12.32; p=0.0043 (main effect of TeTx). F_(89,1068)=2.766, p<0.0001; F_(89,1068)=2.766; p<0.0001 (time x TeTx interaction). (B) Mean food intake from (A) during L, D, and 24-hr periods. Two-way ANOVA: F_(1,12)=16.26; p=0.0017 (main effect of TeTx). (C) Locomotor activity (LMA). Two-way ANOVA: F_(1,12)=36.22; p<0.0001 (main effect of TeTx); F_(89,1068) = 5.197; p<0.0001 (time x TeTx interaction). (D) Mean LMA from (C) during L, D, and 24-hr periods. Two-way ANOVA: F_(1,12)=27.98; p=0.0002 (main effect of TeTx). (E) Heat production. Two-way ANOVA: F_(1,12)=0.5405; p=0.4764 (main effect of TeTx); F_(89,1068)=5.903; p<0.0001 (time x TeTx interaction).