Fig 1.
Gnas deletion in hypothalamic Opn5 neurons.
A. Schematic of conditional Gnas deletion in Opn5 cells. Recombination of loxP sites by Opn5cre excises Gnas Exon 1 in the conditional allele (Gnasfl). The wild-type Gnas allele (Gnas+) does not have loxP sites and is unaffected by cre. B. Representative coronal brain section of Opn5cre; Ai14 mice (scale bar, 1mm). Ai14 (tdTomato, red) expression by Opn5cre is only in the hypothalamic preoptic area (inset; scale bar, 500 μm). C. Representative images showing co-localization of Gnas (green) in tdTomato+ (red) cells in control (Opn5cre;Gnas+/+;Ai14, top row) and mutant (Opn5cre;Gnasfl/fl;Ai14, bottom row) coronal brain sections. Yellow arrows indicate co-localization of Gnas and tdTomato; white arrows show no co-localization. Scale bars, 50 μm. D. Quantification of z-scores of Gnas intensity in Ai14+ cells in Opn5cre/+; Gnas+/+; Ai14+/-, (n = 2 mice, gray), Opn5cre/+; Gnasfl/+; Ai14+/- (n = 2 mice, light blue), and Opn5cre/+; Gnasfl/fl; Ai14+/- (n = 3 mice, blue). Data is presented as mean± s.e.m. and analyzed by one-way ANOVA with Tukey’s HSD. *p<0.05; **p<0.01.
Table 1.
Summary of metabolic analysis of Opn5cre; Gnas+/+ and Opn5cre; Gnasfl/fl mice by two-way ANOVA.
Table 2.
Summary of metabolic analysis of Opn5cre; Gnas+/+ and Opn5cre; Gnasfl/fl mice by CircaCompare.
Fig 2.
Daily energy expenditure of Gnas conditional mutants and controls at different temperatures.
Energy expenditure in Opn5cre; Gnasfl/fl (n = 9, blue) and Opn5cre; Gnas+/+ (n = 7, gray) mice was recorded by indirect calorimetry. Respective temperatures are indicated at the top of each column. For time of day on x-axes, t = 0 represents the start of the light phase at 6AM and t = 12 represents the start of the dark phase at 6PM (light:dark bar above each column). 24-hour energy expenditure profiles (A-C) at thermoneutrality (A), standard ambient temperature (B), and during cold-stress (C). Data points in A-C are presented as mean± s.e.m. for 15-minute measurements and analyzed by two-way ANOVA for each 3-hour interval. Significant p-values are indicated above each interval. Energy-mass relationships (D-F) from 24-hour data in A-C were analyzed by ANCOVA at each temperature with mass as the covariate. P-values of genotype and genotype x mass interaction for D-F are presented below each graph. Cosinor curves (G-I) were generated by CircaCompare for thermoneutrality (G), standard ambient temperature (H), and during cold-stress (I). Estimates of circadian values and p-values (Tables J-L) are listed under each cosinor curve.
Fig 3.
Daily respiratory exchange ratios of Gnas conditional mutants and controls at different temperatures.
Respiratory exchange (vCO2/vO2) in Opn5cre; Gnasfl/fl (n = 9, blue) and Opn5cre; Gnas+/+ (n = 7, gray) mice was recorded by indirect calorimetry. Respective temperatures are indicated at the top of each column. For time of day on x-axes, t = 0 represents the start of the light phase at 6AM and t = 12 represents the start of the dark phase at 6PM (light:dark bar above each column). 24-hour respiratory exchange ratios (A-C) at thermoneutrality (A), standard ambient temperature (B), and during cold-stress (C). Data points in A-C are presented as mean± s.e.m. for 15-minute measurements and analyzed by two-way ANOVA for each 3-hour interval. Significant p-values are indicated above each interval. Cosinor curves (D-F) were generated by CircaCompare for thermoneutrality (D), standard ambient temperature (E), and during cold-stress (F). Estimates of circadian values and p-values (G-I) are listed under each cosinor curve.
Fig 4.
Daily food intake of Gnas conditional mutants and controls at different temperatures.
Food intake in Opn5cre; Gnasfl/fl (n = 9, blue) and Opn5cre; Gnas+/+ (n = 7, gray) mice was recorded by weight-based sensors. Respective temperatures are indicated at the top of each column. For time of day on x-axes, t = 0 represents the start of the light phase at 6AM and t = 12 represents the start of the dark phase at 6PM (light:dark bar above each column). 24-hour food consumption profiles (A-C) at thermoneutrality (A), standard ambient temperature (B), and during cold-stress (C). Data points in A-C are presented as mean± s.e.m. for 15-minute measurements and analyzed by two-way ANOVA for each 3-hour interval. Significant p-values are indicated above each interval. Cosinor curves (D-F) were generated by CircaCompare for thermoneutrality (D), standard ambient temperature (E), and during cold-stress (F). Estimates of circadian values and p-values (G-I) are listed under each cosinor curve.
Fig 5.
Daily water intake of Gnas conditional mutants and controls at different temperatures.
Water intake in Opn5cre; Gnasfl/fl (n = 9, blue) and Opn5cre; Gnas+/+ (n = 6, gray) mice was recorded by weight-based sensors. Respective temperatures are indicated at the top of each column. For time of day on x-axes, t = 0 represents the start of the light phase at 6AM and t = 12 represents the start of the dark phase at 6PM (light:dark bar above each column). 24-hour water consumption profiles (A-C) at thermoneutrality (A), standard ambient temperature (B), and during cold-stress (C). Data points in A-C are presented as mean± s.e.m. for 15-minute measurements and analyzed by two-way ANOVA for each 3-hour interval. Significant p-values are indicated above each interval. Cosinor curves (D-F) were generated by CircaCompare for thermoneutrality (D), standard ambient temperature (E), and during cold-stress (F). Estimates of circadian values and p-values (G-I) are listed under each cosinor curve.
Fig 6.
Daily locomotor activity of Gnas conditional mutants and controls at different temperatures.
Locomotor distance traveled in Opn5cre; Gnasfl/fl (n = 9, blue) and Opn5cre; Gnas+/+ (n = 7, gray) mice was passively recorded by infrared beam breaks. Respective temperatures are indicated at the top of each column. For time of day on x-axes, t = 0 represents the start of the light phase at 6AM and t = 12 represents the start of the dark phase at 6PM (light:dark bar above each column). 24-hour locomotor profiles (A-C) at thermoneutrality (A), standard ambient temperature (B), and during cold-stress (C). Data points in A-C are presented as mean± s.e.m. for 15-minute measurements and analyzed by two-way ANOVA for each 3-hour interval. Significant p-values are indicated above each interval. Cosinor curves (D-F) were generated by CircaCompare for thermoneutrality (D), standard ambient temperature (E), and during cold-stress (F). Estimates of circadian values and p-values (G-I) are listed under each cosinor curve.