with 3 supplements. Ox1R predominantly mediates orexin-induced activation of dopaminergic neurons in the substantia nigra (SN).

Representative images of RNAscope in situ hybridization in SN and paranigral (PN), parainterfascicular (PIF) and parabrachial (PBP) subnuclei of the ventral tegmental area (VTA), of (A) a control and (B) an Ox1RΔDAT mouse. Amplifications of the (C) left and (D) right marked areas in white squares in (A). Amplifications of the (E) left and (F) right marked area in white squares in (B). White, tyrosine hydroxylase (Th); magenta, Ox1R; green, Ox2R. Scale bar: 100 µm (A, B) or 50 µm (C-F). Representative images of an overview in SN and VTA were shown in figure 1—figure supplement 1. Percentages of (G) Ox1R and (H) Ox2R positive neurons in Th positive neurons. IF, interfascicular subnuclei of VTA. Control, n = 3; Ox1RΔDAT, n = 4. Data are represented as means ± SEM. **p < 0.01; ****p < 0.0001; as determined by two-way ANOVA followed by Sidak’s post hoc test. (G) Subnuclei: F(4, 25) = 0.60, p = 0.66; genotype: F (1, 25) = 173.4, p < 0.0001; subnuclei x genotype: F(4, 25) = 1.43, p = 0.25. (H) Subnuclei: F(4, 25) = 17.06, p < 0.0001; genotype: F (1, 25) = 0.50, p = 0.48; subnuclei x genotype: F(4, 25) = 0.90, p = 0.48. (I) Schematic illustration showing the subnuclei of the left VTA and SN. The blue frame indicates the region where panel (A) and (B) are showing and the red frame indicates the region where calcium imaging was performed. (J) Pie charts showing Ox1R and Ox2R expression in dopaminergic neurons in the VTA and SN. 3 control mice were analyzed and N indicates the mean dopaminergic neuron numbers in the respective subregions. Magenta, dopaminergic neurons only expressing Ox1R; green, dopaminergic neurons only expressing Ox2R; yellow, dopaminergic neurons expressing both Ox1R and Ox2R; grey, dopaminergic neurons expressing neither Ox1R or Ox2R. (K, L) Effect of orexin A on dopaminergic SN neurons analyzed by Ca2+ imaging with GCaMP6s. Recordings were performed in acute brain slices from control and Ox1RΔDAT male mice with GCaMP6s expressed in dopaminergic SN neurons. (K) Exemplary heat maps of 5 individual orexin A responsive dopaminergic SN neurons from control (top) and 5 not orexin A-responsive dopaminergic SN neurons of Ox1RΔDAT mice (bottom). The recordings show the responses to 100 nM and 300 nM orexin A. The dashed lines indicate the range where the responses to 100 nM were quantified. Heat maps of all recorded neurons are shown in Figure 1—figure supplement 4. (L) Top: The stacked bar shows the percentage of individual dopaminergic neurons in control mice in which the increase in [Ca2+]i was larger than three times the standard deviation of the baseline fluorescence (3 σ criterion ≙ Z-score of 3), thus defining them as orexin A responsive (see Materials and Methods). The dopaminergic neurons in Ox1RΔDAT mice did not respond to 100 nM orexin A. Bottom: Population Ca2+ responses upon 100 nM orexin A application from all recorded dopaminergic SN neurons of control and Ox1RΔDAT mice. Data are shown as the percentage of the maximal response to high K+ saline. The significance of this mean response was tested for each group (control and Ox1RΔDAT) using one-sample t-tests (control: p < 0.0001, n = 71; Ox1RΔDAT: p = 0.5, n = 86). Bar graphs represent means ± SEM. p-values are provided above the bar graphs. n-values are given in brackets below the bar graphs. The full statistics are provided alongside the source data.

with 5 supplements. Deficiency of Ox1R in dopaminergic neurons increases locomotion and exploration behaviors.

(A) Total traveling distance, (B) ambulating time, (C) vertical activity and (D) stereotypic activity of male mice in the open field test. Control, n = 14; Ox1RΔDAT, n = 15. (E) Total traveling distance, (F) ambulating time, (G) vertical activity and (H) stereotypic activity of female mice in the open field test. Control, n = 14; Ox1RΔDAT, n = 12. Locomotor activity upon intracerebroventricular (ICV) injection of saline (NS) or orexin A in (I) male and (K) female control and Ox1RΔDAT mice. Grey boxes indicate the pre- and post-injection period for the area under curve (AUC) quantification. (J, L) Quantification of AUC pre- and post-saline and orexin A injection to (J) male and (L) female mice. Male (control and Ox1RΔDAT), n = 8; female-control, n = 9; female-Ox1RΔDAT, n = 10. Data are represented as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; as determined by unpaired two-tailed Student’s t-test (A-H), or mixed two-way ANOVA followed by Sidak’s post hoc test (J, L). (J) Treatment: F (1, 14) = 2.34, p = 0.15; genotype: F (3, 42) = 38.59, p < 0.0001; treatment x genotype: F (3, 42) = 3.76, p = 0.018; subject: F (14, 42) = 2.27, p = 0.02. (L) Treatment: F (1, 17) = 3.73, p = 0.084; genotype: F (3, 51) = 45.52, p < 0.0001; treatment x genotype: F(3, 51) = 3.23, p = 0.030; subject: F (17, 51) = 1.55, p = 0.12. . The full statistics are provided alongside the source data.

with 3 supplements. PET imaging studies comparing Ox1RΔDAT and control mice.

3D maps of p-values in PET imaging studies comparing Ox1RΔDAT and control mice, after intracerebroventricular (ICV) injection of (A) saline (NS) and (B) orexin A. Brain areas with significant changes are indicated. Control-NS, n = 8; control-orexin, n = 6; Ox1RΔDAT (NS and orexin), n = 8. Data are compared by unpaired two-tailed Student’s t-test. M2, secondary motor cortex; MPA, medial preoptic area; Pir, piriform cortex; IEn, intermediate endopiriform claustrum; DEn, dorsal endopiriform claustrum; VEn, ventral endopiriform claustrum; LSS, lateral stripe of the striatum; BNST, the dorsal bed nucleus of the stria terminalis; HDB, nucleus of the horizontal limb of the diagonal band; MCPO, magnocellular preoptic nucleus; S1Sh, primary somatosensory cortex, shoulder region; S1HL, primary somatosensory cortex, hindlimb region; S1BF, primary somatosensory cortex, barrel field; S1Tr, primary somatosensory cortex, trunk region; V1, primary visual cortex; V2L, secondary visual cortex, lateral area; SubCV, subcoeruleus nucleus, ventral part; Gi, gigantocellular reticular nucleus; IRt, intermediate reticular nucleus; LPGi, lateral paragigantocellular nucleus; Sp5O, spinal trigeminal nucleus, oral part; Sp5I, spinal trigeminal nucleus, interpolar part; sp5, spinal trigeminal tract. More statistical information is provided in figure 3—figure supplement 1 and 2 and in the source data.

with 3 supplements. c-Fos and dopamine receptors in the lateral paragigantocellular nucleus (LPGi) and the dorsal bed nucleus of the stria terminalis (BNST).

(A) Representative images of c-Fos and tyrosine hydroxylase (Th) staining in LPGi of control and Ox1RΔDAT mice injected (ICV) with saline (NS) and orexin A. Quantification of (B) Th fluorescence and (C) c-Fos positive neurons in LPGi. (D) Representative images of D1 and D2 subtypes of dopamine receptor (DRD1 and DRD2) in LPGi of control and Ox1RΔDAT mice. (E) Quantification of DRD2 fluorescence in LPGi. (F) Representative images of a negative control staining of DRD1 and DRD2 in LPGi of control mice, and (G) a positive control staining around the lateral ventricle (LV). (H) Representative images of c-Fos and Th staining in the dorsal BNST of control and Ox1RΔDAT mice injected (ICV) with saline or orexin A. Quantification of (I) Th fluorescence and (J) c-Fos positive neurons in dorsal BNST. (K) Representative images of DRD1 and DRD2 in the dorsal BNST of control and Ox1RΔDAT mice. In the interest of clarity, the representative images for single channel signal are shown in the Figure 4—figure supplement 3. Quantification of (L) DRD1 and (M) DRD2 fluorescence in dorsal BNST. Scale bar: 200 µm (D, F, G, K), 100 µm (A, H) or 50 µm (insertions in H). Control, n = 3; Ox1RΔDAT, n = 4. Cyan, Th; red, c-Fos (A, H). Magenta, DRD1; cyan, DRD2; blue, dapi (D, F, G, K). Data are represented as means ± SEM. *p < 0.05; ***p < 0.001; as determined by unpaired two-tailed Student’s t-test (E, L, M), or two-way ANOVA followed by Sidak’s post hoc test (B, C, I, J). (B) treatment: F(1, 10) = 1.76, p = 0.21; genotype: F (1, 10) = 2.83, p = 0.12; treatment x genotype: F(1,10) = 0.15, p = 0.71; (C) treatment: F(1, 10) = 27.94, p = 0.0004; genotype: F (1, 10) = 36.42, p = 0.0001; treatment x genotype: F(1,10) = 5.72, p = 0.04; (I) treatment: F(1, 10) = 1.24, p = 0.29; genotype: F (1, 10) = 0.95, p = 0.35; treatment x genotype: F (1,10) = 0.26, p = 0.62; (J) treatment: F (1, 10) = 5.76, p = 0.04; genotype: F (1, 10) = 0.05, p = 0.83; treatment x genotype: F (1,10) = 3.65, p = 0.09.