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. (I) Schematic illustration of 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. (J-L) Effect of orexin A on dopaminergic SN neurons analyzed by Ca2+ imaging with GCaMP6. Recordings were performed in brain slices from control and Ox1RΔDAT male mice with GCaMP6 expressed in dopaminergic SN neurons for Ca2+ imaging. (J) Schematic illustration of the SN. The red frame indicates the region where calcium imaging was performed. (K, L) Orexin A effect on [Ca2+]i of dopaminergic SN neurons. (K) Heat maps of 5 individual dopaminergic SN neurons from control (top) and Ox1RΔDAT mice. The recordings show the responses to 100 nM orexin A. The dashed lines indicate the range where the responses were quantified. (L) Mean Ca2+ responses upon 100 nM orexin A application. Data are shown as the percentage of the maximal response to high K+ saline. Bar graphs represent means ± SEM. The stacked bar shows the percentage of individual dopaminergic neurons in control mice in which the increase in [Ca2+]i was larger than 3 x SD of the control, thus defining them as orexin A responsive (see Materials and Methods). The significance of mean changes in [Ca2+]i during orexin A application were compared using one-sample t-tests. **** p < 0.0001. n values are given in brackets.

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. (J, L) Quantification of the area under curve (AUC) after orexin injection to (J) male and (L) female mice. Male, n = 8; female-control, n = 9; female-Ox1RΔDAT, n = 10. Data are represented as means ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001; as determined by unpaired two-tailed Student’s t-test (A-C, E-H), or two-way ANOVA followed by Sidak’s post hoc test (J, L).

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, n = 8. 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.

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. 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 (L, M), or two-way ANOVA followed by Sidak’s post hoc test (C, J).