Deficiency of orexin receptor type 1 in dopaminergic neurons increases novelty-induced locomotion and exploration
- Max Planck Institute for Metabolism Research, Department of Neuronal Control of Metabolism, Germany
- Department of Biology, Institute for Zoology, University of Cologne, Germany
- Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Germany
- Max Planck Institute for Metabolism Research, Multimodal Imaging of Brain Metabolism Group, Germany
Figures
Figure 1 with 4 supplements
Orexin receptor subtype 1 (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 Ox1R∆DAT 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 panels (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. Three 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; gray, 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 five individual orexin A responsive dopaminergic SN neurons from control (top) and five 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.
Figure 1—figure supplement 1
An overview of orexin receptor expression in the substantia nigra (SN) and the ventral tegmental area (VTA).
Representative images of RNAscope in situ hybridization in SN and interfascicular (IF), paranigral (PN), parainterfascicular (PIF), and parabrachial (PBP) subnuclei of VTA, of (A) a control, and (B) an Ox1RΔDAT mouse. The amplifications of areas marked in blue squares are shown in Figure 1A for the control mouse and in Figure 1B for the Ox1RΔDAT mouse. White, tyrosine hydroxylase (Th); magenta, orexin receptor subtype 1 (Ox1R); green, Ox2R. Scale bar: 200 µm. Control, n=3; Ox1RΔDAT, n=4.
Figure 1—figure supplement 2
Orexin A increases the action potential frequency of dopaminergic neurons in the substantia nigra (SN).
Recordings were performed from dopaminergic SN neurons in acute brain slices of control mice in the perforated patch clamp configuration to preserve intracellular signaling pathways. In all recordings, GABAergic and glutamatergic synaptic input was pharmacologically blocked (see Materials and methods). Orexin A was bath applied at 100 nM and 300 nM for 8 min for each concentration. (A) Top: Schematic illustration of the substantia nigra where the recordings have been performed (red crosshatched). Bottom: Image of a recorded dopaminergic SN neuron, which was filled with biocytin after converting the perforated patch clamp configuration to the whole-cell configuration. The neuron was double-labeled with biocytin-streptavidin (red) and an anti-serum against tyrosine hydroxylase (green). Scale bar: 20 µm. (B) Orexin A effect on action potential firing rate of dopaminergic SN neurons. Rate histograms (bin width: 50 s) (top) and representative sections of the original recordings (bottom). The numbers mark the recording sections displayed in high time resolution as original traces (bottom). (C) Top: The stacked bars show the number of individual neurons in which the increase in action potential frequency was three times larger than the standard deviation (3 σ criterion ≙ Z-score of 3) of the control, thus defining them as responsive (see Materials and methods) (exc, excited; nr, not responsive). Bottom: The population effect of orexin A on the action potential frequency was measured as the orexin A induced change in frequency (Δ frequency) and tested for significance using a one-ample Wilcoxon matched-pairs signed rank test (p=0.016, n=8). The horizontal lines in the box plots show the median. The whiskers were calculated using the Tukey method. Recordings not responding to 100 nM orexin A are indicated in red. n values are given in brackets. SNR, substantia nigra pars reticulata; SNC, substantia nigra pars compacta; VTA, ventral tegmental area; OxA, orexin A.
Figure 1—figure supplement 3
Orexin A and high K+ saline effect on [Ca2+]i of dopaminergic neurons in the substantia nigra (SN) of control and Ox1RΔDAT mice which express GCaMP6s.
Change in GCaMP6 fluorescence induced by orexin A (100 nM and 300 nM) and high (40 mM) K+ saline, to which the orexin A responses were normalized for quantification (see Figure 1L). As described in Materials and methods, the orexin A response can be expressed for each neuron individually as a percentage of its high K+-induced GCaMP6 fluorescence. This value is a solid reference point reflecting the GCaMP6 fluorescence at maximal voltage-activated Ca2+ influx. The Ca2+ concentration at this point is exceptionally high and not typically reached under physiological conditions. Therefore, as shown in (A), the physiologically relevant responses may, at first glance, appear to be relatively small when plotted together with the high K+ response. In (A) and (B), for completeness, different presentation options and scales are exemplified using the five orexin A-responsive dopaminergic SN neurons from control mice and the five not orexin A-responsive dopaminergic neurons of Ox1RΔDAT mice neurons from Figure 1K (Heat maps of all recorded neurons are shown in Figure 1—figure supplement 4). Top: mean responses. Error bars are not displayed for clarity reasons. Bottom: Heat maps showing the Orexin A effect of the individual neurons. On the single-cell level, neurons were considered orexin A-responsive when the change in [Ca2+]i was larger than three times the standard deviation (3 σ criterion ≙ Z-score of 3) of the baseline fluorescence (see Materials and methods). OxA, orexin A.
Figure 1—figure supplement 4
Orexin A and high K+ saline effect on [Ca2+]i of dopaminergic neurons in the substantia nigra (SN) of control and Ox1RΔDAT mice which express GCaMP6s.
(A) Individual heat maps showing the change in GCaMP6s fluorescence induced by 100 nM and 300 nM orexin A from dopaminergic SN neurons from (left) control (n=71) and (right) Ox1RΔDAT mice (n=86). The asterisks indicate the example responses shown in Figure 1K, Figure 1—figure supplement 3. (B) Mean responses of all recorded dopaminergic neurons of control and Ox1RΔDAT mice are shown in (A). Error bars are not displayed for clarity reasons.
Figure 2 with 5 supplements
Deficiency of orexin receptor subtype 1 (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; Ox1R∆DAT, 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; Ox1R∆DAT, n=12. Locomotor activity upon intracerebroventricular (ICV) injection of saline (NS) or orexin A in (I) male and (K) female control and Ox1RΔDAT mice. Gray 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 Ox1R∆DAT), n=8; female-control, n=9; female-Ox1R∆DAT, 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 × 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 × 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.
Figure 2—figure supplement 1
Unaltered mouse spontaneous activity in home cages by deficiency of orexin receptor subtype 1 (Ox1R) in dopaminergic neurons.
Spontaneous activity in home cages of (A) male and (B) female mice. Top: original counts, bottom: area under curve (AUC) was calculated for unpaired two-tailed Student’s t-test. There were no significant changes between groups. Male-control, n=10; male-Ox1RΔDAT, n=12; female-control, n=11; female-Ox1RΔDAT, n=11. Data are represented as means ± SEM. The full statistics are provided alongside the source data.
Figure 2—figure supplement 2
Increased exploratory behaviors of female Ox1RΔDAT in the hole-board (HB) test and the novel object test (NOT).
(A) Nose pokes of male mice in the hole-board test. Control, n=10; Ox1RΔDAT, n=9. (B) Nose pokes of female mice in the hole-board test. Control, n=8; Ox1RΔDAT, n=7. Exploration time of the objects by male mice at (C) acclimation phase and (D) novelty-exposure phase in the novel object test. Control, n=13; Ox1RΔDAT, n=14. Exploration time of the objects by female mice at (E) acclimation phase and (F) novelty-exposure phase in the novel object test. Control, n=15; Ox1RΔDAT, n=11. Data are represented as means ± SEM. *p<0.05; **p<0.01; ***p<0.001; as determined by two-way ANOVA followed by Sidak’s post hoc test. (A) Mixed-effect analysis (matching across time) was used because two-way ANOVA could not be performed due to one missing value in the Ox1RΔDAT group in the last session: time, p<0.0001; genotype, p=0.53; time × genotype, p=0.98. Two-way ANOVA (matching across time or object): (B) time: F (7, 91)=92.57, p<0.0001; genotype: F (1, 13)=7.41, p=0. 02; time × genotype: F (7, 91)=3.07, p=0.006; subject: F (13, 91)=2.15, p=0.02; (C) object: F (1, 25)=0.10, p=0.76; genotype: F (1, 25)=0.04, p=0.55; object × genotype: F (1, 25)=2.50, p=0.13; subject: F (25, 25)=5.94, p<0.0001; (D) object: F (1, 25)=2.45, p=0.13; genotype: F (1, 25)=0.23, p=0.64; object × genotype: F (1, 25)=0.04, p=0.84; subject: F (25, 25)=1.45, p=0.18; (E) object: F (1, 24)=2.98, p=0.10; genotype: F (1, 24)=16.23, p=0.0005; object × genotype: F (1, 24)=0.02, p=0.89; subject: F (24, 24)=1.94, p=0.06; (F) object: F (1, 24)=13.49, p=0.001; genotype: F (1, 24)=3.67, p=0.07; object × genotype: F (1, 24)=0.60, p=0.45; subject: F (24, 24)=1.25, p=0.29. The full statistics are provided alongside the source data.
Figure 2—figure supplement 3
Unaltered anxiety-related behaviors in Ox1RΔDAT mice.
(A) Time spent in the center area and (B) times of entries into the center area by male mice in the open field test (OFT). Control, n=14; Ox1RΔDAT, n=15. (C) Time spent in the center area and (D) times of entries into the center area by female mice in the open field test. Control, n=14; Ox1RΔDAT, n=12. (E) Time spent at the light side and (F) times of entries into the light side by male mice in the dark/light (D/L) box test. Control, n=12; Ox1RΔDAT, n=14. (G) Time spent at the light side and (H) times of entries into the light side by female mice in the dark/light box test. Control, n=8; Ox1RΔDAT, n=5. (I) Time spent in the open arms and (J) times of entries into the open arms by male mice in the 0-maze test. Control, n=13; Ox1RΔDAT, n=16. (K) Time spent in the open arms and (L) times of entries into the open arms by female mice in the 0-maze test. Control, n=8; Ox1RΔDAT, n=5. Data are represented as means ± SEM. No significant differences between groups were detected by unpaired two-tailed Student’s t-test. The full statistics are provided alongside the source data.
Figure 2—figure supplement 4
Unaltered reward-related behaviors in Ox1RΔDAT mice.
Sucrose/sucralose solution preference of male mice expressed as (A) percentages in total daily intake and (B) absolute values of sucrose/sucralose solution consumption in the sucrose/sucralose preference test. Control, n=8–12; Ox1RΔDAT, n=8–10. (C) Conditional place preference (CPP) score of male mice in the CPP test for cocaine. Control, n=7; Ox1RΔDAT, n=6. Sucrose/sucralose solution preference of female mice expressed as (D) percentages in total daily intake and (E) absolute values of sucrose/sucralose solution consumption in the sucrose/sucralose preference test. Control, n=9–12; Ox1RΔDAT, n=9–10. (F) CPP score of female mice in the CPP test for cocaine. Control, n=12; Ox1RΔDAT, n=14. Data are represented as means ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; as determined by one-sample t-test (theoretical value: A and D, 50; C and F, 0). Two-way ANOVA followed by Sidak’s post hoc test: (A) Sweet solution: F (3, 67)=7.29, p=0.0003; genotype: F (1, 67)=0.78, p=0.38; sweet solution × genotype: F (3, 67)=0.23, p=0.87; (B) Sweet solution: F (3, 67)=9.99, p<0.0001; genotype: F (1, 67)=1.05, p=0.31; sweet solution × genotype: F (3, 67)=0.55, p=0.65; (D) Sweet solution: F (3, 67)=2.46, p=0.07; genotype: F (1, 67)=3.65, p=0.06; sweet solution × genotype: F (3, 67)=0.58, p=0.63; (E) Sweet solution: F (3, 67)=8.37, p<0.0001; genotype: F (1, 67)=1.14, p=0.29; sweet solution × genotype: F (3, 67)=0.58, p=0.63. Unpaired two-tailed Student’s t-test: (C) p=0.32, (F) p=0.79. The full statistics are provided alongside the source data.
Figure 2—figure supplement 5
Unaltered energy homeostasis in Ox1RΔDAT mice.
(A) Average body weight (BW) of male mice. Control, n=10; Ox1RΔDAT, n=12. (B) Average body fat content (percentage in BW) of male mice measured by nuclear magnetic resonance. Control, n=10; Ox1RΔDAT, n=16. Phenomaster (PM) analysis of (C) daily food intake, (D) respiratory exchange ratio (RER), and (E) energy expenditure (EE) of male mice. Control, n=10; Ox1RΔDAT, n=11 (C) or 12 (D, E). (F) Insulin tolerance test (ITT) of male mice. Control, n=11; Ox1RΔDAT, n=12. (G) Glucose tolerance test (GTT) of male mice. Control, n=16; Ox1RΔDAT, n=15. (H) Average BW of female mice. Control, n=11; Ox1RΔDAT, n=11. (I) Average body fat content (percentage in BW) of female mice. Control, n=15; Ox1RΔDAT, n=13. PM analysis of (J) daily food intake, (K) RER, and (L) EE of female mice. Control, n=11; Ox1RΔDAT, n=11. (M) ITT of female mice. Control, n=10; Ox1RΔDAT, n=9. (N) GTT of female mice. Control, n=10; Ox1RΔDAT, n=9. Data are represented as means ± SEM. The inserts in D, E, K, and L present the area under curve (AUC) for statistical analysis. No significant differences were found between control and Ox1RΔDAT groups by two-way ANOVA (A, F, G, H, M, N) or unpaired two-tailed Student’s tests (B–E, I–L). (A) Time: F (14, 291)=153.8, p<0.0001; genotype: F (1, 291)=0.13, p=0.72; time × genotype: F (14, 291)=0.21, p>0.99; (F) time: F (3, 84)=17.81, p<0.0001; genotype: F (1, 84)=3.70, p=0.06; time × genotype: F (3, 84)=0.97, p=0.41; (G) time: F (4, 145)=98.95, p<0.0001; genotype: F (1, 145)=1.38, p=0.24; time × genotype: F (4, 145)=0.94, p=0.44; (H) Time: F (14, 247)=102.4, p<0.0001; genotype: F (1, 247)=0.003, p=0.96; time × genotype: F (14, 247)=0.20, p>0.99; (M) time: F (3, 68)=18.57, p<0.0001; genotype: F (1, 68)=0.01, p=0.92; time × genotype: F (3, 68)=0.45, p=0.72; (N) time: F (4, 85)=131.7, p<0.0001; genotype: F (1, 85)=0.002, p=0.96; time × genotype: F (4, 85)=0.20, p=0.94. The full statistics are provided alongside the source data.
Figure 3 with 3 supplements
PET imaging studies comparing Ox1RΔDAT and control mice.
3D maps of p-values in positron emission tomography (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; Ox1R∆DAT (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 supplements 1 and 2 and in the source data.
Figure 3—figure supplement 1
Positron emission tomography (PET) imaging studies comparing Ox1RΔDAT and control mice, shown as p-map images with the original voxel size.
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. Inserted black squares mark the voxels taken as examples to represent the respective area in Figure 3—figure supplement 2, which shows bar graphs with individual dots. 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.
Figure 3—figure supplement 2
Statistical testing for the volumes of interest from brain regions in positron emission tomography (PET) imaging studies.
The cerebral glucose (CE/CP) uptake, as measures for neuronal activity, is compared between Ox1RΔDAT and control (Ctrl) mice, after intracerebroventricular (ICV) injection of saline (NS) or orexin. The volumes of interest are from the brain regions labeled in the respective coronal planes of brain in Figure 3, Figure 3—figure supplement 1. (A) MPA (medial preoptic area); (B) IEn (intermediate endopiriform claustrum) and Pir (piriform cortex); (C) M2 (secondary motor cortex); (D) DEn (dorsal endopiriform claustrum) and Pir, in the coronal planes shown in the second row × second column in Figure 3A and B, Figure 3—figure supplement 1A and B; (E) LSS (lateral stripe of the striatum) and VEn (ventral endopiriform claustrum); (F) BNST (the dorsal bed nucleus of the stria terminalis); (G) MCPO (magnocellular preoptic nucleus) and HDB (nucleus of the horizontal limb of the diagonal band); (H) VEn; (I) S1Sh (primary somatosensory cortex, shoulder region) and S1HL (primary somatosensory cortex, hindlimb region); (J) DEn and Pir, in the coronal planes shown in the third row × second column in Figure 3A and B, Figure 3—figure supplement 1A and B; (K) V2L (secondary visual cortex, lateral area) and V1 (primary visual cortex); (L) Pir; (M) SubCV (subcoeruleus nucleus, ventral part); (N) IRt (intermediate reticular nucleus), Gi (gigantocellular reticular nucleus) and Sp5O (spinal trigeminal nucleus, oral part); (O) LPGi (lateral paragigantocellular nucleus); (P) Sp5I (spinal trigeminal nucleus, interpolar part) and sp5 (spinal trigeminal tract). The row and column location described above the bar figures (D and J) indicates the respective coronal plane in Figure 3, Figure 3—figure supplement 1A and B. Ctrl-NS, n=8; Ctrl-orexin, n=6; Ox1RΔDAT, n=8. Data are represented as means ± SEM. *p<0.05; **p<0.01; ***p<0.001; as determined by unpaired two-tailed Student’s t-test. The full statistics are provided alongside the source data.
Figure 3—figure supplement 3
c-Fos and tyrosine hydroxylase (Th) staining in substantia nigra (SN) and ventral tegmental area (VTA).
(A) Representative images of c-Fos and Th staining in male control and Ox1RΔDAT mice injected (ICV) with saline (NS) or orexin A. (B) Quantification of Th fluorescence in SN and VTA. (C) c-Fos positive non-dopaminergic neurons in SN and VTA. Inserted images at the right up corner of each image are the amplifications of the marked area. Cyan, Th; red, c-Fos. Scale bar: 200 µm or 100 µm (insertions). Control, n=3; Ox1RΔDAT, n=4. Data are represented as means ± SEM. p-value is determined by two-way ANOVA followed by Sidak’s post hoc test. (B) Treatment: F (1, 10)=0.07, p=0.80; genotype: F (1, 10)=0.15, p=0.71; treatment × genotype: F (1, 10)=0.71, p=0.42; (C) treatment: F (1, 10)=8.72, p=0.01; genotype: F (1, 10)=0.07, p=0.79; treatment × genotype: F (1, 10)=0.12, p=0.74. The full statistics are provided alongside the source data.
Figure 4 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 × 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 × 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 × 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 × genotype: F (1,10)=3.65, p=0.09.
Figure 4—figure supplement 1
A schematic illustration of the dorsal bed nucleus of the stria terminalis (dBNST) and lateral paragigantocellular nucleus (LPGi) based on the reference atlas.
Blue text and lines indicate (A) dBNST and (A) LPGi. Unilateral nuclei were analyzed and shown in Figure 4 and its supplements.
Figure 4—figure supplement 2
Dopaminergic fibers innervating the dorsal bed nucleus of the stria terminalis (BNST) and lateral paragigantocellular nucleus (LPGi).
Representative images of GFP staining in (A) substantia nigra (SN) and ventral tegmental area (VTA), (B) the dorsal BNST, and (C) LPGi of DATEYFP mice. Representative images of GFP staining in (D) SN and VTA, (E) the dorsal BNST, and (F) LPGi of DATΔOx1R;EYFP mice. n=3. Scale bar: 500 µm (A, D) or 200 µm (B, C, E, F).
Figure 4—figure supplement 3
Representative images showing dopamine receptors in the dorsal bed nucleus of the stria terminalis (BNST).
(A) Representative images of D1 and D2 subtypes of dopamine receptor (DRD1 and DRD2) in the dorsal BNST of a control (Ctrl) mice. (B) Representative images of D1 and D2 subtypes of dopamine receptor (DRD1 and DRD2) in the dorsal BNST of an Ox1RΔDAT mice. Scale bar: 200 µm. Control, n=3; Ox1RΔDAT, n=4. Magenta, DRD1; cyan, DRD2.
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Deficiency of orexin receptor type 1 in dopaminergic neurons increases novelty-induced locomotion and exploration
eLife 12:RP91716.
https://doi.org/10.7554/eLife.91716.4
