Dissociating orexin-dependent and -independent functions of orexin neurons using novel Orexin-Flp knock-in mice
- Nagoya University, Japan
- CREST, JST, Honcho Kawaguchi, Japan
- Niigata University, Japan
- The University of Tokyo, Japan
- Kyoto University, Japan
Figures
Figure 1
Generation of OF mice.
(A) Schematic representations of the hypocretin gene, targeting vector, and targeted gene. To achieve orexin neuron-specific expression of Flp recombinase, we inserted EGFP-2A-Flp just behind the translation initiation site of the hypocretin gene in-frame. Viral T2A peptide is cleaved just after translation, and EGFP and Flp recombinase localize independently. DT, diphtheria toxin; Neo, neomycin-resistant gene expression cassette. (B) Structure of the reporter gene in the presence of Flp. Orexin-Flp; FSF-mTFP1 bigenic mice were generated to express mTFP1 in orexin neurons. (C) Representative pictures from coronal brain sections of Orexin-Flp; FSF-mTFP1 bigenic mice. Arrow indicates mTFP1 and/or EGFP expressing orexin neurons and the arrowhead indicates the position of the MCH neuron. Orexin-IR, orexin-immunoreactive; MCH-IR, MCH-immunoreactive. Inset scale bar: 20 µm. (D) Summary of the co-expression analysis (n = 4 mice). The p values were determined using one-way ANOVA test followed by a post-hoc Tukey analysis. Data represent the mean ± SEM.
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Figure 1—source data 1
Source data for Figure 1D.
- https://doi.org/10.7554/eLife.44927.003
Figure 2
Gene expression control using OF mice to target different cell types within the same brain region.
(A) Schematic showing Cre (left) and Flp (right) recombinase-dependent gene expression control. (B and E) The breeding scheme for Orexin-Flp; MCH-Cre and Orexin-Flp; Gad67-Cre bigenic mice, respectively. (C and F) Schematic drawings showing micro-injection of the AAV cocktail into the LHA of bigenic mice. (D and G) Representative coronal brain sections showing the segregated expression of tdTomato and hrGFP in a Cre and Flp recombinase-dependent manner, respectively.
Figure 3 with 1 supplement
Immunohistochemical confirmation of OF mice.
(A and B) Representative coronal brain sections showing the expression of EGFP (green), orexin (red) and dynorphin (blue). GFP-IR, GFP-immunoreactive; Dynorphin-IR, Dynorphin-immunoreactive; Orexin-IR, orexin-immunoreactive.
Figure 3—figure supplement 1
Immunohistochemical expression analysis in OF (KI/-) and OF (KI/KI) mice.
(A) Co-expression analysis showing dynorphin-positive neurons are also positive for orexin immunoreactivity in OF (KI/-) and OF (KI/KI) mice (n = 3 mice each). (B) Bar diagram showing the co-expression analysis in OF (KI/-) mice (n = 3 mice). The p value was determined using two-tailed unpaired student’s t-test. Data represent mean ± SEM.
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1A and B.
- https://doi.org/10.7554/eLife.44927.007
Figure 4
Electrophysiological properties of orexin neurons with/without orexin.
(A-C) show representative traces recorded from OF (KI/-) (i), OF (KI/KI) (ii) and OE (iii) mice. (A) Membrane potential in whole-cell current clamp recordings. (B) Spontaneous firing in cell-attached recordings. (C) Step current injection-induced membrane potential changes. Panel iv is a summary of the data in panel i to iii. (D) Representative images showing EGFP expression in acute coronal brain slices during electrophysiological recording. (E) Cell capacitance from whole-cell current clamp recording. The p values were determined using one-way ANOVA test followed by a post-hoc Tukey analysis. Data represent the mean ± SEM.
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Figure 4—source data 1
Source data for Figure 4Aiv, 4Biv, 4Civ and 4E.
- https://doi.org/10.7554/eLife.44927.009
Figure 5
Orexin neurons receive fewer glutamatergic inputs in the absence of orexin.
(A) Representative sEPSC traces recorded from EGFP-expressing neurons at a holding potential of −60 mV. (B-C) Cumulative probability plot for the representative traces shown in A. Bar diagrams in D and E summarize the sEPSC data. (D) Inter-event interval. (E) amplitude (n = 25–29 cells). F, Representative sIPSC traces recorded from EGFP-expressing neurons at a holding potential of −60 mV. (G-H) Cumulative probability plot for the representative traces shown in F. Bar diagrams in (I and J) summarize the sIPSC data. (G) Inter-event interval. (H) amplitude (n = 17–22 cells). The p values were calculated by one-way ANOVA followed by a post-hoc Tukey test. Data represent the mean ± SEM.
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Figure 5—source data 1
Source data for Figure 5D, E and I, J.
- https://doi.org/10.7554/eLife.44927.011
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Figure 5—source data 2
Source data for Figure 5D, E and I, J.
- https://doi.org/10.7554/eLife.44927.012
Figure 6
Selective chemogenetic activation of orexin neurons.
(A) Intracranial injection of AAV in OF mice to achieve Flp-dependent expression of hM3Dq-mCherry fusion protein in orexin neurons. (B) Representative images showing the expression of mCherry in orexin-immunoreactive neurons in heterozygous OF (KI/-) mice. (C) c-Fos expression after i.p. administration of either saline or CNO in both OF (KI/-) and OF (KI/KI) mice. (D) Summary of the immunostaining data shown in C. CNO administration can significantly increase neuronal activity in both heterozygous and homozygous mice (n = 3 mice per group). (E) Schematic showing the schedule of i.p. administration during sleep recording. The p values were determined by a two-tailed student’s t-test; data represent the mean ± SEM.
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Figure 6—source data 1
Source data for Figure 6D.
- https://doi.org/10.7554/eLife.44927.015
Figure 7 with 1 supplement
Chemogenetic activation of orexin neurons resulted in altered sleep/wakefulness based on the availability of orexin.
(A) Line graph with symbols showing the time spent in wakefulness (i), REM sleep (ii), NREM sleep (iii), and cataplexy (iv) during each hour for the 6 hr following CNO or saline administration during light period. B, Bar graph showing the 2 hr average of the total time spent in wakefulness (i), REM sleep (ii), NREM sleep (iii), and cataplexy (iv) following CNO or saline injection during the light period. C, Scatter plot showing the averaged bout and duration in wakefulness (i), REM sleep (ii), NREM sleep (iii), and cataplexy (iv). The data in D-F are shown similar to the representation in A-C, respectively, recorded during the dark period (OF (KI/-): n = 9 mice and OF (KI/KI): n = 8 mice). Data represent the mean ± SEM in both the line and bar graph. The p values were determined by either two-way ANOVA followed by a post-hoc Bonferroni test or paired student’s t-test (cataplexy).
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Figure 7—source data 1
Source data for Figure 7A, B, C, E and F.
- https://doi.org/10.7554/eLife.44927.018
Figure 7—figure supplement 1
Representative hypnogram of OF mice after i.p. administration of either saline or CNO.
(A and B) hypnograms showing vigilance state change for 2 hr after i.p. administration during either light (A) or dark (B) period in OF (KI/-, i and ii) or OF (KI/KI, iii and iv) mice. C: cataplexy, W: wakefulness, N: NREM sleep, R: REM sleep.
Figure 8
CNO-induced cataplexy was not different from naïve cataplexy.
(A) hypnogram of 2 hr after saline or CNO administration. (B) typical traces showing EEG signal, EEG power spectrum and EMG of cataplexy episode. (C) line graph (left) and bar graph (right) showing relative power spectrum of EEG during cataplexy episode in the light period (n = 8 mice). Data represent the mean ± SEM in both the line and bar graph.
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Figure 8—source data 1
Source data for Figure 8C.
- https://doi.org/10.7554/eLife.44927.020
Author response image 1
Bouts and duration of wakefulness in OF (KI/-) (n = 4) and OF (KI/KI) (n = 7) mice after saline injection for 12 hr).
Data represent mean ± S.E.M.
Author response image 2
Number of wakefulness bouts of short duration (~5 min), middle duration (5 min-1 hr) and long duration (1 hr~) in WT mice and OF (KI/KI) mice during the dark period.
Data represent mean ± S.E.M.
Tables
Table 1
Vigilance state parameters recorded from OF (KI/KI) and WT animals.
The table shows total time spent in individual states in minutes, duration of respective states in seconds and number of episodes (bouts) observed in either 24 hr, or only in light or dark period in OF (KI/KI, n = 7 mice; WT, n = 8 mice). *, p<0.05. The p values were determined using two-tailed unpaired student’s t-test. Values are represented as the mean ± SEM.
| REM | Cataplexy | NREM | Wake | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Orexin-Flp (KI/KI) | WT | Orexin-Flp (KI/KI) | WT | Orexin-Flp (KI/KI) | WT | Orexin-Flp (KI/KI) | WT | ||
| 24 hr | Total time (min) | 96.0 ± 5.7 | 92.2 ± 4.6 | 18.7 ± 6.3 | 584.6 ± 19.1 | 555.7 ± 14.8 | 740.7 ± 18.3 | 792.1 ± 19.1 | |
| Duration (sec) | 55.7 ± 4.2 | 57.3 ± 2.4 | 61.5 ± 12.1 | 81.4 ± 7.0 | 94.8 ± 5.8 | 102.1 ± 8.4* | 189.9 ± 16.8 | ||
| Bouts | 110.7 ± 8.5 | 96.8 ± 7.5 | 17.4 ± 5.2 | 442.3 ± 37.4* | 359.3 ± 20.8 | 453.0 ± 36.4* | 358.6 ± 20.8 | ||
| Light period | Total time (min) | 57.3 ± 3.8* | 68.4 ± 2.9 | 2.1 ± 0.8 | 355.1 ± 13.5* | 390.8 ± 4.9 | 305.5 ± 14.6* | 260.8 ± 7.2 | |
| Duration (sec) | 48.4 ± 3.9* | 58.9 ± 1.8 | 48.9 ± 13.0 | 93.4 ± 7.5 | 94.6 ± 5.0 | 80.1 ± 4.6* | 63.1 ± 4.2 | ||
| Bouts | 73.4 ± 7.6 | 70.0 ± 4.4 | 2.0 ± 0.7 | 234.6 ± 17.3 | 251.8 ± 14.1 | 231.0 ± 17.6 | 251.1 ± 13.9 | ||
| Dark period | Total time (min) | 38.7 ± 4.8* | 23.8 ± 2.5 | 16.6 ± 5.6 | 229.5 ± 12.4* | 164.9 ± 10.5 | 435.2 ± 11.3* | 531.3 ± 12.7 | |
| Duration (sec) | 63.0 ± 6.2 | 55.6 ± 4.0 | 74.1 ± 19.8 | 69.3 ± 8.2* | 94.9 ± 7.6 | 124.0 ± 12.7* | 316.6 ± 30.9 | ||
| Bouts | 37.3 ± 4.3* | 26.8 ± 3.5 | 15.4 ± 4.7 | 207.7 ± 21.1* | 107.5 ± 10.5 | 222.0 ± 20.6* | 107.5 ± 10.6 | ||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background | AAV(9)-CMV-dFRT-hM3Dq-mCherry | This paper | NA | Titer: 1.0 × 1012particles/ml |
| Strain, strain background | AAV(DJ)-CMV-dFRT-hrGFP | This paper | NA | Titer: 3.7 × 1012particles/ml |
| Strain, strain background | AAV(DJ)-CMV-FLEX-tdTomato | This paper | NA | Titer: 2.0 × 1012particles/ml |
| Genetic reagent (Mus musculus) | Orexin-Flippase | This paper | Orexin-Flp | Knock-in mice; EGFP-2A-Flp was placed just behind the translational initiation site of the hypocretin gene in-frame |
| Genetic reagent (Mus musculus) | Glutamic acid decarboxylase 67-Cre | Higo et al., 2009 | Gad67-Cre | |
| Genetic reagent (Mus musculus) | R26-CAG-FRT-STOP-FRT-mTFP1 | Imayoshi et al., 2012 | FSF-mTFP1 | |
| Genetic reagent (Mus musculus) | Melanin-concentrating hormone-Cre | Kong et al., 2010 | MCH-Cre | |
| Genetic reagent (Mus musculus) | Orexin-enhanced green fluorescence protein | Yamanaka et al., 2003 | Orexin-EGFP | |
| Antibody | goat polyclonal anti-orexin | Santa Cruz Biotechnology | sc-8070 | (1/1000) |
| Antibody | rabbit polyclonal anti-MCH | Sigma-Aldrich | M8440 | (1/2000) |
| Antibody | rabbit polyclonal anti-prodynorphin | Merck Millipore | AB5519 | (1/100) |
| Antibody | mouse monoclonal anti-c-Fos | Santa Cruz Biotechnology | E-8 | (1/500) |
| Antibody | mouse monoclonal anti-DsRED | Santa Cruz Biotechnology | sc-390909 | (1/1000) |
| Antibody | mouse monoclonal anti-GFP | Fujifilm Wako Pure Chemical Corporation | mFX75 | (1/1000) |
| Sequence-based reagent (primer) | forward primer: 5’-CTCATTAGTACTCGGAAACTGCCC-3’ | This paper | NA | Primer for PCR genotyping of orexin-Flp mouse tail DNA |
| Sequence-based reagent (primer) | reverse primer: 5’-AAGCACTATCATGGCCTCAGTAGT-3’ | This paper | NA | Primer for PCR genotyping of orexin-Flp mouse tail DNA |
| Chemical compound, drug | clozapine-N-oxide (CNO) | Enzo Life Sciences | BML-NS105-0025 | |
| Chemical compound, drug | Picrotoxin | Sigma-Aldrich | P1675 | |
| Chemical compound, drug | tetrodotoxin (TTX) | Alomone Labs | Cat# T-550 | |
| Chemical compound, drug | D-2-Amino-5-phosphopentanoic acid (AP5) | Alomone Labs | Cat# D-145 | |
| Chemical compound, drug | 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) | Sigma-Aldrich | Cat# C127 | |
| Chemical compound, drug | Lidocaine N-ethyl bromide (QX-314 bromide) | Alomone Labs | Cat# Q-100 | |
| Software, algorithm | Origin 2017 | Lightstone | Origin 2018 | |
| Software, algorithm | SleepSign | Kissei Comtec | Version 3 | |
| Software, algorithm | pClamp 10.5Software and Algorithms | Molecular Devices | RRID:SCR_011323 | |
| Software, algorithm | ImageJ | https://imagej.nih.gov/ij/ | RRID:SCR_003070 | |
| Other | DAPI stain | Thermo Fisher Scientific | Cat# D1306 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.44927.021
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Dissociating orexin-dependent and -independent functions of orexin neurons using novel Orexin-Flp knock-in mice
eLife 8:e44927.
https://doi.org/10.7554/eLife.44927
