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Dissociating orexin-dependent and -independent functions of orexin neurons using novel Orexin-Flp knock-in mice

  1. Srikanta Chowdhury
  2. Chi Jung Hung
  3. Shuntaro Izawa
  4. Ayumu Inutsuka
  5. Meiko Kawamura
  6. Takashi Kawashima
  7. Haruhiko Bito
  8. Itaru Imayoshi
  9. Manabu Abe
  10. Kenji Sakimura
  11. Akihiro Yamanaka  Is a corresponding author
  1. Nagoya University, Japan
  2. CREST, JST, Honcho Kawaguchi, Japan
  3. Niigata University, Japan
  4. The University of Tokyo, Japan
  5. Kyoto University, Japan
Research Article
Cite this article as: eLife 2019;8:e44927 doi: 10.7554/eLife.44927
10 figures, 2 tables and 1 additional file


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.

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.

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.

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.

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.

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).

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.

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.

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.



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.

Orexin-Flp (KI/KI)WTOrexin-Flp (KI/KI)WTOrexin-Flp (KI/KI)WTOrexin-Flp (KI/KI)WT
24 hrTotal time (min)96.0 ± 5.792.2 ± 4.618.7 ± 6.3584.6 ± 19.1555.7 ± 14.8740.7 ± 18.3792.1 ± 19.1
Duration (sec)55.7 ± 4.257.3 ± 2.461.5 ± 12.181.4 ± 7.094.8 ± 5.8102.1 ± 8.4*189.9 ± 16.8
Bouts110.7 ± 8.596.8 ± 7.517.4 ± 5.2442.3 ± 37.4*359.3 ± 20.8453.0 ± 36.4*358.6 ± 20.8
Light periodTotal time (min)57.3 ± 3.8*68.4 ± 2.92.1 ± 0.8355.1 ± 13.5*390.8 ± 4.9305.5 ± 14.6*260.8 ± 7.2
Duration (sec)48.4 ± 3.9*58.9 ± 1.848.9 ± 13.093.4 ± 7.594.6 ± 5.080.1 ± 4.6*63.1 ± 4.2
Bouts73.4 ± 7.670.0 ± 4.42.0 ± 0.7234.6 ± 17.3251.8 ± 14.1231.0 ± 17.6251.1 ± 13.9
Dark periodTotal time (min)38.7 ± 4.8*23.8 ± 2.516.6 ± 5.6229.5 ± 12.4*164.9 ± 10.5435.2 ± 11.3*531.3 ± 12.7
Duration (sec)63.0 ± 6.255.6 ± 4.074.1 ± 19.869.3 ± 8.2*94.9 ± 7.6124.0 ± 12.7*316.6 ± 30.9
Bouts37.3 ± 4.3*26.8 ± 3.515.4 ± 4.7207.7 ± 21.1*107.5 ± 10.5222.0 ± 20.6*107.5 ± 10.6
Key resources table
Reagent type
(species) or
DesignationSource or
Strain, strain backgroundAAV(9)-CMV-dFRT-hM3Dq-mCherryThis paperNATiter: 1.0 × 1012particles/ml
Strain, strain backgroundAAV(DJ)-CMV-dFRT-hrGFPThis paperNATiter: 3.7 × 1012particles/ml
Strain, strain backgroundAAV(DJ)-CMV-FLEX-tdTomatoThis paperNATiter: 2.0 × 1012particles/ml
Genetic reagent (Mus musculus)Orexin-FlippaseThis paperOrexin-FlpKnock-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-CreHigo et al., 2009Gad67-Cre
Genetic reagent (Mus musculus)R26-CAG-FRT-STOP-FRT-mTFP1Imayoshi et al., 2012FSF-mTFP1
Genetic reagent (Mus musculus)Melanin-concentrating hormone-CreKong et al., 2010MCH-Cre
Genetic reagent (Mus musculus)Orexin-enhanced green fluorescence proteinYamanaka et al., 2003Orexin-EGFP
Antibodygoat polyclonal anti-orexinSanta Cruz Biotechnologysc-8070(1/1000)
Antibodyrabbit polyclonal anti-MCHSigma-AldrichM8440(1/2000)
Antibodyrabbit polyclonal anti-prodynorphinMerck MilliporeAB5519(1/100)
Antibodymouse monoclonal anti-c-FosSanta Cruz BiotechnologyE-8(1/500)
Antibodymouse monoclonal anti-DsREDSanta Cruz Biotechnologysc-390909(1/1000)
Antibodymouse monoclonal anti-GFPFujifilm Wako Pure Chemical CorporationmFX75(1/1000)
Sequence-based reagent (primer)forward primer: 5’-CTCATTAGTACTCGGAAACTGCCC-3’This paperNAPrimer for PCR genotyping of orexin-Flp mouse tail DNA
Sequence-based reagent (primer)reverse primer: 5’-AAGCACTATCATGGCCTCAGTAGT-3’This paperNAPrimer for PCR genotyping of orexin-Flp mouse tail DNA
Chemical compound, drugclozapine-N-oxide (CNO)Enzo Life SciencesBML-NS105-0025
Chemical compound, drugPicrotoxinSigma-AldrichP1675
Chemical compound, drugtetrodotoxin (TTX)Alomone LabsCat# T-550
Chemical compound, drugD-2-Amino-5-phosphopentanoic acid (AP5)Alomone LabsCat# D-145
Chemical compound, drug6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)Sigma-AldrichCat# C127
Chemical compound, drugLidocaine N-ethyl bromide (QX-314 bromide)Alomone LabsCat# Q-100
Software, algorithmOrigin 2017LightstoneOrigin 2018
Software, algorithmSleepSignKissei ComtecVersion 3
Software, algorithmpClamp 10.5Software and AlgorithmsMolecular DevicesRRID:SCR_011323
Software, algorithmImageJhttps://imagej.nih.gov/ij/RRID:SCR_003070
OtherDAPI stainThermo Fisher ScientificCat# D1306

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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