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Dual orexin and MCH neuron-ablated mice display severe sleep attacks and cataplexy

  1. Chi Jung Hung
  2. Daisuke Ono
  3. Thomas S Kilduff
  4. Akihiro Yamanaka  Is a corresponding author
  1. Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Japan
  2. Department of Neural Regulation, Nagoya University Graduate School of Medicine, Japan
  3. CREST, JST, Honcho Kawaguchi, Japan
  4. Center for Neuroscience, Biosciences Division, SRI International, United States
Research Article
Cite this article as: eLife 2020;9:e54275 doi: 10.7554/eLife.54275
10 figures, 3 videos and 3 tables

Figures

Figure 1 with 1 supplement
Dual ablation of orexin and MCH neurons using the tetracycline tTA/TetO system.

(A) Schematic illustrating generation of Hcrt-tTA (Tg/-); Pmch-tTA (Tg/-); TetO DTA (Tg/-) mice. (B) Schematic showing use of the tetracycline-controlled Tet-off gene expression system. In the presence of DOX (light blue circle), DOX binds to tTA (purple oval) and DTA expression is blocked. The absence of DOX allows tTA to bind TetO, allowing transcription of the diphtheria toxin A (DTA) subunit which results in cell death. (C) Experimental protocols for sleep recording and immunostaining. Protocol I is the control (DOX(+)) condition; Protocol II is the experimental (DOX(-) for 4 weeks) condition. EEG and EMG surgeries were performed at age 10 weeks in the sleep-recording group; the mice used for immunostaining did not undergo surgery. Light blue and white bars represent the periods of DOX chow (DOX(+)) and normal chow (DOX(-)) availability, respectively. Gray bars indicate sleep recordings; red arrows indicate when mice were sacrificed for immunostaining. (D) Immunostaining of orexin (brown) and MCH (black) neurons in the LH at DOX(-) 0 week (i and v), 2 weeks (ii and vi), 4 weeks (iii and vii), and DOX(+) 4 weeks (iv and viii). Black and brown arrowheads indicate typical examples of orexin and MCH neurons, respectively. Panels v-viii are magnifications of the areas delineated by the squares in Panels i-iv. Scale bars: i-iv, 500 µm; v-viii, 100 µm. (E) The number of orexin and MCH neurons in OXMC mice from the DOX(+) and (-) conditions (n = 3–6). Values are mean ± SEM. *p<0.05 vs. DOX(+). Data were analyzed by unpaired t test.

Figure 1—figure supplement 1
In situ RNA hybridization of orexin, MCH, vGlut2 and vGAT: orexin- and MCH-positive cells were colocalized with vGlut2 but not with vGAT.

The dotted circle indicates the area used for cell counting. The area enclosed by the rectangle area within the dotted circle is magnified in the 3 insets on the right of each panel. Arrowheads indicate co-expressing neurons. Bar graphs show the ratio of neurons (n = 3–5). Values are means ± SEM. Scale bars: 300 µm and 30 µm (inset). Ox: orexin, ZI: zona incerta. mGP: medial globus pallidus. VMH: ventromedial hypothalamus. LH: lateral hypothalamus.

Time course of orexin and MCH nerve terminal ablation in projection areas.

(A) Immunostaining of MCH nerve terminals (black) in the medium septum (MS). (B) Immunostaining of orexin nerve terminals (black) and tryptophan hydroxylase (Tph; brown) in the raphe nucleus. (C) Immunostaining of orexin nerve terminals (black) and tyrosine hydroxylase (TH; brown) in the locus coeruleus (LC). Photos indicate DOX(-) condition at week 0 (i and v), 2 weeks (ii and vi), 4 weeks (iii and vii) and the DOX(+) condition at 4 weeks (iv and viii). Panels v-viii are magnifications of the areas delineated by the squares in Panels i-iv. Scale bars: i-iv, 500 µm; v-viii, 100 µm.

Criteria used to classify DT sleep.

(A) Representative EEG and EMG traces and EEG power spectral density during NREM and REM sleep for OXMC mice after 4 weeks in the DOX(+) and DOX(-) conditions and for OX mice after 4 weeks in the DOX(-) condition. (B) Representative EEG and EMG traces and EEG power spectral density during cataplexy in OXMC DOX(-) and OX DOX(-) mice. (C) Representative EEG and EMG traces and EEG power spectral density during DT sleep in OXMC DOX(-) mice. DT sleep was characterized by low EMG amplitude with high EEG spectral power in the δ (1–5 Hz) and θ (6–10 Hz) bandwidths. (D) Relative EEG power during NREM, REM, cataplexy and DT sleep in OXMC DOX(-) mice (n = 6–8) from 0 to 4 weeks DOX(-). The greatest EEG power across all stages was set at 100% for each mouse. Values are presented as mean ± SEM. (E) Relative EEG power in the standard frequency bands (δ, θ, α and β) for each state. The sum of each band is 100%. Values are presented as mean ± SEM. *p<0.05. Data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. Despite comparable EMG levels, DT sleep has significantly greater spectral power in the δ range and less power in the θ range than either REM sleep or cataplexy.

DOX removal from the diet in OXMC mice does not affect relative EEG power distribution or the EMG integral.

(A) Relative EEG power in three strains of mice in the DOX(+) condition (before DOX removal) during the dark phase. Upper graphs show relative EEG power at 1 Hz resolution; lower graphs present the standard EEG frequency bands. (B) As in (A) but for the light phase. (C) Relative EEG power of OXMC mice measured during the dark phase from 0 to 4 weeks in the DOX(-) condition. Upper graphs show relative EEG power at 1 Hz resolution; lower graphs present the standard EEG frequency bands. (D) As in (C) but for the light phase. (E) EMG integral of OXMC mice in the DOX (-) condition. n = 6–8, Values are mean ± SEM.

Effects of dual orexin- and MCH-neuron ablation on sleep/wake architecture in OXMC mice.

(A) Hypnograms from OXMC DOX(+), OXMC DOX(-) and OX DOX(-) mice before (0 week) and during dual orexin and MCH neuron ablation (1–4 weeks). Clock time and light-dark phase are shown below the hypnograms. D, DT sleep; C, cataplexy; W, wake; NR, NREM sleep; R, REM sleep. (B) Hypnograms for the 1 hr period after dark onset indicated by magenta rectangles in the 4 weeks hypnogram in A. (C) Total duration (upper panels), number of bouts (middle) and mean bout duration (lower panels) for wake, NREM, REM, cataplexy and DT sleep during the dark phase. (D) As in C but for the light phase. Values are means ± SEM. (OXMC DOX(+): (n = 7), OXMC DOX(-): (n = 8), OX DOX(-): (n = 4)). *p<0.05 vs OXMC DOX(+). #p<0.05 vs OX DOX(-). Data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test.

DT sleep is distinct from cataplexy.

(A) Upper panels show representative traces of EEG and EMG when tactile stimulation (arrowheads) was performed during cataplexy, DT sleep and NREM sleep in OXMC DOX(-) mice after 4 weeks in the DOX(-) condition. Lower panels show the percentage of each state 1 min before and 1 min after tactile stimulation. Vigilance states were determined in 4 s epochs. Bar graphs shows the percentage of wakefulness during the first 3 epochs (12 s) after tactile stimulation during cataplexy, DT sleep (DT) and NREM sleep (NR). *p<0.05, one-way ANOVA followed by the Bonferroni post hoc test (OXMC DOX(-) (n = 6)). (B) Schematic drawing of home cage (upper left) showing location of nest and water bottle and corresponding picture (lower left). Bar graphs show the percentage of DT sleep (DT), cataplexy (C), NREM (NR), and REM sleep (R) occurring in the nest area in OXMC (-) mice. Dashed line in the bar graph indicates chance level (25%). *p<0.05, one-way ANOVA followed by the Bonferroni post hoc test (OXMC DOX(-) (n = 7)). (C) Bar graphs indicate the behaviors observed before cataplexy and DT sleep. Values are mean ± SEM. *p<0.05, paired t-test (OXMC DOX(-) (n = 7)). (D) Total time, number of bouts and mean bout duration of cataplexy and DT sleep during the dark and light phases in OX DOX(-) (n = 4) mice (left) and OXMC DOX(-) (n = 7) mice (right). *p<0.05, paired t-test.

Effects of chocolate availability on DT sleep and cataplexy in OXMC mice.

(A) Schematic showing the experimental protocol of chocolate availability in OXMC DOX(-) mice (n = 8). To avoid further neuron degeneration during the test, DOX chow was re-introduced after 4 weeks in the DOX(-) condition; light blue bar indicates this DOX(+) condition. Light-dark phases are indicated as white and black bars. Chocolate was made available for 15 min prior to light offset (19:45-20:00). (B) Hourly amounts of time, number of bouts and mean bout duration for each vigilance state during the first 6 hr of the dark phase after chocolate administration. Values are mean ± SEM. (C) Total time, number of bouts and mean bout duration for each vigilance state during the first 6 hr of the dark phase after chocolate administration. *p<0.05, vs control or vehicle. Values are mean ± SEM. Data were analyzed by paired t-test.

Effects of clomipramine on DT sleep and cataplexy in OXMC DOX(-) mice.

(A) Schematic showing the experimental protocol for clomipramine administration (15 mg/kg, i.p.) in OXMC DOX(-) mice (n = 8). To avoid further neuron degeneration during the test, DOX chow was re-introduced after 4 weeks in the DOX(-) condition; light blue bar indicates this DOX(+) condition. Light-dark phases are indicated as white and black bars. Clomipramine or vehicle was administered by i.p. injection prior to dark onset (19:45-20:00) as indicated by the arrows. (B) Hourly amounts of time, number of bouts and mean bout duration for each vigilance state during the first 6 hr of the dark phase after clomipramine administration. Values are mean ± SEM. (C) Total time, number of bouts and mean bout duration for each vigilance state during the first 2 hr of the dark phase after clomipramine administration. *p<0.05, vs control or vehicle. Values are mean ± SEM. Data were analyzed by paired t-test.

Effects of modafinil on DT sleep and cataplexy in OXMC DOX(-) mice.

(A) Schematic showing the experimental protocol for modafinil administration (100 mg/kg, i.p.) in OXMC DOX(-) mice (n = 8). To avoid further neuron degeneration during the test, DOX chow was re-introduced after 4 weeks in the DOX(-) condition; light blue bar indicates this DOX(+) condition. Light-dark phases are indicated as white and black bars. Modafinil or vehicle was administered by i.p. injection prior to dark onset (19:45-20:00) as indicated by the arrows. (B) Hourly amounts of time, number of bouts and mean bout duration for each vigilance state during the first 6 hr of the dark phase after modafinil administration. Values are mean ± SEM. (C) Total time, number of bouts and mean bout duration for each vigilance state during the first 3 hr of the dark phase after modafinil administration. *p<0.05, vs control or vehicle. Values are mean ± SEM. Data were analyzed by paired t-test.

Comparison of EEG spectral power during DT sleep to the transition from NREM to REM sleep.

(A) Typical traces of EEG and EMG during the transition from NREM to REM sleep (upper) and during DT sleep (lower). Three epochs during the transition from NREM sleep to REM sleep were analyzed. (B) Comparison of relative EEG power during the transition from NREM to REM sleep in OXMC DOX(-) mice during ablation (0, 3, and 4 weeks DOX(-)).The upper graph shows relative EEG power distribution. The lower bar graph shows the relative EEG power in each spectral band (δ, θ, α and β). Values are mean ± SEM. Data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. (C) Comparison of relative EEG power and EMG integral (inset) during DT sleep and the transition from NREM to REM sleep in OXMC DOX(-) mice. Upper graph shows relative EEG power distribution; lower bar graph shows relative EEG power in each band (δ, θ, α and β). Values are mean ± SEM. Paired t-test.

Videos

Video 1
Typical behavior for DT sleep in orexin- and MCH neuron-ablated mice.
Video 2
Tactile stimulation during cataplexy in orexin- and MCH neuron-ablated mice.
Video 3
Tactile stimulation during DT sleep in orexin- and MCH neuron-ablated mice.

Tables

Table 1
Comparison of parameters between orexin neuron-ablated mice and orexin MCH-blated mice.
ParameterOrexin neuron-ablated miceOrexin and MCH-ablated mice- fold Difference (OXMC/OX)P value
Earliest detection timing of cataplexy2 weeks post-ablation2 weeks post-ablationN/AN/A
Time-of-day occurrence at 4 weeksBoth light and dark phasesBoth light and dark phasesN/AN/A
Total time in wakefulness during dark phase at 4 weeks418.9 ± 20.2 min571.0 ± 22.2 min1.361.4E-03
Total time in NREM sleep during dark phase at 4 weeks258.3 ± 16.2 min90.5 ± 19.8 min0.352.8E-04
Total time in REM sleep during dark phase at 4 weeks25.6 ± 0.9 min2.7 ± 1.7 min0.104.3E-06
Total time in wakefulness during light phase at 4 weeks278.1 ± 19.7 min322.6 ± 7.3 min1.163.1E-02
Total time in NREM sleep during light phase at 4 weeks389.8 ± 17.4 min340.1 ± 8.1 min0.871.6E-02
Total time in REM sleep during light phase at 4 weeks46.3 ± 3.4 min20.8 ± 4.2 min0.452.7E-03
Total time in cataplexy in dark phase at 4 weeks11.0 ± 0.6 min31.2 ± 3.6 min2.832.6E-03
Number of cataplexy bouts in dark phase at 4 weeks14.8 ± 2.016.9 ± 2.11.140.53
Mean bout duration in dark phase at 4 weeks53.3 ± 7.6 sec113.0 ± 9.6 sec2.122.2E-03
Total time in cataplexy in light phase at 4 weeks5.4 ± 0.9 min24.2 ± 4.5 min4.451.4E-02
Number of cataplexy bouts in light phase at 4 weeks8.3 ± 1.415.4 ± 3.11.870.13
Mean bout duration in light phase at 4 weeks50.0 ± 11.4 sec96.3 ± 4.8 sec1.931.7E-03
Table 2
Comparison of parameters between DT sleep and cataplexy.
ParameterDT sleepCataplexy
Neuron dependenceLoss of both MCH and orexin neurons necessaryOnly loss of orexin neurons required; loss of MCH neurons exacerbates
EMG amplitudeLowLow
Delta EEG spectral powerHighLow
Theta EEG spectral powerHighHigh
Earliest detection1 week post-ablation2 weeks post-ablation
Progressive increase as neurodegeneration proceeds?NoYes
Time-of-day occurrenceBoth light and dark phasesPrimarily in dark phase in OX mice; both phases in OXMC mice
OccurrenceSpontaneousSpontaneous
Mean bout durationSimilar during both light and dark phases:~15 sSimilar during both light and
dark phases:~50 s in OX mice but ~ 100 s in OXMC mice
Response to tactile stimulationHigh; immediate arousalLow
Position in home cage during occurrenceRandomRandom
Grooming more likely to precede occurrenceYesNo
Running more likely to precede occurrenceNoYes
Response to chocolateNo effectIncreased
Response to clomipramineNo effectDecreased
Response to modafinilDecreasedNo effect
Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (Mus musculus)Hcrt-tTATabuchi et al., 2014RRID:IMSR_APB:7778
Genetic reagent (Mus musculus)Pmch-tTATsunematsu et al., 2014RRID:IMSR_RBRC05844
Genetic reagent (Mus musculus)TetO DTAJackson labRRID:IMSR_JAX:008468
Genetic reagent (Mus musculus)Hcrt-tTA;TetO DTATabuchi et al., 2014RRID:MGI:5583048
Genetic reagent (Mus musculus)Hcrt-tTA; Pmch-tTA; TetO DTAThis paperMating of the Hcrt-tTA; TetO DTA with the Pmch-tTA mice
AntibodyRabbit anti-Pmch antibodySigma-AldrichRRID:AB_260690(1:1000)
AntibodyGoat polyclonal anti-HcrtSanta Cruz BiotechnologyRRID:AB_653610(1:1000)
AntibodyMouse anti-tryptophan hydroxylase antibodySigma-AldrichRRID:AB_261587(1:500)
AntibodyRabbit anti-tyrosine hydroxylase antibodyMerckRRID:AB_390204(1:1000)
AntibodyBiotinylated horse anti-goat IgG antibodyVector LaboratoriesRRID:AB_2336123(1:1000)
AntibodyBiotinylated goat anti-rabbit/mouse IgG antibodyVector LaboratoriesRRID:AB_2313606(1:1000)
Sequence-based reagentHcrtACD BioCat No. 490461-C2; RRID:SCR_012481(1:1500)
Sequence-based reagentPmchACD BioCat No. 478721-C1; RRID:SCR_012481(1:1500)
Sequence-based reagentSlc32a1 (vGAT)ACD BioCat No. 319191-C4; RRID:SCR_012481(1:750)
Sequence-based reagentSlc17a6 (vGlut2)ACD BioCat No. 319171-C3; RRID:SCR_012481(1:750)
Chemical compound, drugOpal520PerkinElmerRRID:SCR_012163(1:1000)
Chemical compound, drugOpal620PerkinElmerRRID:SCR_012163(1:1000)
Chemical compound, drugChocolateMeijiMilk chocolate(1.7–1.9 g/once)
Chemical compound, drugClomipramineSigma-AldrichC7291-1G; RRID:SCR_008988(15 mg/kg)
Chemical compound, drugModafinilCephalon Inc (Teva Pharmaceutical Industries Ltd.)(100 mg/kg)
Software, algorithmOrigin 2017LightstoneRRID:SCR_014212; version 2017
Software, algorithmSleepSignKissei Comtec
Software, algorithmImageJhttps://imagej.nih.gov/ij/RRID:SCR_002285

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