Cortical layer 6b mediates state-dependent changes in brain activity and effects of orexin on waking and sleep
- Department of Physiology, Anatomy and Genetics, University of Oxford, United Kingdom
- Sleep and Circadian Neuroscience Institute, University of Oxford, United Kingdom
- Kavli Institute for Nanoscience Discovery, University of Oxford, United Kingdom
- University Hospital of Psychiatry and Psychotherapy, University of Bern, Switzerland
- Department of Pharmacology, University of Oxford, United Kingdom
Figures
Figure 1 with 1 supplement
Dopamine receptor 1a (Drd1a) Cre-positive cells are preferentially localised in layer 6b (L6b) across the entire cortical mantle.
Drd1a (TdTom) distributions across prefrontal cortex (a–a’’), primary motor cortex (b–b’’), primary somatosensory cortex (c–c’’), and primary visual cortex (d–d’’) in a single example animal, with cell densities averaged across multiple animals (a’’’–d’’’). (a, b, c, d) Hemisection for reference, with DAPI staining for tissue structure (blue), Drd1a-Cre+ cells identified by TdTom expression (magenta). Tbr1 immunostaining (cyan) labels layer 6 and is used to identify the layer 5–6 boundary. Cpxl3 immunostaining (yellow) labels L6b and is used to distinguish L6a and L6b. Tiled images. (a’, b’, c’, d’) Hemisection for reference with only the Drd1a-TdTom channel shown. White boxes indicate the cortical region of interest enlarged in (a’’,b’’,’c’’,d’’). Tiled images. (a’’, b’’, c’’, d’’) Magnified image of the cortical region of interest. (a’’’, b’’’, c’’’, d’’’) Laminar cell densities of Drd1a-Cre;TdTom-positive cells. Within-region between-layers comparisons showed that greatest densities are found in L6b, followed by L6a, with sparse to no Drd1a expression in upper layers (cortical layer, F(6,112)=319.6, p<0.0001) in each cortical region analysed. Please refer to Figure 1—source data 1 and 2 for exact combinatoric inter-layer and inter-region test results, respectively. Data represented as mean ± SEM. Scale bar, a–d and a’–d’, 1000 µm. Scale bar, a’’–d’’, 200 µm. Experimental replicates, PFC (n=5), M1 (n=6), S1 (n=6), V1 (n=3). For each animal, three technical replicates were used. Images were obtained with a spinning-disk confocal microscope.
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Figure 1—source data 1
Inter-layer pairwise comparisons.
- https://cdn.elifesciences.org/articles/106992/elife-106992-fig1-data1-v1.docx
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Figure 1—source data 2
Inter-region pairwise comparisons.
- https://cdn.elifesciences.org/articles/106992/elife-106992-fig1-data2-v1.docx
Figure 1—figure supplement 1
Absence of dopamine receptor 1a (Drd1a)-Cre-positive cells in the suprachiasmatic nucleus.
Representative images from an individual Drd1a-Cre; Ai14 animal. Tissue structure is demarcated with DAPI (blue) and Drd1a-Cre cells are marked by TdTom expression (magenta). (A) Overview (1.6×) with white square indicating the area magnified in B. Scale bar, 1000 µm. (B) No Cre expressing cells are present in the suprachiasmatic nucleus (SCN). Scale bar, 100 µm.
Figure 2
Daily sleep-wake architecture is unchanged in layer 6b (L6b)-silenced animals.
(a) Position of electrodes. The frontal and occipital electroencephalogram (EEG) (expressed as mm distance from Bregma in midline [ML] and anteroposterior [AP] directions) were referenced against a cerebellar screw. Two EMGs were implanted in the nuchal musculature and referenced against one another. (b) Representative frontal EEG, occipital EEG, and EMG traces during the respective vigilance states in an L6b-silenced and a control animal show similar patterns of activity, allowing blinded scoring of vigilance states. (c) 24 hr profiles of slow-wave activity (SWA) in the frontal EEG (% of mean), EMG activity (arbitrary units), and vigilance-specific spectrograms in a representative L6b-silenced animal and control animal. The bar on top represents the duration of the light phase (yellow) and dark phase (dark blue). (d) Hypnograms for all individual animals. (e) Daily time course of wakefulness, non-rapid eye movement (NREM), and rapid eye movement (REM) sleep were comparable in L6b-silenced and control animals. Controls n=7, L6b silenced n=9.
Figure 3 with 1 supplement
Vigilance state-specific electroencephalogram (EEG) spectra are changed in layer 6b (L6b)-silenced animals.
(a) EEG spectral power in the frontal and occipital derivation during wake, non-rapid eye movement (NREM), and rapid eye movement (REM) sleep in L6b-silenced and control animals. Filled dots show bins with significant differences between genotype groups after comparison with post hoc tests in 0.25 Hz bins when there was a significant genotype × frequency interaction with two-way analysis of variance (ANOVA). Frontal EEG, controls n=7, L6b silenced n=9. Occipital EEG, controls n=6, L6b silenced n=9. (b) EEG spectral power in the 2 min preceding and 1 min following NREM-REM transitions averaged across all NREM-REM transitions across 24 hr, with average power in L6b-silenced animals relative to control animals in percentages. Top, frontal EEG, controls n=6, L6b silenced n=9. Bottom, occipital EEG, controls n=6, L6b silenced n=9. (c) EEG spectral power during NREM sleep in the 32 s preceding the NREM-REM transition relative to the EEG power in NREM sleep across 24 hr, in the frontal (top, controls n=7, L6b silenced n=9) and occipital (bottom, controls n=6, L6b silenced n=9) EEG. (d) Enlarged representation of the occipital EEG spectral power shown in (a) during wakefulness (top) and REM sleep (bottom). Filled dots mark significant genotype differences in 0.25 Hz bins with post hoc tests when there was significant genotype × frequency bin interaction in the two-way ANOVAs. Controls n=6, L6b silenced n=9. Dotted lines illustrate the EEG spectral power and frequency of the theta peak. (e) Peak theta frequency in the occipital EEG during wakefulness (top) and REM sleep (bottom) for control and L6b-silenced animals. Asterisks mark significant differences between genotypes. Controls n=6, L6b silenced n=9.
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Figure 3—source data 1
Comparison of electroencephalogram (EEG) spectra from layer 6b (L6b)-silenced and control animals across the full frequency range recorded.
Comparisons were made between spectra 0.0–128 Hz in 0.25 Hz bins, after exclusion of bins 0.0–0.5 Hz and 49–51.5 Hz (electrical noise), using two-way analyses of variance (ANOVAs). Results are shown for genotype × frequency interaction. Frontal EEG, controls n=7, L6b silenced n=9; occipital EEG, controls n=6, L6b silenced n=9.
- https://cdn.elifesciences.org/articles/106992/elife-106992-fig3-data1-v1.docx
Figure 3—figure supplement 1
Spectral power density across wake, non-rapid eye movement (NREM), and rapid eye movement (REM) across the full frequency range.
(a) Frontal electroencephalogram (EEG) power during the respective vigilance states in layer 6b (L6b)-silenced (n=9) and control (n=7) animals. Filled circles depict significant differences between genotypes in 0.25 Hz bin power spectra in post hoc tests when two-way analyses of variance (ANOVAs) showed a significant genotype × frequency interaction. (b) Occipital EEG during the respective vigilance states in L6b-silenced (n=9) and control (n=6) animals. Filled circles depict significant differences between genotypes in 0.25 Hz bin power spectra in post hoc tests when two-way ANOVAs showed a significant genotype × frequency interaction.
Figure 4
The response to sleep deprivation is altered in layer 6b (L6b)-silenced animals.
(a) 24 hr profiles of slow-wave activity (SWA) (0.5–4.0 Hz) after sleep deprivation in a representative control and L6b-silenced animal, with hypnograms plotted below. The bar on top marks the duration of the light phase (yellow) and dark phase (dark blue). (b) Electroencephalogram (EEG) power during sleep deprivation in L6b-silenced and control animals, normalised to wakefulness spectra on baseline day. (c) EEG spectral power in the sixth (final) hour of sleep deprivation normalised to the first hour of sleep deprivation. Filled circles represent 0.25 Hz bins where post hoc tests showed a significant genotype difference, when two-way analysis of variance (ANOVA) showed a significant genotype × frequency interaction. (d) Spectral power in the frontal EEG during the first 30 min of non-rapid eye movement (NREM) sleep following sleep deprivation. (e) Time course of SWA (0.5–4.0 Hz) during NREM sleep across the 6 hr following sleep deprivation. (f) The rate of SWA decline was approximated with an exponential fit, and the method is shown for the frontal EEG from a representative individual animal. (g) Absolute exponent coefficients for an exponential fit across only the first 2 hr following sleep deprivation. (h) Absolute exponent coefficients for an exponential fit across only the first 6 hr following sleep deprivation. Frontal EEG, controls n=7, L6b silenced n=9. Occipital EEG, controls n=6, L6b silenced n=8.
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Figure 4—source data 1
One-sample t-test and Wilcoxon test of wake during sleep deprivation (SD) frontal electroencephalogram (EEG) mean controls.
- https://cdn.elifesciences.org/articles/106992/elife-106992-fig4-data1-v1.csv
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Figure 4—source data 2
One-sample t-test and Wilcoxon test of wake during sleep deprivation (SD) frontal electroencephalogram (EEG) mean L6b silenced.
- https://cdn.elifesciences.org/articles/106992/elife-106992-fig4-data2-v1.csv
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Figure 4—source data 3
One-sample t-test and Wilcoxon test of wake during sleep deprivation (SD) occipital electroencephalogram (EEG) mean controls.
- https://cdn.elifesciences.org/articles/106992/elife-106992-fig4-data3-v1.csv
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Figure 4—source data 4
One-sample t-test and Wilcoxon test of wake during sleep deprivation (SD) occipital electroencephalogram (EEG) mean L6b silenced.
- https://cdn.elifesciences.org/articles/106992/elife-106992-fig4-data4-v1.csv
Figure 5 with 3 supplements
Intracerebroventricular infusion of orexin A promotes wakefulness in layer 6b (L6b)-silenced and control animals.
(a) Histological and schematic overview of the intracerebroventricular infusion canula and electroencephalogram (EEG)/EMG implant, with position of the cannula and electrodes defined as coordinates from bregma in the anterioposterior (AP) direction and midline (ML) direction, and cannula dorsoventral position (DV) from the surface of the dura. Scale bar, 100 µm. (b) Schematic overview of the infusion procedure with tubing front-filled with orexin or vehicle solution and backfilled with saline. (c) Time course of slow-wave activity (SWA) in the 3 hr following the infusion of vehicle and 0.6 nmol orexin A in a representative control and L6b-silenced animal. (d) Time course of vigilance states in the first 6 hr after infusion of vehicle (saline), a lower dose of orexin A (0.3 nmol), a higher dose of orexin A (0.6 nmol), orexin B (0.6 nmol) in L6b-silenced and control animals. Controls n=4, L6b silenced n=7. (e) Total amount of wake, non-rapid eye movement (NREM), and rapid eye movement (REM) sleep in the first 3 hr after infusion of orexin A or orexin B. Controls n=4, L6b silenced n=7. (f) The latency to consolidated NREM sleep was increased after infusion of orexin A (left column) but not ORXB (right column). Controls n=4, L6b silenced n=7.
Figure 5—figure supplement 1
Effects of orexin A on electroencephalogram (EEG) spectral power in a representative individual animal.
(a) Representative epoch of the frontal EEG (top), occipital EEG (middle), and EMG (bottom) channel in a representative control animal during wake, non-rapid eye movement (NREM), and rapid eye movement (REM). The left column shows traces after vehicle infusion, the right column shows traces after the higher dose (0.6 nmol) of ORXA infusion. (b) EEG power spectra for the frontal EEG (top) and occipital EEG (bottom) after vehicle infusion (left column) and ORXA (0.6 nmol) infusion in a representative control animal, averaged across all wake, NREM, and REM epochs during 24 hr.
Figure 5—figure supplement 2
Hypnograms of all animals after the different infusions.
Each row represents a 6 hr hypnogram from light onset for an individual animal after (a) vehicle infusion, (b) infusion of the lower dose of orexin A (0.3 nmol), (c) infusion of the higher dose of orexin A (0.6 nmol), and (d) infusion of orexin B (0.6 nmol). The red arrowheads indicate the start of the infusion, the black arrowheads represent the closure of the recording chamber. Hypnograms are split out per genotype, with controls on the left and layer 6b (L6b)-silenced animals on the right.
Figure 5—figure supplement 3
Homeostatic regulation of sleep after orexin infusion.
(a) Time spent in wake, non-rapid eye movement (NREM), and rapid eye movement (REM) after vehicle (saline) and ORXA infusion (dose 0.3 nmol and 0.6 nmol, respectively). (b) Time spent in wake, NREM, and REM after vehicle (saline) and ORXB (dose 0.6 nmol) infusion. (c) Slow-wave activity rebound during NREM sleep following ORXA infusion (0.6 nmol) in the frontal electroencephalogram (EEG) and occipital EEG. Controls n=5, L6b silenced n=8.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/106992/elife-106992-mdarchecklist1-v1.pdf
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Source data 1
Dataset supplements for Meijer et al. with links and explanations.
- https://cdn.elifesciences.org/articles/106992/elife-106992-data1-v1.docx
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Cortical layer 6b mediates state-dependent changes in brain activity and effects of orexin on waking and sleep
eLife 14:RP106992.
https://doi.org/10.7554/eLife.106992.3
