Drd1a Cre positive cells are preferentially localized in 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 Supplemental table T1 and T2 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, 3 technical replicates were used. Images were obtained with a spinning-disk confocal microscope.

Daily sleep-wake architecture is unchanged in L6b silenced animals.

a. Position of electrodes. The frontal and occipital 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 other. b. Representative frontal EEG, occipital EEG and EMG traces during the respective vigilance states in a L6b silenced and a control animal show similar patterns of activity, allowing blinded scoring of vigilance states. c. 24-hour 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, NREM and REM sleep were comparable in L6b silenced and control animals. Controls n=7, L6b silenced n=9.

Vigilance state specific EEG spectra are changed in L6b silenced animals.

a. EEG spectral power in the frontal and occipital derivation during Wake, NREM and 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 x frequency interaction with two-way 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 minutes preceding and 1 minute following NREM-REM transitions averaged across all NREM-REM transitions across 24 hours, 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 seconds preceding the NREM-REM transition relative to the EEG power in NREM sleep across 24 hours, 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 x 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.

The response to sleep deprivation is altered in L6b silenced animals.

a. 24-hour profiles of 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. 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 ANOVA showed a significant genotype x frequency interaction. d. Spectral power in the frontal EEG during the first 30 minutes of NREM sleep following sleep deprivation. e. Time course of SWA (0.5-4.0 Hz) during NREM sleep across the six hours following sleep deprivation. f. The rate of SWA decline was approximated with an exponential fit, 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 two hours following sleep deprivation. h. Absolute exponent coefficients for an exponential fit across only the first six hours following sleep deprivation. Frontal EEG, controls n=7, L6b silenced n=9. Occipital EEG, controls n=6, L6b silenced n=8.

Intracerebroventricular infusion of orexin A promotes wakefulness in L6b silenced and control animals

a. Histological and schematic overview of the intracerebroventricular infusion canula and 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. b. Schematic overview of the infusion procedure with tubing front-filled with orexin or vehicle solution and backfilled with saline. c. Time course of SWA in the 3 hours following 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 hours 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, NREM and REM sleep in the first 3 hours 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.