GoKawiilMice
Mice were maintained on a 12 h:12 h light:dark cycle with ad libitum access to food and water, at 22 ± 2 °C and 55 ± 10% humidity. All experiments were performed in accordance with the Swiss National Institutional Guidelines on Animal Experimentation and were approved by the cantonal Veterinary Office Committees for Animal Experimentation. Male C57BL6/J mice were used for whole-brain FOS mapping during circadian and sleep-deprivation time points, and both males and females were used for all other experiments. Age-matched mice (2–6 months) of the appropriate genotype were randomly distributed into experimental groups. Knock-in and transgenic lines were obtained from the Jackson Laboratory, including the TRAP2 knock-in line (Fos-2A-iCreERT2, strain 030323)40, Ai14 Cre reporter (RCL-tdT, strain 007914), Sert-Cre (Slc6a4tm1(cre)Xz, strain 014554)77, Vgat-IRES-Cre (Slc32a1tm2(cre)Lowl, strain 028862)78 and Vglut2-IRES-Cre (Slc17a6tm2(cre)Lowl, strain 016963)78.
Sleep deprivation and sleep attempts
Nesting material was removed from the cage at the beginning of sleep deprivation, and replaced at the end of the deprivation period. Mice were housed individually for all deprivation experiments. For grooming-based sleep deprivation, mice were finely misted with Milli-Q water to elicit grooming behaviour every 15 min for up to 6 h. Sleep attempts were interrupted by opening the cage door or applying an additional water mist. For novel-object-based sleep deprivation, a new object was placed in the cage every 15 min to maintain behavioural engagement for up to 6 h. Sleep attempts were interrupted by opening the cage door and displacing the object. Sleep attempts were defined as the adoption of a sleep-like posture and the concurrent emergence of NREM-like slow-wave activity in live EEG recordings, which reverted to wake-like EEG after cage-door opening, water misting or novel object displacement, and could be detected post hoc as sleep bouts by automated state classification (see Extended Data Fig. 7 and Methods sections below).
Whole-brain immunostaining and imaging
For all mapping experiments, mice were housed individually and allowed to habituate to the home cage for several days. For circadian mapping, whole brains were collected every 3 h during the unperturbed light–dark cycle. For deprivation mapping, whole brains were collected 1.5 h, 3 h, 5 h and 6 h into sleep deprivation, as well as 1.5 h and 3 h into the recovery period after 6 h deprivation. Six to seven brains per time point were collected for the circadian experiment and light-phase sleep deprivation, and four brains per time point were collected for dark-phase sleep deprivation. All samples were immunostained and cleared according to the iDisco79 workflow (https://idisco.info/) and imaged in horizontal orientation on a MesoSPIM system (Center for Microscopy and Image Analysis, University of Zurich) with corresponding control software (https://github.com/mesoSPIM/mesoSPIM-control) and a Hamamatsu Orca camera, using the following settings: 1.25× zoom, z_stepsize 5.0 μm, pixel size 5.26 μm, 2,048 × 2,048 resolution. Immunostaining was performed using a polyclonal rabbit anti-FOS antibody (Synaptic Systems, 226 003) at 1:2,000 and donkey anti-rabbit Alexa Fluor 647 (Invitrogen, A31573) at 1:800. Autofluorescence from the 561-nm channel was collected for overall brain morphology.
Whole-brain FOS mapping
Whole-brain light-sheet data were registered to an average mouse-brain template using the SHIELD-based workflow24. For each brain sample, striping artefacts were removed from the raw autofluorescence channel before resizing to the dimensions of a 461 × 471 × 323-pixel light-sheet-optimized average brain template25 (https://github.com/Gubra-ApS/LSFM-mouse-brain-atlas) and performing automated non-rigid alignment with sitk-align. Alignment results were inspected and manually corrected by reiteratively adjusting data:atlas correspondences and re-warping images in nuggt (Neuroglancer ground truth; https://github.com/chunglabmit/nuggt, master) using the nuggt-align function24. In parallel, FOS+ nuclei were automatically detected in three-dimensional (3D) stacks using a Laplacian of Gaussian filter (skimage.feature.blob_log, scikit-image v.0.19.3, https://scikit-image.org/). Identical alignment parameters were then applied to FOS+ nuclei from the same sample. To create heat maps of FOS+ cell density for each brain sample, Gaussian blur (sigma = 2.25) was applied to aligned FOS nuclei and the signal was averaged between the left and right brain hemispheres, before performing batch correction based on the ComBat approach80. To evaluate distinct patterns of brain activation across experimental conditions, averages for mice within the same experimental time point were normalized across time points within each experiment by dividing by the total activation per voxel. We then segregated each experimental dataset by peak activation time point, resulting in four categories per experimental condition: late wake (zeitgeber time (ZT)15–ZT21 circadian, sleep deprivation (SD) time points SD5h and SD6h for deprivation), sleep (ZT03–ZT09 circadian, recovery (R) time points R1.5h–R3h for deprivation), early wake (ZT12 circadian, SD1.5h–SD3h deprivation) and transition (ZT00 circadian, ZT12 dark deprivation). Finally, hierarchical clustering (clustering depth of 20) was performed for each category, and each cluster was manually categorized as type 1–type 3 on the basis of its mean response pattern during sleep deprivation and recovery. Whole-brain datasets can be browsed at: https://sleep-wake-atlas.scicore.unibas.ch/, and code for all analyses is available at https://gitlab.com/ceda-unibas/sleep-brain-atlas. For Pearson correlation analyses using unperturbed circadian data (Extended Data Fig. 3), voxel intensity correlations with wake duration were calculated for a 2-h time window preceding each experimental time point.
EEG analyses
For EEG implantation, electrodes were stereotactically placed over the right cerebral hemisphere at anteroposterior (AP) −2.25; lateral (L) +1.7 and AP +1.5, L +1.2 relative to bregma. Custom EEG implants were fixed to the skull using superglue and orthodontic resin (Paladur, Kulzer). All mice were allowed to recover from surgery for at least one week in their home cages. Mice were then connected to a flexible recording cable with a commutator and allowed to habituate to custom-made behaviour cages for at least 3 days, before EEG and video tracking were collected continuously for the duration of each experiment. Data were acquired using a 16-channel AC amplifier (A-M Systems, model 3500), filtered (0.3–300 Hz) with a gain of 500, digitized at 200 Hz and recorded with Spike2 (v.9.09a). The EEG signal was downsampled to 100 Hz and partitioned into 2-s epochs. Behavioural states were classified as high-theta wake, low-theta wake, NREM sleep and REM sleep using custom software (offline electroencephalography state space analysis (OESSA); https://github.com/VBits/oessa). EEG recording was used to quantify sleep behaviour in all cases. For the sleep-deprivation FOS-mapping experiments in Fig. 1, only a subset of representative mice was recorded given continuous wake maintenance by manual sleep deprivation. EEG power spectra in the 0–50 Hz range were calculated using a fast Fourier transform at 0.25-Hz resolution. To account for inter-individual variability in EEG signal, spectral data for each mouse were normalized to the total power for each vigilance state and shown as a percentage. Delta power (0.25–4 Hz) time-course data were normalized to mean baseline values from the last four hours of the light phase, when delta power is lowest2,81. To evaluate the relationship between wake duration and NREM delta power, a three-term filter for wake-enriched episodes42 was applied to EEG data from a 5-day undisturbed baseline period: NREM ≥ 10 min preceding wake ≥ 15 min, immediately followed by NREM ≥ 15 min. NREM segments allowed brief wake or REM intrusions of 1 min or less, and wake-enriched segments allowed brief NREM intrusions of 3 min or less.
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