GoKawiilMouse models
All animal experiments at the University of California Los Angeles (UCLA) were conducted in accordance with the US National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Chancellor’s Animal Research Committee at UCLA. The UCLA ARC protocol numbers for the experiments were 2009-043, 2020-002, 2017-039 and 2022-016. All mice were housed with food and water available ad libitum in a 12-h light–dark environment at temperatures of 20–22 °C with 40–60% humidity. All mice were 2–5 months old when used for experiments, were healthy with no obvious behavioural phenotype, were not involved in previous studies and were euthanized during the light cycle. Wild-type C57BL/6N mice were maintained in an in-house breeding colony or purchased from The Jackson Laboratory (JAX) or Taconic Biosciences. Approximately equal numbers of male and female mice were used and no differences were observed between sexes. RiboTag mice (B6N.129-Rpl22tm1.1Psam/J, JAX stock 011029) were acquired from JAX and crossed with Aldh1l1cre/ERT2 BAC mice (B6N.FVB-Tg(Aldh1l1-Cre/ERT2)1Khakh/J, JAX stock 029655) fully backcrossed to C57BL/6NTac background in an in-house breeding colony. GCaMP6f mice (Ai95D (C57BL/6J), JAX stock 028865) were acquired from JAX and crossed with Aldh1l1cre/ERT2 BAC mice (B6N.FVB-Tg(Aldh1l1-Cre/ERT2)1Khakh/J, JAX stock 029655) in an in-house breeding colony. Ponesimod behavioural experiments were performed at ONO Pharmaceutical and were approved by the Institutional Animal Care and Use Committee. Arl13bf/f;Aldh1l1cre/ERT2 mice were group housed (4–5 mice of the same sex per cage) in a controlled environment (12-h light–12-h dark cycle at 21 °C) with unrestricted access to water and a standard chow diet at the University of Calgary, and experiments were approved by the University of Calgary Health Sciences Animal Care Committee, Health Sciences Animal Care Committee, and all experiments were completed in accordance with the Canadian Council of Animal Care guidelines. Arl13bf/f and Ift88f/f mice72,73 were a gift from J. Guo and were maintained in an in-house breeding colony at UCLA. All mice were assigned to experimental groups at random. Because of the experimental design and availability of resources, blinding was not always feasible. For behavioral experiments, we minimized subjectivity as much as possible by using automated analysis software whenever possible.
Ethics statement
Human postmortem brain tissue samples were obtained from the Harvard Brain Tissue Resource Center, a NIH NeuroBioBank repository. All procedures were approved by the Mass General Brigham Institutional Review Board and conducted in accordance with institutional guidelines, NIH regulations and the Health Insurance Portability and Accountability Act. Written informed consent for brain donation was obtained from the legal next-of-kin or legally authorized representative. All samples were de-identified before distribution.
Tamoxifen injections for inducible gene expression
To selectively express RPL22–HA in astrocytes, hemizygous Aldh1l1cre/ERT2;RiboTag mice74,75 were injected with 75 mg kg–1 tamoxifen (Sigma-Aldrich, T5648) dissolved in corn oil once per day for 5 consecutive days at 6–8 weeks of age and were euthanized 2–3 weeks after tamoxifen injections for astrocyte RNA-seq experiments. Arl13bf/f;Aldh1l1cre/ERT2 mice were administered tamoxifen (200 μg per mouse per day) from postnatal days 7–9 and were euthanized 4 weeks later for RNA-seq experiments.
CRS
Mice were weighed and placed in restrainers (diameter of 2.5 cm × 9.5 cm, 551-BSRR, Plas-Labs) for 6 h each day (from 10:00 to 16:00) for 14 consecutive days. US mice were weighed and kept in their home cages. Light intensity during restraint stress was maintained at approximately 50 lux. All mice were returned to their home cages at the end of the stress period.
WAS
Mice were weighed and placed on a platform (diameter of 5 cm × 10 cm) attached to the bottom of a plastic tank (45-cm long × 25-cm wide × 25-cm high) for 2 h each day (from 10:00 to 12:00) for 14 consecutive days. The plastic tank was filled with water (25 °C) up to 1 cm below the top of the platform. US mice were weighed and kept in their home cages. Light intensity during WAS was maintained at approximately 50 lux. All mice were returned to their home cages at the end of the stress period.
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