Animals
All animal experiments were approved by the Salk Institute Institutional Animal Care and Use Committee (IACUC). Rats and mice were typically housed with a standard 12 h:12 h light:dark cycle in the Salk Institute animal facilities, with lights on at 06:00 and lights off at 18:00. Dark-reared mice were housed with a 24 h dark cycle since birth. Mice and rats were provided access to food and water ad libitum. Humidity ranged from 38–62% and temperature from 20 to 22 °C.
Mice
For bulk RNA=sequencing experiments, astrocyte-Ribotag mice were generated by crossing Gfap-cre hemizygous females (B6.Cg-Tg (GFAP-cre)73.12Mvs/J, Jax 012886) to homozygous flox-Rpl22-HA males (B6N.129-Rpl22tm1.1Psam/J, Jax 011029). Male mice hemizygous for cre and heterozygous for flox-Rpl22-HA (Rpl22-HA+;Gfap-cre+) were used for all experiments. Wild-type C57Bl6/J mice were used (Jax 000664) for experiments. For snRNA-seq, male wild-type mice were used. For smFISH experiments (Fig. 1h) validating the bulk RNA sequencing, male mice were used. For adult knockout experiments, Ccn1fl/fl mice were a gift from L. Lau51 and were maintained on a C57Bl6/J background. These mice were crossed to mice expressing tamoxifen-inducible Cre recombinase under an astrocytic promoter for temporal elimination (Aldh1;creERT2, Jax 029655 (ref. 52)). Experimental mice were homozygous for the Ccn1 floxed allele and either cre− or cre+ (wild type or Ccn1-cKO). For adult cKO experiments, mice were injected intraperitoneally with 75 mg kg−1 of tamoxifen (MP Biomedicals 156738) for 5 consecutive days at 1 month of age. For juvenile cKO experiments, mice were injected intraperitoneally once at P3–4 with 100 mg kg−1 of tamoxifen. Mice of both sexes were used and sexes were noted. Sample sizes were chosen on the basis of power analyses (80% power) and literature review. Experimenter was blinded to genotype or manipulation when analysing data.
Rats
Sprague-Dawley rats (Charles Rivers) were used at P1–2 for the preparation of primary cortical astrocyte cultures.
Surgical procedures
Juvenile viral injections
For adeno-associated virus (AAV) injections at P14–15, P11–12, or 3 months of age, C57Bl6/J mice were used. In brief, mice were administered pre-operative carprofen (5 mg kg−1) subcutaneously and anaesthetized using isoflurane. Stereotaxic coordinates for the binocular zone were 2.25 mm lateral and 0.5 mm anterior from lambda. Virus was injected at 3 sites at a depth of 500–600 µm from just below the skull surface. The pipette was kept in the brain for 3 min after each injection to allow the virus to diffuse. TdT, CCN1 and CCN1(D125A) viruses were injected for a total titre of ~2 × 108 viral genomes (vg) per ml. For snRNA-seq and western blotting, bilateral injections of both binocular zones were performed. After injection, mice were sutured and placed back with the dam if pre-weaning.
Monocular enucleation and monocular deprivation
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