Nineteen mice (60–240 days old) of either sex were used, with fifteen for imaging and behavioral experiments and four for behavioral data only. The genotype was CamK2a-tTA × Ai94 (TITL-GCaMP6s) × slc17a7 IRES Cre, providing widespread GCaMP6s expression in excitatory cortical neurons. Mice were housed on a 12:12 reverse light:dark cycle with behavioral experiments during the dark phase and maintained at 21±1°C and 50±10% relative humidity. Circular 3-mm craniotomies were centered over ALM. ST-ChrimsonR was expressed via AAV injections 400 µm below the dura at multiple sites. Cranial windows comprised three glass layers (450 µm total) and a customized headbar was attached. Water restriction (1 ml/day) began 3–4 weeks after viral expression.
"In brief, circular (3-mm diameter) craniotomies were centred over ALM (2.5 mm anterior and 1.5 mm lateral from Bregma). We expressed the soma-targeted opsin ST-ChrimsonR in excitatory neurons by injecting a virus (10 12 titre; AAV2/2 camKII-KV2.1-ChrimsonR-FusionRed; Addgene, plasmid, catalogue no. 102771) into the craniotomy, 400 µm below the dura (five to ten sites, 20-30 nl each), centred in the craniotomy and spaced by approximately 500 μm between injection sites."
"The craniotomy was covered by a cranial window composed of three layers of circular glass (total thickness 450 μm). The diameter of the bottom two layers was 2.5 mm. The top layer was 3 mm or 3.5 mm and rested on the skull. The window was cemented using cyanoacrylate glue and dental acrylic (Lang Dental). A customized headbar was attached using cyanoacrylate glue and dental cement."
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