In 3 male and 1 female CaMKII-tTA × tetO-GCaMP6s mice, AAV1-CaMKIIα0.4-NLS-tdTomato-WPRE (custom made by Vigene Biosciences, MD, USA) was injected during the cranial window procedure. For Scnn1a-Cre mice, AAV1-Syn-Flex-GCaMP6s-WPRE-SV40 (a gift from Douglas Kim & GENIE Project (Addgene viral prep # 100845-AAV1; http://addgene.org/100845 ; RRID: Addgene_100845)) was injected. We pulled a glass capillary (Wiretrol® II, Drummond) into 10–20 μm in tip diameter using a micropipette puller (Model P-97, Sutter Instrument), and beveled the tip to about 30°. After filling the capillary with mineral oil (M5904, Sigma-Aldrich), we withdrew virus solution (titrated to 1012 GC/mL using 0.001% Pluronic F-68 in saline) from the tip using a plunger. Care was taken not to introduce an air gap between mineral oil and virus solution. We injected virus solution at the 4 corners of a 200 μm square whose center matched to that of identified C2 region, 50 nL per site over 5 min. We waited for 2 min after each virus injection before withdrawing. Depth of the pipette tip was 400 μm for Scnn1a-Cre mice, and 200 and 400 μm for others.
Aav1 syn flex gcamp6s wpre sv40
Manufactured by Addgene
AAV1-Syn-Flex-GCaMP6s-WPRE-SV40 is a recombinant adeno-associated virus (rAAV) construct that expresses the GCaMP6s calcium indicator under the control of the synapsin promoter. The construct includes a WPRE element and SV40 polyadenylation signal.
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2 protocols using aav1 syn flex gcamp6s wpre sv40
1
Specific Virus Transduction in Mouse Brain
2
Imaging Whisker Cortical Responses in Mice
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Mice (2.5–3 months old) were anesthetized with isoflurane (1–1.5% in O2), and a stainless-steel head holder with 6 mm aperture was affixed to the skull using cyanoacrylate glue and dental cement. D1, D2 and D3 whisker columns were localized using transcranial intrinsic signal optical imaging61 (link),62 (link). A 3 mm diameter craniotomy was made centered on the D2 column. For viral GCaMP6s expression, AAV1-Syn-Flex-GCaMP6s-WPRE-SV40 (Addgene #100845-AAV1) was injected at 3–4 locations surrounding the D2 column at 250 µm and 350 µm depth. A chronic cranial window (3 mm diameter glass coverslip, #1 thickness, CS-3R, Warner Instrument) was attached with dental cement. Before surgery, mice received dexamethasone (2 mg/kg), enrofloxican (5 mg/kg), and meloxicam (10 mg/kg). Post-operative buprenorphine (0.1 mg/kg) was administered. For viral expression of nucleus-targeted GCaMP6s, mice were co-injected with AAV1-syn-H2B-GCaMP6s43 (a gift from Dr. Na Ji, Depts. of Physics and Molecular & Cell Biology, University of California, Berkeley) and AAV1-mDlx-NLS-mRuby2 (Addgene #99130-AAV1), which drives mRuby2 expression to mark inhibitory interneurons63 (link).
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