Pgp aav cag flex jgcamp7f wpre

To transduce the GECI jGCaMP7f in RGCs, pGP-AAV-CAG-Flex-jGCaMP7f-WPRE (Addgene #104496) was obtained from Addgene (Watertown, MA). The plasmid was then modified by the University of Michigan Vector Core to create the final vector pGP-AAV-CAG-jGCaMP7f-WPRE. Mice were anesthetized with intraperitoneal injection of ketamine (100 mg kg⁻¹) and xylazine (10 mg kg⁻¹). Pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride. Topical tetracaine hydrochloride was applied for local anesthesia. A pilot hole was created through the sclera, choroid, and retina 1 – 2 mm posterior to the corneal limbus using a 30-gauge needle. A 5 μl Hamilton syringe (Hamilton Robotics, Reno, NV) with a 32-gauge blunt needle was used to inject 1 μl (1.83 × 10¹² vg/ml) of pGP-AAV-CAG-jGCaMP7f-WPRE in the vitreous area. Injection was done slowly over 30 seconds and left in place for another 30 seconds after injection and slowly retracted to minimize leakage. Antibiotic eye ointment was used on the injection site to prevent infection.

Young adult (postnatal day 42–98) male C57BL/6J (Jackson Labs) mice were anaesthetized using isoflurane (2.5% for induction and 1.5% during surgery). A circular craniotomy (diameter of 3 mm) above medial crus I (2 mm left and 1 mm posterior to the midline junction of the interparietal and occipital bones). Viral suspension (200 nl) was injected in two locations near the centre point at a depth of 300–400 μm. Constructs injected included: AAV2/1-CAG-FLEx-jGCaMP8 constructs (pGP-AAV-CAG-FLEx-jGCaMP8f-WPRE, Addgene plasmid #162382; pGP-AAV-CAG-FLEx-jGCaMP8m-WPRE, Addgene plasmid #162381; pGP-AAV-CAG-FLEx-jGCaMP8s-WPRE, Addgene plasmid #162380; pGP-AAV-CAG-FLEx-jGCaMP7f-WPRE, Addgene plasmid #104496; and pGP-AAV-CAG-FLEx-jGCaMP6f-WPRE, Addgene plasmid #100835; all viruses were diluted to 4 × 10¹² GC per millilitre titre).
Purkinje cell-specific expression was induced by co-injection of virus expressing Cre under control of a promoter fragment from the Purkinje cell protein 2 (Pcp2; also known as L7) gene (AAV2/1-sL7-Cre, 5.3 × 10¹⁰ GC per millilitre titre)^{68 (link)}. A 3-mm diameter circular coverslip glued to a donut-shaped 3.5-mm diameter coverslip (no. 1 thickness, Warner Instruments) was cemented to the craniotomy using dental cement (C&B Metabond, Parkell). A custom titanium head post was cemented to the skull.