In Vivo Ca2+ Imaging Using GRIN Lens

For in vivo Ca²⁺ imaging, mice underwent a single surgery in which 400 nl of AAV8-CamkII-GCamp6f virus were injected unilaterally with a 10 μl NanoFil syringe and a 33g beveled needle (World Precision Instruments, Sarasota, FL) at a constant speed of 100 nl/min prior to implanting a GRIN lens over the injection site. GRIN lenses were 6.1 mm long with a 0.5 mm diameter for vDG imaging (Inscopix, Palo Alto, CA). GRIN lens implantation was performed as previously described^{27 (link),32 (link)}. Briefly, a craniotomy centered at the lens implantation site was made and dura was carefully removed from the brain surface and cleaned with a stream of sterile saline and absorptive spears (Fine Science Tools, Foster City, CA). No brain tissue was aspirated prior to lowering the GRIN lens. Three 1/16” microscrews (Antrin Miniature Specialities, Inc) were inserted in evenly spaced locations around the implantation site. The lens was lowered in 0.1 mm DV steps and then fixed to the skull with dental cement (Dentsply, Sinora, PA). Viral injections coordinates were −3.6 mm AP, ±2.8 mm ML, −3.2 mm DV (from brain). Lens placement coordinates were −3.6 mm AP, ±2.8 mm ML, −2.9 mm DV (from brain). At the completion of surgery, the lens was protected with liquid mold rubber (Smooth On, Lower Macungie, PA), and imaging experiments commenced 4 weeks later.