Targeted GCaMP3 Expression in Hippocampal Astrocytes

Postnatal day 49–63 (P49–P63) male and female C57BL/6 mice were used in all experiments in accordance with institutional guidelines. All surgical procedures were conducted under general anesthesia using continuous isoflurane (induction at 5%, maintenance at 1–2.5% vol/vol). Depth of anesthesia was monitored continuously and adjusted when necessary. After induction of anesthesia, the mice were fitted into a stereotaxic frame, with their heads secured by blunt ear bars and their noses placed into an anesthesia and ventilation system (David Kopf Instruments). Mice were administered 0.05 ml buprenorphine (0.1 mg/ml; Buprenex) subcutaneously before surgery. The surgical incision site was then cleaned three times with 10% povidone iodine and 70% ethanol. Skin incisions were made, followed by craniotomies of 2–3 mm in diameter above the left parietal cortex using a small steel burr (Fine Science Tools) powered by a high speed drill (K.1070; Foredom). Saline (0.9%) was applied onto the skull to reduce heating caused by drilling. Unilateral viral injections were performed by using stereotaxic apparatus (David Kopf Instruments) to guide the placement of beveled glass pipettes (World Precision Instruments) into the left hippocampus (2 mm posterior to bregma, 1.5 mm lateral to midline, and 1.6 mm from the pial surface). Either 2 µl AAV2/5 gfaABC₁D Lck-GCaMP3 (1.2 × 10¹³ gc/ml), 1.5 µl AAV2/5 gfaABC₁D GCaMP3 (1.5 × 10¹³ gc/ml), 1.5 µl AAV2/5 gfaABC₁D Lck-GFP (2.41 × 10¹³ gc/ml), or 1.0 µl AAV2/5 gfaABC₁D tdTomato (2.5 × 10¹³ gc/ml) was injected using a syringe pump (Pump11 PicoPlus Elite; Harvard Apparatus). Glass pipettes were left in place for at least 10 min. Surgical wounds were closed with single external 5–0 nylon sutures. After surgery, animals were allowed to recover overnight in cages placed partially on a low voltage heating pad. Buprenorphine was administered two times per day for up to 2 d after surgery. In addition, trimethoprim sulfamethoxazole (40 and 200 mg, respectively, per 500 ml water) was dispensed in the drinking water for 1 wk. Mice were killed 12–20 d after surgery for imaging (typically 13–15 d). We chose this period because generally it takes ∼2 wk to achieve GECI expression in cells by AAV infection and because it has been suggested that long-term expression (>3 wk after AAV injection) can cause toxicity in neurons (Akerboom et al., 2012 (link)).