Targeted expression of EGFP or ChR2 in Cre-neurons

Conditional expression of EGFP or of the H134R variant of ChR2 in Cre-containing neurons was achieved using recombinant adeno-associated viruses (AAVs) encoding a double-floxed inverted open reading frame (DIO) of ChR2-mCherry or EGFP, as described previously [19] (link), [20] (link). Briefly, for intracranial injections, mice (postnatal day 30–40) were anesthetized with isoflurane and placed in a small animal stereotaxic frame (David Kopf Instruments). After exposing the skull under aseptic conditions, a small burr hole was drilled for microinjections. All microinjections were performed through a pulled glass pipette using a UMP3 microsyringe pump (World Precision Instruments). Virus (AAV-DIO-EGFP or AAV-DIO-ChR2-mCherry) was injected into V1 (0.3 mm lateral from Lambda, and 0.6 mm below pia) at the rate of 50 nL per minute (500–750 nL total volume). Fluorescent latex microspheres (Red Retrobeads (RRB), Lumafluor) were injected into dorsomedial striatum (0.9 mm anterior and 1.5 mm lateral from Bregma, 2.0 mm below pia) at the rate of 100 nL per minute (150 nL total volume). Following the injection, the skin was sutured using Vicryl 7.0 silk sutures (Ethicon) and mice were returned to their home cage for at least 21 days for AAV injections and 7 days for experiments using fluorescent retrobeads.