0.7 μl of pAAV9-EF1a-DIO hChR2(C128S/D156A)-EYFP or AAV2/1.CAG.FLEX. EGFP.WPRE.bGH was bilaterally injected into the midbrain region (from bregma: AP −3.2, ML0.5, DV4.4mm) of Arc−/−;TH-Cre animals within postnatal days P21-P25. Around postnatal days P35-P42, a cranial window was opened in above the frontal cortex (from Bregma: AP 1.0–2.5 mm, ML 1mm across both sides of the midline) in animals anesthetized with isofluorane (~1.5%). The cranial window was filled with silicone gel, covered with a glass cover slip, and sealed with dental cement. A head bar was glued on the skull for fixation during light activation. Animals were allowed to wake and recover for at least 2 hrs. Animals were then head fixed and an optical fiber (200 μm in diameter, Thor Labs) connected to a 473 nm solid-state laser diode (CrystaLaser) with ~10 mW output from the fiber was used to deliver 2 s light pulses to the frontal cortex. Three spots separated by around 200 μm anterior posterior in the center of the window were activated in each hemisphere. The light activation was repeated for two more days. For one-day-after tests, animals were tested in the Y-maze 1 day after the last injection day. For adulthood tests, animals of 2~3-month age were first tested in the Y-maze, then tested for amphetamine induced locomotion as described above. After behavior testing, animals were perfused, and SSFO expression was confirmed in the midbrain regions. Animals that did not show VTA labeling were excluded from further analysis.
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