SSFO expression in midbrain dopamine neurons and validation of cortical light activation

0.7 µl of pAAV9-EF1a-DIO-hChR2(C128S/D156A)-EYFP (titer 5×10¹³ copies/ml diluted 1:20 in PBS, Penn Vector Core) or AAV2/1.CAG.FLEX.EGFP.WPRE.bGH (titer 8×10¹² copies/ml, Penn Vector Core) was injected into the midbrain region (from bregma: AP –3.2, ML 0.5, DV 4.4 mm) and 0.7 µl of AAV1.Syn.Flex.GCaMP6s.WPRE.SV40 (titer 8×10¹²copies/ml, Penn Vector Core) was injected into the frontal cortex (from bregma: AP 1.7, ML 0.7, DV 0.4 mm) of Th-Cre adult animals.

After ~2 weeks, cranial window (from Bregma: AP 1.0–2.5 mm, ML 1 mm) opening and two-photon imaging was carried out as described above. Animals were imaged before and 30 min after light activation with 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. Three spots separated by around 200 µm anterior-posterior in the center of the window were activated with a 2 s light pulse for each spot. After imaging, animals were perfused, and SSFO expression was confirmed in the midbrain regions. Images were analyzed as described above. Three min times series images of spontaneous activity before and after CNO were analyzed. Baseline fluorescence (F0) was defined as the average of the fluorescent signals (Ft) in the time series. The standard deviation of the (ΔF/F) was used as a quantitative measure of overall cortical activity.