0.7 μl of pAAV8-hSyn-DIO-hM3D(Gq)-mCherry (6×1012 copies/ml, UNC Vector Core) or pAAV8-hSyn-DIO-mCherry (3×1012 copies/ml, UNC Vector Core) was injected into the right hemisphere of the midbrain region (from bregma: AP −3.2, ML0.5, DV4.4mm) and 0.7μl of AAV1.Syn.Flex.GCaMP6s.WPRE.SV40 (2×1012 copies /ml, Penn Vector Core) was injected into the right hemisphere of the frontal cortex (from bregma: AP 1.7, ML 0.7, DV 0.4mm) of TH-Cre adult animals.
After ~2 weeks, a cranial window was opened above the AAV-GCaMP6 injected region in the frontal cortex (from Bregma: AP 1.0–3.0 mm, ML 0.3–1.3 mm, covering the M2 region) 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 plate was glued on the skull for fixation during imaging. The animals were then taken off the anesthesia and allowed to recover for ~1hr before imaging. A two-photon microscope (FV1000, Olympus) was used to image the brain under the cranial window (excitation laser: 900 nm) using a 20x water immersion lens (NA 0.95) in the head fixed awake animal. Animals were imaged before and 1hr after CNO (1mg/kg, ip) injection. After imaging, animals were perfused, and DREADD-Gq expression was confirmed in the midbrain regions. Animals that did not show VTA labeling were excluded from further analysis.
Images were analyzed using NIH ImageJ. Two 2-min movies of spontaneous activity before and after CNO were analyzed. The mean pixel intensity in each image frame of the movie was calculated as Ft. Baseline fluorescence (F0) was defined as the average of the fluorescent signals (Ft) in the time series. Changes in calcium signals (ΔF/F) are calculated as (Ft-F0)/F0. The standard deviation of the (ΔF/F) was used as a quantitative measure of overall cortical activity.
After ~2 weeks, a cranial window was opened above the AAV-GCaMP6 injected region in the frontal cortex (from Bregma: AP 1.0–3.0 mm, ML 0.3–1.3 mm, covering the M2 region) 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 plate was glued on the skull for fixation during imaging. The animals were then taken off the anesthesia and allowed to recover for ~1hr before imaging. A two-photon microscope (FV1000, Olympus) was used to image the brain under the cranial window (excitation laser: 900 nm) using a 20x water immersion lens (NA 0.95) in the head fixed awake animal. Animals were imaged before and 1hr after CNO (1mg/kg, ip) injection. After imaging, animals were perfused, and DREADD-Gq expression was confirmed in the midbrain regions. Animals that did not show VTA labeling were excluded from further analysis.
Images were analyzed using NIH ImageJ. Two 2-min movies of spontaneous activity before and after CNO were analyzed. The mean pixel intensity in each image frame of the movie was calculated as Ft. Baseline fluorescence (F0) was defined as the average of the fluorescent signals (Ft) in the time series. Changes in calcium signals (ΔF/F) are calculated as (Ft-F0)/F0. The standard deviation of the (ΔF/F) was used as a quantitative measure of overall cortical activity.
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