Fiber photometry experiments were performed at least 3 weeks after AAV-GCaMP6m injection. The implanted fiber was connected to Fiber Optic Meter (ThinkerTech, Nanjing, China) through an optical fiber patch cord (200 μm, 0.37 NA, Inper, Hangzhou, China). To record fluorescence signals, a beam from a 480 LED was reflected with a dichroic mirror, focused with a lens coupled to an CMOS detector (Thorlabs, Inc. DCC3240M). The LED power at the tip of the patch cord was less than 50 μW. A Labview program was used to control the CMOS camera which recorded calcium signals at 50 Hz.
To record PVT→NAc or PVT→CeA pathway calcium activity, mice infected with GCaMP6m was trained with the identical CPP protocol as described above. To minimize photobleaching and potential fiber damage, pathway calcium activity was collected for 20 min during CPP acquisition and retrieval session. Analysis of the signal was done with custom-written MATLAB software. The fluorescence change (ΔF/F) was calculated as (F-F0)/F0, where F0 is the baseline fluorescence signal. Events was identified as the peak that exceeded the mean by one standard deviation. The area under the curve (AUC) was calculated as mean ΔF/F of the event.
To record PVT→NAc or PVT→CeA pathway calcium activity, mice infected with GCaMP6m was trained with the identical CPP protocol as described above. To minimize photobleaching and potential fiber damage, pathway calcium activity was collected for 20 min during CPP acquisition and retrieval session. Analysis of the signal was done with custom-written MATLAB software. The fluorescence change (ΔF/F) was calculated as (F-F0)/F0, where F0 is the baseline fluorescence signal. Events was identified as the peak that exceeded the mean by one standard deviation. The area under the curve (AUC) was calculated as mean ΔF/F of the event.