Figure 3.
The Oprm1-Cre line is amenable to optogenetics. Oprm1-CreVTA-VTA::ChR2 photostimulation causes behavioral avoidance. a, Diagram showing viral delivery of AAV2.EF1a.DIO.ChR2-mCherry (channelorhodopsin) or AAV2.EF1a.DIO.mCherry (control) into the VTA and fiber-optic implantation above the VTA, a well as a timeline for the experimental procedure. b, Representative image of viral expression (red), optic fiber implantation, and VTA dopamine cells immunostained with anti-tyrosine hydroxylase (TH)/Alexa Fluor 647 (blue). Tissues were observed on an inverted epifluorescence microscope. c, Confocal imaging of VTA sections stained with GABA antibody show overlapping with viral expression. Scale bar, 10 μm. d, Occupancy plots for representative individual at 20 Hz. Mice were free to explore the two- chamber RTPT apparatus for 20 min. Mice then received a blue stimulation when entering the light-paired side at 0, 10, 20, and 40 Hz (473 nm, 10 mW, 10 ms pulse width) on 4 consecutive days, as described by Seo et al. (2016). e, Activation of VTA MOR neurons produces place avoidance. Light stimulation in Oprm1-CreVTA-VTA::ChR2 mice induced significant behavioral avoidance to the light-paired side compared with control group (n = 7–8/group). The graph shows frequency responses of mice receiving a blue stimulation when entering the light-paired side (473 nm, 10 mW, 10 ms pulse width at 0, 10, 20, and 40 Hz; n = 9–10, control vs ChR2). Data are expressed as the mean ± SEM. **p < 0.01, ***p < 0.001. f, Further analysis of the 20 Hz simulation condition indicated significant avoidance for the light-paired side without affecting the total distance traveled (p > 0.05).