Fig. 1.

Optogenetic stimulation of DA axons in mPFC had no immediate effect on licking, but facilitated subsequent learning for the DA-paired stimulus. (A) Schematic diagram showing injection of TH-Cre mice with either DIO-ChR2-EYFP or DIO-EYFP constructs in VTA, and bilateral placement of optic fibers in mPFC. (Insets) Coronal sections with DAPI nuclear staining (blue) and EYFP expression (green). VTA axons in mPFC (Left) and VTA (Right) are shown. (B) Three-phase training protocol. Phase I: Mice were conditioned to lick at the end of a tone for water rewards (CS₁, 1 s; reward period, 1 s). Phase II: A new tone (CS₂) was introduced, and licking within the 1-s period following CS₂ resulted in optical stimulation of VTA fibers in mPFC. Phase III: The tone and water association was switched from CS₁ to CS₂. (C, Left) Learning in phase I: progression of licking rate in response to CS₁ on d1, d3, and d8 in the same mouse. The yellow area is the duration of CS₁ and anticipatory behavior (aLicks), and the gray area is the reward period. Progression of aLicks (C, Middle) and the percentage of rewarded CS₁ (C, Right) are shown, plotted as mean ± SEM for the experimental (ChR2/EYFP, solid blue circles; n = 5 mice) and control (EYFP, black circles; n = 7 mice) groups. No significant difference was found between the two groups [percentage of rewarded CS₁, F(1,10) = 1.8, P = 0.4; aLicks F(1,10) = 4.2, P = 0.2]. (D) Data from phase II in the same mice as in phase I. (Left to Right) Example of licking responses to CS₁ (thick lines) and CS₂ (thin dotted lines) for experimental (blue) and control (black) mice; development of aLicks for CS₂, CS₁, and percentage of CS₂-laser pairs across sessions (mean ± SEM). Note similar extinction of CS₂ aLicks, and percentage of CS₂-laser pairs in the two groups [F(1,10) = 4.1 and 1.2, P = 0.2 and P = 0.3, respectively]. (E) Data from phase III. (Left to Right) Example of licking behavior for experimental and control mice (same as in D); aLicks for CS₂, CS₁, and percentage of rewarded CS₂ (mean ± SEM across days, color-coded as in D). Note the faster acquisition of aLicks and higher percentage of rewarded CS₂ in the ChR2/EYFP group.