Figure 3. PPO inhibits somatodendritic excitability to suppresses reward behaviors.

(A) Schematic of PPO coupling to GIRK channels to inhibit neuronal activity.

(B) Experimental strategy for expressing Cre-dependent PPO-Venus (AAV5:Ef1α:DIO:PPO-Venus) in DAT-Cre⁺ dopamine neurons in the ventral tegmental area (VTA).

(C) Horizontal slice depicting the VTA (purple shaded region). Overlayed IR-DIC and fluorescence images of PPO-Venus in acute horizontal slices. Patch pipette is in dashed lines. Scale=100 μm

(D) 40x IR-DIC images (top) and PPO-Venus (bottom) in cell bodies of the VTA. Scale=10 μm

(E) Plot of normalized holding currents in response to blue LED stimulation (10 Hz, 10 ms, 10 mW/mm²) n=6

(F) Voltage traces from PPO-expressing VTA DA neurons in response to step current injections. Neurons were held at −60 mV.

(G) Same neuron in (F) during photostimulation with 10 Hz blue LED light.

(H) Inhibition was reversed by bath application of the GIRK channel blocker Ba²⁺ (1 mM)

(I-J) Summary graph of the change in rheobase (I) and input resistance (J) in response to 10 Hz LED stimulation and Ba²⁺. n=6, Paired t-tests of baseline to LED stimulation, p<0.05

(K) Plot of AP frequency to depolarizing current injections before (purple), during 10 Hz LED (blue) and after bath application of Ba²⁺ (red).

(L) Summary graph of input-output gain (AP# divided by the cumulative current injected). n=6, paired t-test p<0.05

(M) Current elicited by a voltage ramp from −40 to −140 mV at baseline (purple), during 10 Hz LED (blue) and after Ba²⁺ (red).

(N) Experimental strategy for expressing Cre-dependent PPO-Venus in DAT-Cre+ DA neurons in the VTA. Optical fibers were implanted bilaterally above the VTA to stimulate cell bodies.

(O) Confocal micrograph of PPO-Venus expression in VTA DA neurons. Scale=400 μm.

(P) Experimental timeline for operant task training for sucrose rewards. Mice were trained on fixed ratio (FR)-1 and FR-3 schedules.

(Q) Cartoon of an operant chamber. Mice nose poke into an active port in response to a light cue to receive a sucrose reward.

(R) Experimental design of the operant task where a sucrose reward is given for nose pokes in the active port after the light cue. No reward is given for pokes in the inactive port. FR-1 requires 1 nose poke for reward while FR-3 requires 3 nose pokes.

(S) Summary graphs of operant behaviors. 10 Hz pulsed laser light (473 nm, 10 ms, 5–8 mW) decreased both the number of nose pokes (left) and rewards (right). n=9 mice each, paired t-test **p<0.01