Figure 3. DR_VGLUT3 inputs to VTA activate NAcLat-projecting DA neurons and promote reward.

(A) Schematic of experimental design.

(B) Anatomical distribution of vNAcMed- (left) and NAcLat-projecting (right) starter cells. Note the clear anatomical separation of the two subtypes and their locations in the medial VTA (mVTA) and lateral VTA (lVTA), respectively (green: RV-ΔG-GFP, red: TVA-mCherry, blue: TH; Scale bar 25 μm).

(C) Horizontal and sagittal views of processed whole brains displaying brain-wide inputs to vNAcMed- (left) and NAcLat- (right) projecting DA neurons.

(D) Quantification of inputs to vNAcMed- (light blue) and NAcLat- (dark blue) projecting DA neurons. Data are presented as a percentage of total input (px) counted in each individual brain. Color code indicates different brain structures shown in C. Abbreviations shown in legend of Figure S3 (Data represent means ± SEM).

(E) Schematic of experimental design.

(F) ChR2-eYFP expressing DR_VGLUT3 terminals (green) are more frequently detected in the lVTA adjacent to retrogradely labeled (beads, red) TH-immunopositive (blue) cells projecting to NAcLat (left) than in the mVTA (right; scale bars 10 μm).

(G) Mean fluorescence intensity of ChR2-eYFP expression in lVTA and mVTA (* p < 0.05; data represent means ± SEM).

(H, I) EPSCs generated by stimulation of DR_VGLUT3 inputs in retrogradely labeled (beads, red) VTA neurons projecting to (H) vNAcMed or (I) NAcLat. Cells were filled with neurobiotin (NB, green) and are TH-immunopositive (blue; scale bars: 50 pA/10 ms, 10 μm; data represent means ± SEM).

(J) Mean EPSC amplitudes and response probabilities generated by light stimulation of DR_VGLUT3 inputs (*** p < 0.001; data represent means ± SEM).

(K) Spontaneous firing in vNAcMed- (top) and NAcLat-projecting (bottom) DA neurons and 4 Hz stimulation of DR_VGLUT3 inputs (scale bar: 20 mV/1 s).

(L) Relative increase in firing rate during 4 Hz and 20 Hz DR_VGLUT3 terminal stimulation for vNAcMed- and NAcLat-projecting DA neurons (* p < 0.05; data represent means ± SEM).

(M) Schematic of experimental design.

(N) Schematic of real-time place preference assay.

(O) Trajectory of an animal that received 4 Hz light stimulation in one compartment (Phase 1, blue, top panel) for the initial 10 min period followed by stimulation in the other compartment (Phase 2, blue, lower panel) for an additional 10 min.

(P) Mean time mice spent in the compartment paired with 4 Hz light stimulation and the compartment that was not paired with light stimulation for mice expressing ChR2 or eYFP in LH_VGLUT2 neurons. (** p < 0.01; data represent means ± SEM).