Figure 6. Behavioral Function of the Anterior Cortex→VTA-DA→NAcLat Circuit.
(A) Schematic of experimental setup. An AAV expressing either ChR2-eYFP or eYFP was injected into the anterior cortex (medial and lateral sites) and optical fibers were implanted above the VTA so that anterior cortex axon terminals innervating the VTA could be optically stimulated. A guide cannula was also implanted targeting the NAcLat in all mice for drug delivery in some experiments. All manipulations were bilateral.
(B) Top, ChR2-eYFP expression following virus injection into anterior cortex. Scale, 1 mm. Bottom, ChR2-eYFP-expressing axon terminals in the VTA of the same mouse. Blue lines indicate optical fiber tips. Scale, 100 μm.
(C) Schematic of ICSS task. Mice were allowed to respond freely at 5 nosepoke ports for 1 hr per day over 5 training days. A response at 4 ports resulted in the delivery of a 2s train of light pulses at varying frequencies (1, 5, 10, 20 Hz); an LED in the back of the port was concurrently illuminated to serve as a visual cue indicating ongoing stimulation. Responses at a 5th control port (0 Hz) had no consequence. For the first training session, all nosepokes were baited to facilitate initial investigation.
(D) Mice expressing ChR2-eYFP (n=7) developed a strong preference for the 20 Hz port as compared to the 0 Hz control port (1-way Friedman repeated-measures ANOVA on ranks, main effect of nosepoke type p<0.006 on days 3–5; 20 Hz vs 0 Hz p<0.05, Tukey post-hoc tests).
(E) Mice expressing eYFP (n=8) responded at similarly low levels at all nosepoke ports (p>0.351, days 1–5).
(F) On training day 5, ChR2-eYFP mice made significantly more nosepokes at the 20 Hz port than eYFP mice (Mann-Whitney Rank-Sum test with Bonferroni correction, p=0.005), indicating that optical activation of the anterior cortex-VTA projection is reinforcing.
(G–I) Infusion of the non-selective dopamine receptor antagonist flupenthixol into the NAcLat dose-dependently decreased responding for 20 Hz stimulation of anterior cortex→VTA projections in ChR2-eYFP-expressing mice (n=5) (G; 1-way repeated-measures ANOVA, main effect of drug p<0.001; 10 Ag and 5 Ag flupenthixol vs. saline vehicle p<0.001 and p=0.01 respectively, Holm-Sidak post-hoc tests). Doses indicate amount of drug delivered per hemisphere in 0.25 μl saline. While locomotion was also concurrently decreased by flupenthixol (H; 1-way repeated-measures ANOVA, main effect of drug p<0.001; 10 μg, 5 μg and 2.5 μg flupenthixol vs. saline vehicle, p<0.001, p<0.001 and p=0.003 respectively, Holm-Sidak post-hoc tests) (H), the latency to make the first response was unaffected (I; p=0.482), indicating that mice were physically capable of performing nosepoke responses during behavioral sessions where flupenthixol was administered.
See Figure S6 for related data.