Positive reinforcement mediated by midbrain dopamine neurons requires D1 and D2 receptor activation in the nucleus accumbens

Abstract

The neural basis of positive reinforcement is often studied in the laboratory using intracranial self-stimulation (ICSS), a simple behavioral model in which subjects perform an action in order to obtain exogenous stimulation of a specific brain area. Recently we showed that activation of ventral tegmental area (VTA) dopamine neurons supports ICSS behavior, consistent with proposed roles of this neural population in reinforcement learning. However, VTA dopamine neurons make connections with diverse brain regions, and the specific efferent target(s) that mediate the ability of dopamine neuron activation to support ICSS have not been definitively demonstrated. Here, we examine in transgenic rats whether dopamine neuron-specific ICSS relies on the connection between the VTA and the nucleus accumbens (NAc), a brain region also implicated in positive reinforcement. We find that optogenetic activation of dopaminergic terminals innervating the NAc is sufficient to drive ICSS, and that ICSS driven by optical activation of dopamine neuron somata in the VTA is significantly attenuated by intra-NAc injections of D1 or D2 receptor antagonists. These data demonstrate that the NAc is a critical efferent target sustaining dopamine neuron-specific ICSS, identify receptor subtypes through which dopamine acts to promote this behavior, and ultimately help to refine our understanding of the neural circuitry mediating positive reinforcement.

Figure 1. ChR2-YFP expression is limited to Th::Cre+ rats.

(A) ChR2-YFP and TH staining in the striatum of representative Th::Cre+ (top) and Th::Cre- (bottom) rats. Both rats received identical virus injections targeted to the VTA. (B) ChR2-YFP and TH staining in the midbrain of representative Th::Cre+ (top) and Th::Cre- (bottom) rats. Both rats received identical virus injections targeted to the VTA. In A-B, scale bar  = 1 mm.

Figure 2. Example and group histology.

(A) Top; representative striatal ChR2-eYFP expression and NAc optical fiber placement for experiment 1 (blue dot indicates fiber tip). Bottom; histological reconstruction of optical fiber tip placements for subjects receiving intra-NAc optical stimulation. Blue dots indicate tip placement in Th::Cre+ rats; black dots indicate tip placement in Th::Cre- rats. (B) Top; representative striatal ChR2-eYFP expression and NAc infuser tip placement for experiment 2 (red dot indicates infuser tip). Bottom; histological reconstruction of infuser tip placements for subjects receiving intra-NAc drug infusions. (C) Top; representative VTA ChR2-eYFP expression and optical fiber placement for experiment 2 (blue dot indicates fiber tip). Bottom; histological reconstruction of optical fiber tip placements for subjects receiving intra-VTA optical stimulation. In A-C scale bars  = 500 µm.

Figure 3. Optical stimulation of VTA dopamine efferents to NAc supports self-stimulation.

(A) Virus was infused into the VTA, and an optical fiber was implanted targeting the NAc. (B) Schematic of ICSS task. A response at the active nosepoke port was reinforced with optical stimulation (20 pulses, 20 Hz, 5 ms duration, 473 nm) on an FR1 schedule. Responses at the inactive nosepoke port had no consequence. (C) Active and inactive nosepoke responding for Th::Cre+ and Th::Cre- rats during 4 days of FR1 training (120 min sessions). Th::Cre+ rats performed significantly more active than inactive nosepokes on all 4 days (2-tailed Wilcoxon Rank test with Bonferroni correction, *p<0.05) (D) YFP fluorescence in the VTA of Th::Cre+ rats correlates with the log of FR1 response rate on training day 4 (p = .026; r² = 0.482).

Figure 4. Self-stimulation driven by VTA dopamine neurons is attenuated by intra-NAc D1 and D2 receptor antagonists.

(A) Virus was injected into the VTA and an optical fiber was targeted to this region; cannulae were targeted to the NAc. (B) Schematic of ICSS task with drug infusions. A 60 min baseline ICSS session was administered where responses at the active nosepoke port were reinforced with optical stimulation (20 pulses, 20 Hz, 5 ms duration, 473 nm) on an FR1 schedule. After intra-NAc drug infusion, a 60 min test ICSS session was administered that was identical to the baseline session. (C) Active nosepoke responding during baseline (pre-drug) sessions. There were no differences in responding (Friedman one-way repeated measures ANOVA, main effect of treatment χ²(6) = 6.771, p = 0.343) (D) Active nosepokes during test (post-drug) sessions quantified as a percentage of baseline responding. Relative to saline, all drug treatments significantly reduced responding (one-way repeated measures ANOVA, main effect of treatment p<0.001, **post-hoc test vs. saline p<0.01). (E) Cumulative active nosepokes made during the 60 min test session, with the corresponding value from baseline sessions subtracted to highlight differential responding. Note that responding from saline sessions remains close to the baseline value while responding after drug treatment steadily decreases. Data represent the mean of all rats (n = 5), SEM not shown. Inset, total number of active nosepokes made in the first 5 minutes of each test session without baseline subtraction. There were no differences in this measure (Friedman one-way repeated measures ANOVA, main effect of treatment χ²(6) = 5.829, p = 0.443).

Figure 5. ICSS elicits bilateral c-Fos expression in NAc neurons.

(A) C-Fos immunohistochemical staining in the NAc of a Th::Cre+ rat sacrificed immediately after an ICSS session. Black boxes correspond to areas magnified in B and C. (B) High-magnification view showing c-Fos+ cells both ipsilateral and contralateral (C) to the optical fiber. (D) Quantification of c-Fos+ cells in the NAc (n = 8 sections per rat, n = 7 rats). There were more c-Fos+ cells in Th::Cre+ rats in both hemispheres of the NAc as compared to Th::Cre- controls (***p<0.001 ipsi, **p<0.01 contra, Student-Newman-Keuls post-hoc test). Th::Cre+ rats also had stronger c-Fos expression in the ipsilateral as compared to contralateral hemisphere (**p<0.01, Student-Newman-Keuls post-hoc test) Ipsi/contra designation refers to the location of the optical fiber in the VTA. Scale bar  = 500 µm in A and 50 µm in B–C.

Figure 6. C-Fos expression in VTA neurons after ICSS.

(A) C-Fos immunohistochemical staining and optical fiber placement in the VTA of a Th::Cre+ rat sacrificed immediately after an ICSS session. Blue line indicates location of optical fiber tip. Black boxes correspond to areas magnified in B and C. (B) High-magnification view of c-Fos staining showing c-Fos+ cells both ipsilateral and contralateral (C) to the optical fiber. (D) Quantification of c-Fos+ cells in the VTA (n = 4 sections per rat, n = 7 rats). Although there was a trend for greater c-Fos expression in Th::Cre+ rats (two-way repeated measures ANOVA, main effect of genotype p = 0.083), no comparison reached statistical significance. (E) High-magnification view of ChR2-eYFP and TH immunohistochemical staining in a c-Fos+ neuron showing colocalization of all three proteins. Scale bar  = 500 µm in A; 50 µm in B–C; and 10 µm in E.