Absence of NMDA receptors in dopamine neurons attenuates dopamine release but not conditioned approach during Pavlovian conditioning

Abstract

During Pavlovian conditioning, phasic dopamine (DA) responses emerge to reward-predictive stimuli as the subject learns to anticipate reward delivery. This observation has led to the hypothesis that phasic dopamine signaling is important for learning. To assess the ability of mice to develop anticipatory behavior and to characterize the contribution of dopamine, we used a food-reinforced Pavlovian conditioning paradigm. As mice learned the cue-reward association, they increased their head entries to the food receptacle in a pattern that was consistent with conditioned anticipatory behavior. D1-receptor knockout (D1R-KO) mice had impaired acquisition, and systemic administration of a D1R antagonist blocked both the acquisition and expression of conditioned approach in wild-type mice. To assess the specific contribution of phasic dopamine transmission, we tested mice lacking NMDA-type glutamate receptors (NMDARs) exclusively in dopamine neurons (NR1-KO mice). Surprisingly, NR1-KO mice learned at the same rate as their littermate controls. To evaluate the contribution of NMDARs to phasic dopamine release in this paradigm, we performed fast-scan cyclic voltammetry in the nucleus accumbens of awake mice. Despite having significantly attenuated phasic dopamine release following reward delivery, KO mice developed cue-evoked dopamine release at the same rate as controls. We conclude that NMDARs in dopamine neurons enhance but are not critical for phasic dopamine release to behaviorally relevant stimuli; furthermore, their contribution to phasic dopamine signaling is not necessary for the development of cue-evoked dopamine or anticipatory activity in a D1R-dependent Pavlovian conditioning paradigm.

Fig. 1.

Pavlovian conditioning elicits reward-associated CA behavior in C57BL/6 mice. (A) Our paradigm consisted of an 11-s lever presentation (CS) paired with the delivery of a 20-mg food pellet at t = 10 s. Mice received 25 CS–US pairings at a variable ITI averaging 60 s. (B and C) Mice selectively increased their HE rate during CS presentation relative to baseline (ITI) when it was paired (B) but not when it was unpaired (C) with US delivery (mean ± SEM, two-way ANOVA, rate x session; F_(5,110) = 7.7; ***P < 0.001). (D) Only the mice in the paired group increased their CA score during training (mean ± SEM, two-way ANOVA, group x session; F_(5,110) = 18.9; ***P < 0.001).

Fig. 2.

D1R-KO mice were unable to learn in this Pavlovian conditioning paradigm. (A and B) Control (A) but not D1R-KO mice (B) increased their CS-elicited HE rate relative to baseline (mean ± SEM, two-way ANOVA, rate x session; F_(6,60) = 3.0; *P < 0.05). (C) This trend resulted in increase in CA score only in the control mice (mean ± SEM, two-way ANOVA, genotype x session; F_(6,54) = 3.1; *P < 0.05).

Fig. 3.

D1R antagonism blocks both the acquisition and performance of Pavlovian CA. (A) Saline-treated C57BL/6 mice significantly increased their HE rate during the CS relative to baseline (mean ± SEM, two-way ANOVA, rate x session; F_(5,60) = 4.0; **P < 0.01). On day 7, the selective increase in CS-elicited HE rate remained (Fisher post hoc analysis; ^##P < 0.01); however, in the presence of the D1R antagonist on day 8, there was no difference between CS and ITI HE rate (Fisher post hoc analysis; P = 0.52). (B) Mice treated with 0.1 mg/kg D1R antagonist had an intermediate level of increased HE rate during CS relative to baseline (mean ± SEM, two-way ANOVA, CS vs. ITI; F_(1,10) = 19.9; ⁺⁺P < 0.01). On day 7, the selective increase in CS-elicited HE rate remained (Fisher post hoc analysis; ^##P < 0.01); these mice still showed an elevation in HE rate during the CS when tested in the presence of the high dose of D1R antagonist (Fisher post hoc analysis; ^#P < 0.05). (C) Mice treated with 0.3 mg/kg D1R antagonist had no increase in HE rate during the CS. When tested in the absence of the antagonist on day 7, there still was no difference between CS and baseline HE rate (mean ± SEM). (D) CA score increased in saline- and low-dose–, but not high-dose–treated mice [mean ± SEM, days 1–6, two-way ANOVA; treatment vs. session (saline vs. SCH0.3), F_(5,50) = 6.7; treatment vs. session (SCH0.1 vs. SCH0.3), F_(5,45) = 7.1; **P < 0.01]. Only the group treated with the highest dose of D1R antagonist still had no CA on day 7 when all groups were tested in the absence of the antagonist (Fisher post hoc analysis; ⁺⁺P < 0.01). Saline-treated mice had a significant decrease in CA score in the presence of the high dose of D1R antagonist on day 8 (Fisher post hoc analysis, day 7 vs. day 8; ^###P < 0.01).

Fig. 4.

NMDARs in dopamine neurons are not necessary to acquire Pavlovian CA. (A) Control mice selectively increased their CS-elicited HE rate during training (mean ± SEM, two-way ANOVA, rate x session; F_(6,228) = 4.6; ***P < 0.001). (B) NR1-KO mice also increased their CS-elicited HE rate relative to baseline (mean ± SEM, two-way ANOVA, rate x session; F_(6,180) = 3.2; **P < 0.01). (C) CA scores increased comparably in control and KO mice (mean ± SEM).

Fig. 5.

Phasic dopamine release in response to an unexpected food reward is attenuated in NR1-KO mice. (A) Cresyl violet-stained 30-μm brain section revealing electrode track and site of postexperimental electrolytic lesion. Dashed black line indicates the AcbC. (B) Representative dopamine traces from control and NR1-KO mice in response to the delivery of an unexpected food reward. (C) Representative cyclic voltammograms from control and NR1-KO mice obtained from a current in response to unexpected reward delivery. (D) Peak phasic dopamine release elicited by the delivery of the first unexpected food pellet each day was attenuated in NR1-KO (n = 5) relative to control (n = 6) mice (mean ± SEM, two-way ANOVA, control vs. NR1-KO; F_(1,9) = 14.7; ^##P < 0.01).

Fig. 6.

Phasic dopamine release develops to the CS and persists to the US during Pavlovian conditioning in control and NR1-KO mice. (A) US-evoked dopamine responses were decreased in NR1-KO (n = 5) compared with control (n = 6) mice across all seven training sessions [mean area under the curve (AUC) ± SEM, two-way ANOVA, control vs. NR1-KO; F_(1,9) = 10.8; ^##P < 0.01]. dopamine release at the time of reward retrieval rather than delivery was used for session 1. (B) CS-evoked dopamine responses were decreased in NR1-KO mice but increased significantly during training in both groups (mean AUC ± SEM, two-way ANOVA, genotype x session; F_(6,54) = 3.4; ^##P < 0.01; Fisher post hoc analysis; *P < 0.05; **P < 0.01; ***P < 0.001). (C) CA scores increased similarly in the control and NR1-KO mice used in the FSCV experiment (mean ± SEM). (D) Average dopamine traces form control and NR1-KO mice in response to CS and US presentations on days 1, 2, and 7. Inset shows mean dopamine traces at the time of reward retrieval during session 1. (E and F) Three-dimensional representations summarize the phasic dopamine release to the CS and US for control (E) and NR1-KO (F) across all training days (five-trial blocks).