Indirect Medium Spiny Neurons in the Dorsomedial Striatum Regulate Ethanol-Containing Conditioned Reward Seeking

Abstract

Adenosine 2A receptor (A_2AR)-containing indirect medium spiny neurons (iMSNs) in the dorsomedial striatum (DMS) contribute to reward-seeking behaviors. However, those roles for ethanol-seeking behaviors remain unknown. To investigate ethanol-seeking behaviors, we used an ethanol-containing reward (10% ethanol and 10% sucrose solution; 10E10S). Upon conditioning with 10E10S, mice that initially only preferred 10% sucrose, not 10E10S, showed a stronger preference for 10E10S. Then, we investigated whether the manipulation of the DMS-external globus pallidus (GPe) iMSNs circuit alters the ethanol-containing reward (10E10S) seeking behaviors using the combination of pharmacologic and optogenetic approaches. DMS A_2AR activation dampened operant conditioning-induced ethanol-containing reward, whereas A_2AR antagonist abolished the effects of the A_2AR agonist and restored ethanol-containing reward-seeking. Moreover, pre-ethanol exposure potentiated the A_2AR-dependent reward-seeking. Interestingly, mice exhibiting ethanol-containing reward-seeking showed the reduction of the DMS iMSNs activity, suggesting that disinhibiting iMSNs decreases reward-seeking behaviors. In addition, we found that A_2AR activation reversed iMSNs neural activity in the DMS. Similarly, optogenetic stimulation of the DMS-GPe iMSNs reduced ethanol-containing reward-seeking, whereas optogenetic inhibition of the DMS-GPe iMSNs reversed this change. Together, our study demonstrates that DMS A_2AR and iMSNs regulate ethanol-containing reward-seeking behaviors.SIGNIFICANCE STATEMENT Our findings highlight the mechanisms of how operant conditioning develops the preference of ethanol-containing conditioned reward. Mice exhibiting ethanol-containing reward-seeking showed a reduction of the indirect medium spiny neuronal activity in the dorsomedial striatum. Pharmacological activation of adenosine A_2A receptor (A_2AR) or optogenetic activation of indirect medium spiny neurons dampened operant conditioned ethanol-containing reward-seeking, whereas inhibiting this neuronal activity restored ethanol-containing reward-seeking. Furthermore, repeated intermittent ethanol exposure potentiated A_2AR-dependent reward-seeking. Therefore, our finding suggests that A_2AR-containing indirect medium spiny neuronal activation reduces ethanol-containing reward-seeking, which may provide a potential therapeutic target for alcohol use disorder.

Keywords: A2AR; dorsomedial striatum; ethanol; medium spiny neuron; reward.

Figure 1.

Effects of operant conditioning and A_2AR activation on the preference of ethanol-containing reward in male C57BL/6J mice. A, Experimental scheme. B, Nose-poking behavior and (C) latency to the magazine during the sessions (n = 14/group). D, We presented tap water, 10S, and 10E10S on each arm of the Y-maze. E, The heat map displays mice tracking patterns for 15 min on the Y-maze. F, Time spent and (G) zone entries of each arm, and (H) total distance traveled for 15 min in the Y-maze (n = 10/group). Data represented as mean ± SEM. *p < 0.05, comparing (B) active and inactive nose-poke, (C) first CRF session, (F, G) the water zone of the same treatment group; ^#p < 0.05 comparing the 10S zone of the same treatment group. B, Two-way repeated-measures ANOVA followed by Bonferroni's multiple-comparisons test, (C, F, G) one-way repeated-measures ANOVA followed by Tukey's multiple-comparisons test (H) one-way ANOVA followed by Tukey's multiple-comparisons test. MT, Magazine training; CGS, 0.1 mg/kg, i.p.

Figure 2.

Effects of operant conditioning with the sucrose reward on the preference of ethanol-containing outcome in the Y-maze. A, B, Experimental scheme. C, Time spent and (D) zone entries of each arm of the Y-maze for 15 min (n = 9/group). Data are represented as mean ± SEM. *p < 0.05, comparing the water zone of the same treatment group. One-way repeated-measures ANOVA followed by Tukey's multiple-comparisons test.

Figure 3.

Effects of pharmacological A_2AR modulations in ethanol-containing reward-seeking in C57BL/6J mice. A, B, Experimental scheme. C, Effects of CGS (0.1 mg/kg, i.p.), KW (1 mg/kg, i.p.), or CGS + KW on nose-poke in water presentation. D, G, Nose-poking changes between water (W) and 10E10S presentations. E, Effects of CGS (0.1 mg/kg, i.p.) on total distance traveled for 30 min in the open-field. F, Effects of DMS microinjections of CGS (0.05, 0.5 pmol/0.5 μl) on nose-poke in water presentation. Data represented as mean ± SEM; (C, D) n = 7/VEH, CGS, CGS + KW groups, n = 10/KW group; (E) n = 9/group; (F, G) n = 9/group. *p < 0.05, comparing each group. C, F, One-way ANOVA followed by Tukey's multiple-comparisons test, (D, G) paired Student's t test, (E) unpaired Student's t test.

Figure 4.

Effects of ethanol pre-exposure with A_2AR activation on ethanol-containing reward-seeking in C57BL/6J mice. A, Behavior schedule. B, The number of entries, and (C) spontaneous alternation (%) in the Y-maze. D, Magazine entries in the magazine training session and (E) differences of nose-poking behavior during operant conditioning between air and ethanol vapor exposure. F, Effects of ethanol pre-exposure and pharmacological A_2AR modulations (CGS: 0.1 mg/kg, i.p.; KW: 1 mg/kg, i.p.; or CGS + KW) on nose-poke in water presentation. G, Nose-poking changes between water (W) and 10E10S presentations. Data represented mean ± SEM; (B, C) n = 11/air group, n = 22/ethanol group; (D, E) n = 16/air group, n = 29/ethanol group; (F, G) n = 14/air + VEH group; n = 11/ethanol + VEH group; n = 11/ethanol + CGS group; n = 11/ethanol + CGS + KW group. *p < 0.05, comparing each group. B–D, Unpaired Student's t test, (E) two-way repeated-measures ANOVA followed by Bonferroni's multiple-comparisons test, (F) one-way ANOVA followed by Tukey's multiple-comparisons test, (G) paired Student's t test.

Figure 5.

Effects of A_2AR activation on iMSNs activities in the DMS of mice which exhibited ethanol-containing reward-seeking behavior. A, Experimental scheme. D₂R promoter-driven eGFP (green) and DAPI (blue) in the DMS. DAPI indicates nuclei. Scale bar, 20 μm. Effects of operant conditioning and CGS (10 μm) on (B) the traces, (C) number of the neuronal spike, and (D) the resting membrane potential of the DMS iMSNs. E, F, Effects of A_2AR activation on the normalized eEPSC amplitude and charge transfer in the DMS. Black line, VEH treatment; blue line, CGS treatment. The gray line shows AP5 (selective NMDA receptor antagonist; 50 μm) and DNQX (non-NMDA receptor antagonist; 20 μm) treatment group. G, H, Effects of A_2AR activation on mEPSC frequency, amplitude, and charge transfer in the DMS iMSNs. Data represented mean ± SEM; *p < 0.05 (C) comparing the “no conditioning + VEH group” at the same current point, (F, H) comparing the VEH group; (C) #p < 0.05 comparing the “conditioning + VEH” group at the same current point. C, Two-way ANOVA repeated-measures followed by Bonferroni's multiple-comparisons test, (D) one-way ANOVA followed by Tukey's multiple-comparisons test, (F, H) paired Student's t test. C, D, No conditioning (12 cells from 5 mice), conditioning (14 cells from 4 mice), conditioning + CGS (10 cells from 4 mice); (F) 6 cells from 2 mice; (H) 5 cells from 3 mice.

Figure 6.

Electrophysiological features of DMS iMSNs. A, E, I, Experimental schemes. B, C, Neuronal firing without optogenetic stimulation in the A_2AR-expressing iMSNs of the DMS. D, Light pulse frequency-dependent change of neuronal firing and fidelity with optogenetic stimulation in the iMSNs of the DMS (4 cells from 2 mice). F, G, Effects of optogenetic activation of DMS iMSNs on GPe neuronal firing (n = 10/group from 4 mice). H, oIPSCs in the GPe of A_2AR-Cre mice. Black line, aCSF; gray line, picrotoxin treatment; GABA_A receptor antagonist, 100 μm. J, Effects of optical inhibition of A_2AR-expressing neurons on evoked firing in the DMS. K, L, Effects of optical inhibition of DMS A_2AR-expressing neurons on the spontaneous firing of the GPe (n = 5/group from 3 mice). Data are presented mean ± SEM. *p < 0.05 comparing each group. G, L, One-way repeated-measures ANOVA by Tukey's multiple-comparisons test.

Figure 7.

Optogenetic manipulations of DMS-GPe iMSNs in ethanol-containing reward-seeking behaviors. A–D, Injection of Cre-dependent ChR2-expressing virus and implantation of optic fiber into the DMS. B, M, Effects of blue light stimulation on nose-poke in water presentation. C, N, P, Nose-poking changes between water (W) and 10E10S presentations. D, Effects of blue light stimulation on locomotor activity. E–H, M, N, Injection of Cre-dependent ChR2-expressing virus into the DMS and implantation of optic fiber into the GPe. Effects of light stimulation on (F, J) active nose-poke, (G, K) magazine entry, and (H, L) time spent in the magazine during CRF session. I–L, O, P, Injection of Cre-dependent eNpHR-expressing virus into the DMS and implantation of optic fiber into the GPe. O, Effects of yellow light stimulation on nose-poke in water presentation. Data represented mean ± SEM; (B–D) n = 5/group; (F–H, J–N) n = 6/group; (O, P) n = 7/group. *p < 0.05 comparing each group. B, C, F–H, J–N, P, Paired Student's t test, (D) two-way repeated-measures ANOVA followed by Bonferroni's multiple-comparisons test, (O) one-way ANOVA followed by Tukey's multiple-comparisons test.

Figure 8.

Schematics summarizing the results of this present study. DMS iMSNs activity reduction increases GPe neuronal activities and ethanol-containing reward-seeking behaviors with a conditioned stimulus (CS; operant chamber). However, increased DMS iMSNs activity inhibits neuronal activities in the GPe and ethanol-containing reward-seeking behaviors.