Habits Are Negatively Regulated by Histone Deacetylase 3 in the Dorsal Striatum

Abstract

Background: Optimal behavior and decision making result from a balance of control between two strategies, one cognitive/goal-directed and one habitual. These systems are known to rely on the anatomically distinct dorsomedial and dorsolateral striatum, respectively. However, the transcriptional regulatory mechanisms required to learn and transition between these strategies are unknown. Here we examined the role of one chromatin-based transcriptional regulator, histone modification via histone deacetylases (HDACs), in this process.

Methods: We combined procedures that diagnose behavioral strategy in rats with pharmacological and viral-mediated HDAC manipulations, chromatin immunoprecipitation, and messenger RNA quantification.

Results: The results indicate that dorsal striatal HDAC3 activity constrains habit formation. Systemic HDAC inhibition following instrumental (lever press → reward) conditioning increased histone acetylation throughout the dorsal striatum and accelerated habitual control of behavior. HDAC3 was removed from the promoters of key learning-related genes in the dorsal striatum as habits formed with overtraining and with posttraining HDAC inhibition. Decreasing HDAC3 function, either by selective pharmacological inhibition or by expression of dominant-negative mutated HDAC3, in either the dorsolateral striatum or the dorsomedial striatum accelerated habit formation, while HDAC3 overexpression in either region prevented habit.

Conclusions: These results challenge the strict dissociation between dorsomedial striatum and dorsolateral striatum function in goal-directed versus habitual behavioral control and identify dorsostriatal HDAC3 as a critical molecular directive of the transition to habit. Because this transition is disrupted in many neurodegenerative and psychiatric diseases, these data suggest a potential molecular mechanism for the negative behavioral symptoms of these conditions and a target for therapeutic intervention.

Keywords: Chromatin; Decision making; Epigenetic; HDAC3; Instrumental conditioning; Learning; Reward.

Figure 1. Effect of post-training HDAC inhibition on instrumental learning, behavioral strategy, and acetylation at histone H4

(A) Schematic representation of procedures (B–D) Instrumental (lever press→reward) training performance for rats given limited (B; N=11/group), intermediate (C; N=11–12/group), or extended (D; N=9/group) instrumental training. (data presented as mean + s.e.m). CRF (continuous reinforcement) on the first training day lever pressing was continuously reinforced with food-pellet delivery. A RI-30s reinforcement schedule was in place thereafter. (E–G) Lever presses during the subsequent devaluation tests normalized to total presses across both tests for the Valued [Valued state presses/(Valued + Devalued state presses)] and Devalued [Devalued state presses/(Valued + Devalued state presses)] states for rats that received limited (E), intermediate (F), or extended (G) training. Dashed line indicates point of equal responding between tests. (data presented as mean + scatter). (H) Schematic representation of procedures, (I) Representative immunofluorescent image and quantification (J) of acetylation of H4K8 (H4K8Ac; N=4–6/condition) 1 hr following instrumental training/drug treatment. Scale bar = 20 μm. Data normalized to vehicle control (dashed line). *P<0.05; ** P<0.01.

Figure 2. Effect of training and post-training HDAC inhibition on HDAC3 occupancy at learning-related gene promoters and gene expression in the dorsolateral striatum

(A) Schematic representation of procedures. (B–D) ChIP was performed with anti-HDAC3 followed by qPCR to identify HDAC3 binding to the Bdnf1 (B), Nr4a1 (C), or Nr4a2 (D) promoters in the DLS of home cage (HC) controls or following either intermediate (INT) or extended training (EXT) in vehicle-treated rats, or NaBut treatment post-intermediate training. Data presented as fold change relative to IgG (% Input/IgG). (E–G) mRNA expression of Bdnf1 (E), Nr4a1 (F), and Nr4a2 (G) in the DLS. *P<0.05, **P<0.01, between groups; ^##P<0.01 relative to HC.

Figure 3. Effect of HDAC3 manipulation in dorsolateral striatum on habit formation

(A) Schematic representation of procedures. (B) Top, schematic representation of injector tips in the DLS. Numbers to the lower right of each section represent distance (mm) anterior to bregma. Coronal section drawings taken from (96). Middle, representative immunofluorescent images of H4K8Ac in DMS (left) and DLS (right) 1 hr following instrumental training/intra-DLS vehicle (top) or RGFP966 (bottom) infusion. Scale bar = 20 μm. (F, J) Top, schematic representation of HDAC3 point mutant (F), or HDAC3 (J) expression in the DLS for all subjects. Middle, representative immunofluorescent images of V5-tagged HDAC3 point mutant (F) or HDAC3 (J) expression in the DLS. Bottom, Representative immunofluorescent images of H4K8Ac in rats expressing the HDAC3 point mutant (F) or HDAC3 (J) either outside (left) or inside (right) of expression zone. (C, G, K) Quantification of H4K8Ac for rats receiving intra-DLS vehicle or RGFP966 infusion (C; N=5/group), intra-DLS empty vector (EV) or HDAC3 point mutant (HDAC3pm) (G; N=6/group), or intra-DLS empty vector or HDAC3 overexpression (K; N=5–6/group). (data presented as mean + scatter). (D, H, L) Instrumental training performance for rats given post-training intra-DLS vehicle or RGFP966 infusions (D; N=10–12/group), intra-DLS empty vector or HDAC3 point mutant (H; N=8/group), or intra-DLS empty vector or HDAC3 (L; N=11–13/group). (data presented as mean + s.e.m). CRF (continuous reinforcement) on the first training day lever pressing was continuously reinforced with food-pellet delivery. (E, I, M) Normalized lever presses during the subsequent devaluation tests for rats given post-training intra-DLS vehicle or RGFP966 infusions (E), intra-DLS empty vector (EV) or HDAC3 point mutant (I), or intra-DLS empty vector (EV)or HDAC3 (M). *P<0.05; **P<0.01; ***P<0.001.

Figure 4. Effect of HDAC3 manipulation in dorsomedial striatum on habit formation

(A) Schematic representation of procedures. (B) Top, schematic representation of injector tips in the DMS. Numbers to the lower right of each section represent distance (mm) anterior to bregma. Middle, representative immunofluorescent images of H4K8Ac in DMS (left) and DLS (right) 1 hr following instrumental training/intra-DMS vehicle (top) or RGFP966 (bottom) infusion. Scale bar = 20 μm. (F, J) Top, schematic representation of HDAC3 point mutant (F), or HDAC3 (J) expression in the DMS for all subjects. Middle, representative immunofluorescent images of HDAC3 point mutant (F) or HDAC3 (J) expression in the DMS. Bottom, Representative immunofluorescent images of H4K8Ac in rats expressing the HDAC3 point mutant (F) or HDAC3 (J) either outside (left) or inside (right) of the expression zone. (C, G, K) Quantification of H4K8Ac for rats receiving intra-DMS vehicle or RGFP966 infusion (C; N=4–5/group), intra-DMS empty vector (EV) or HDAC3 point mutant (G; N=4–5/group), or intra-DMS empty vector or HDAC3 overexpression (K; N=4–6/group). (data presented as mean + scatter). (D, H, L) Instrumental training performance for rats given post-training intra-DMS vehicle or RGFP966 infusions (D; N=9/group), intra-DMS empty vector or HDAC3 point mutant (H; N=11–12/group), or intra-DMS empty vector or HDAC3 (L; N=12/group). (data presented as mean + s.e.m). CRF (continuous reinforcement) on the first training day lever pressing was continuously reinforced with food-pellet delivery. (E, I, M) Normalized lever presses during the subsequent devaluation tests for rats given post-training intra-DMS vehicle or RGFP966 infusions (E), intra-DMS empty vector (EV) or HDAC3 point mutant (I), or intra-DMS empty vector (EV) or HDAC3 (M). *P<0.05; **P<0.01.

Figure 5. Effect of training and post-training HDAC inhibition on HDAC3 occupancy at learning-related gene promoters and gene expression in the dorsomedial striatum

(A) Schematic representation of procedures. (B–D) ChIP was performed with anti-HDAC3 followed by qPCR to identify HDAC3 binding to the Bdnf1 (B), Nr4a1 (C), or Nr4a2 (D) promoters in the DMS of home cage (HC) controls or following either intermediate training (INT) or extended training (EXT) in vehicle-treated rats, or NaBut treatment post-intermediate training. Data presented as fold change relative to IgG (% Input/IgG (e–g) mRNA expression of Bdnf1 (E), Nr4a1 (F), and Nr4a2 (G) in the DMS. **P<0.01, between groups; ^##P<0.01 relative to homecage control.