Mammalian target of rapamycin complex 1 and its downstream effector collapsin response mediator protein-2 drive reinstatement of alcohol reward seeking

Abstract

Alcohol use disorder is a chronic relapsing disease. Maintaining abstinence represents a major challenge for alcohol-dependent patients. Yet the molecular underpinnings of alcohol relapse remain poorly understood. In the present study, we investigated the potential role of the mammalian target of rapamycin complex 1 (mTORC1) in relapse to alcohol-seeking behavior by using the reinstatement of a previously extinguished alcohol conditioned place preference (CPP) response as a surrogate relapse paradigm. We found that mTORC1 is activated in the nucleus accumbens shell following alcohol priming-induced reinstatement of alcohol place preference. We further report that the selective mTORC1 inhibitor, rapamycin, abolishes reinstatement of alcohol place preference. Activation of mTORC1 initiates the translation of synaptic proteins, and we observed that reinstatement of alcohol CPP is associated with increased protein levels of one of mTORC1's downstream targets, collapsin response mediator protein-2 (CRMP2), in the nucleus accumbens. Importantly, the level of mTORC1 activation and CRMP2 expression positively correlate with the CPP score during reinstatement. Finally, we found that systemic administration of the CRMP2 inhibitor, lacosamide, attenuates alcohol priming-induced reinstatement of CPP. Together, our results reveal that mTORC1 and its downstream target, CRMP2, contribute to mechanisms underlying reinstatement of alcohol reward seeking. Our results could have important implications for the treatment of relapse to alcohol use and position the Food and Drug Administration approved drugs, rapamycin and lacosamide, for the treatment of alcohol use disorder.

Keywords: CRMP2; alcohol; mTOR.

Figure 1. Alcohol priming induces reinstatement of alcohol place preference. (a) Experimental timeline depicting the acquisition, extinction and reinstatement of alcohol-induced CPP.

(b–c) Mice were systemically administered with saline or alcohol (0.9 or 1.8 g/kg). One hour after the i.p. injection, the NAc were dissected and phosphorylation levels of S6 were determined by western blot analysis. ImageJ was used for optical density quantification. Data are expressed as the mean ratio ± SEM of phospho-S6 to S6 and are expressed as percentage of the saline control. (d) CPP score on the post-acquisition test. During the conditioning phase (6 d), Mice were administered (i.p.) by alcohol (0.9 or 1.8 g/kg) or saline solution and were then _confined in the drug-paired or non-drug-paired compartment. One day after the sixth session, the post-acquisition test was con- ducted for 15 minutes. (e) CPP score of the post-acquisition (Post-acq.), post-extinction (Post-ext.) and reinstatement (Reinst.) tests. In each test day, mice were placed in the central neutral area and allowed to explore both compartments of the apparatus for 15 minutes. In the reinstate- ment test day, mice previously conditioned with saline- (Sal) or alcohol- (Alc, 1.8 g/kg) received a priming injection of alcohol (0.9 g/kg, i.p.) or saline immediately prior to the beginning of the test session. Data are represented as mean percentage ± SEM of time spent in the drug-paired compartment during the post-acquisition, post-extinction and reinstatement tests minus time spent in the same compartment during the pre- acquisition session. (b–c) n = 5, (d) n = 10–11, (e) n = 13–18. (c) ***P < 0.001 versus all other groups. (d) **P < 0.01 and ***P < 0.001 versus saline-conditioned mice; $P < 0.05 Alc 0.9 versus Alc 1.8. (e) ###P < 0.001 versus saline-conditioned mice; ***P < 0.001 versus all other groups. CPP, conditioned place preference

Figure 2. mTORC1 is activated in the nucleus accumbens during reinstatement of alcohol place preference.

(a) Mice underwent acquisition, extinction and reinstatement of alcohol place preference as depicted in Figure 1. DMS, DLS and NAc of mice were dissected 60 minutes after the end of the reinstatement test. (b) Schematic drawing of a coronal section of the mouse brain showing the sectioned DMS, DLS and NAc at bregma DV = +1.10/+0.70. (c–f) Phosphorylation level of S6 by saline- (Sal) or alcohol- (Alc) in conditioned and primed mice was determined by western blot analysis in the NAc (c), DMS (e) and DLS (f). ImageJ was used for optical density quantification. Data are expressed as the mean ratio ± SEM of phospho-S6 to S6 and are expressed as percentage of the control sal/sal group. (d) Scatter plot showing the relationship between CPP score on the reinstatement test and phospho-S6 in the NAc. Centerline is the linear regression and dashed lines are the 95% _confidence interval. n = 4, *P < 0.05 versus all other groups. CPP, conditioned place preference; DLS, dorsolateral striatum; DMS, dorsomedial striatum; mTORC1, mammalian target of rapamycin complex 1; NAc, nucleus accumbens

Figure 3. mTORC1 activation following priming-induced reinstatement of alcohol-CPP is restricted to the NAc shell.

(a) Mice underwent ac- quisition, extinction and reinstatement of alcohol place preference as depicted in Figure 1. Mice were euthanized 30 minutes after the end of the reinstatement test. (b) Schematic drawing of a coronal section of the mouse brain showing the shell and core portions of the NAc. (c–e) Phospho-S6 levels in the NAc shell (c) and core (e) (bregma DV = +1.10/+0.70) by saline- (Sal) or alcohol- (Alc) conditioned and primed mice were determined by immunohistochemistry. Left panels, Representative images of mouse NAc shell (c) and core (e) labeled with phospho-S6 in red and NeuN in green. Scale bar 100 _μm. Right panels, Phospho-S6 labeled neurons are expressed as percentage of NeuN positive cells. (d) Scatter plot showing the relationship between CPP score on the reinstatement test and phospho-S6+ neurons in the NAc shell. Centerline is the linear regression and dashed lines are the 95% confidence interval. Sal/Sal n = 7, Sal/Alc n = 9, Alc/Sal n = 7, Alc/Alc n = 10. ***P < 0.001 versus all other groups. CPP, conditioned place preference; mTORC1, mammalian target of rapamycin complex 1; NAc, nucleus accumbens

Figure 4. Systemic administration with the mTORC1 inhibitor, rapamycin, blocks priming-induced reinstatement of alcohol place preference.

(a) Mice underwent acquisition and extinction of alcohol place preference as depicted in Figure 1. On day 14, mice were pre-treated with vehicle (Veh) or rapamycin (Rapa, 10 mg/kg, i.p.). Three hours later, mice received a priming injection of saline or alcohol (0.9 g/kg, i.p.) and underwent the reinstatement test. (b) CPP scores on the post-acquisition (Post-acq.), post-extinction (Post-ext.) and reinstatement (Reinst.) tests. Data are rep- resented as mean percentage ± SEM of time spent in the drug-paired compartment during the tests minus time spent in the same compartment on the pre-acquisition session. Veh/Sal n = 13, Rapa/Sal n = 14, Veh/Alc n = 13, Rapa/Alc n = 13. ^###P < 0.001 versus saline-conditioned mice and *P < 0.05 versus all other groups. CPP, conditioned place preference; mTORC1, mammalian target of rapamycin complex 1

Figure 5. CRMP2 levels are increased in the NAc following priming-induced reinstatement of alcohol place preference.

Homer (a), PSD95 (b), GluA1 (c), Arc (d), Prosapip1 (e) and CRMP2 (f) levels in the NAc of saline- (Sal) or alcohol- (Alc) conditioned and primed mice was determined by western blot analysis. NAc samples are identical to the tissue samples used in Figure 2c–d. (g) Scatter plot showing the relationship between CPP score on the reinstatement test and CRMP2 levels in the NAc. Centerline is the linear regression and dashed lines are the 95% _confidence interval. (h) CRMP2 levels in the DMS were determined by western blot analysis. ImageJ was used for optical density _{quantification.} Data are represented as the mean ratio ± SEM of protein to GAPDH and are expressed as percentage of the control sal/sal group. n = 4, **P < 0.01 versus all other groups. CPP, conditioned place preference; CRMP2, collapsin response mediator protein-2; DMS, dorsomedial striatum; NAc, nucleus accumbens

Figure 6. Systemic administration of the CRMP2 inhibitor lacosamide prevents reinstatement of alcohol place preference and does not affect spontaneous locomotor activity.

(a) Mice underwent acquisition and extinction of alcohol place preference as depicted in Figure 1. On day 14, mice were pre-treated with vehicle (Veh) or lacosamide (LCM, 10 mg/kg, i.p.). Ninety minutes later, mice received a priming injection of saline or alcohol (0.9 g/kg, i.p.) and underwent the reinstatement test. (b) CPP score on the post-acquisition (Post-acq.), post-extinction (Post-ext.) and re- instatement (Reinst.) tests. Data are represented as mean percentage ± SEM of time spent in the drug-paired compartment during the tests mi- nus time spent in the same compartment on the pre-acquisition session. (c) Mice were injected with either Veh or LCM (10 mg/kg, i.p.) 90 minutes prior to the beginning of the locomotion test. Data are represented as mean percentage ± SEM of spontaneous locomotor activity measured during 30 minutes. (b) Veh/Sal n = 11, LCM/Sal n = 10, Veh/Alc n = 12, LCM/Alc n = 14, (c) n = 5. (b) ^###P < 0.001 versus saline- conditioned mice and *P < 0.05 versus Veh/Alc group. CPP, conditioned place preference