Crystallin Alpha B Inhibits Cocaine-Induced Conditioned Place Preference via the Modulation of Dopaminergic Neurotransmission

Abstract

Nonneuronal cells mediate neurotransmission and drug addiction. However, the role of oligodendrocytes in stress-induced cocaine relapses remains unclear. In the present study, we investigated the role of the oligodendrocyte-abundant molecule crystallin alpha B (CRYAB) in cocaine-induced conditioned place preference (CPP) relapsed by restraint stress. RNA sequencing (RNA-seq) was performed to identify oligodendrocytes and stress-associated molecules in the nucleus accumbens (NAcc) of both drug users and cocaine-treated animals. Further, we studied which cell subtypes in the brain express CRYAB. The effects of stress hormones and cocaine on CRYAB expression were evaluated in vitro in human oligodendrocytes. CRYAB is upregulated in the NAcc of both cocaine-treated animals and drug users. CRYAB levels in the NAcc of mice increased during CPP development but decreased following stress-induced relapse. Interestingly, CRYAB is expressed in oligodendrocytes in the NAcc of mice. Extracellular CRYAB levels are regulated by cocaine and stress hormone treatments in oligodendrocyte cultures. Dopamine levels in the NAcc and CPP development of CPP are significantly increased by cocaine in CRYAB knockout (KO) mice. Further, we demonstrated that CRYAB binds to the excitatory amino acid transporter 2 (EAAT2) in the NAcc of mice treated with cocaine. We suggest that oligodendrocyte-derived CRYAB regulates dopamine neurotransmission and stress-evoked cocaine reward behaviour via the modulation of EAAT2 in the NAcc.

Keywords: cocaine relapse; crystallin alpha B; drug addiction; excitatory amino acid transporter 2; nucleus accumbens; oligodendrocytes.

FIGURE 1

Quantification of gene expression levels in rats exhibiting cocaine‐induced sensitization of locomotor activity and comparison with gene expression in human. (A, B) Rats are injected with either saline‐control or cocaine 15 mg/kg (i.p.). (A) Locomotor activity is measured at 10‐min intervals for 120 min. Data are expressed as the mean distance travelled ± SE (n = 20) and are analysed using two‐way repeated measure ANOVA followed by the Holm–Sidak post hoc t test (**p < 0.01 and *p < 0.05 vs. control [at the same time point]). (B) Total locomotor activity is measured for 120 min. Data are expressed as the mean total distance travelled ± SE (n = 20) and are analysed using Student's t test (**p < 0.01 vs. control). (C, D) The mRNA expression level of the (C) Gpd1 and (D) Cryab detected in the microarray is confirmed using qPCR with each specific primer. The expression of each gene is normalized to the relative amplification of each Gapdh. Data are expressed as the mean ± SE (n = 4, Student's t test, *p < 0.05 vs. control). (E) Differently expressed genes both in the human past and present drug user groups are anatomically and functionally categorized. The differently expressed genes in the cocaine‐sensitized rats are detected via hierarchical clustering. The genes detected in the two different species are compared to identify the target proteins associated with cocaine relapse. All samples are extracted from the NAcc of the human and rat brains.

FIGURE 2

Restraint stress induces relapse in cocaine‐conditioned place preference and alters the expression pattern of CRYAB. (A) Diagram illustrating the timeline of procedures on cocaine‐induced CPP development phase, extinction phase, and restraint stress‐induced reinstatement phase in C57BL/6N wild‐type mice. Postrecording was performed at three different stages of addiction: development phase, on each day of the extinction phase and immediately after restraint stress was applied in the reinstatement phase. The time that each mouse spent in each compartment is recorded and used to determine the preference for each compartment for 15 min. On Day 21, 1 day after the last extinction day, restraint stress was applied for 30 min by restraining the mouse in a 50‐mL conical tube, and then, the CPP was immediately tested. (B) Before starting the extinction, the mice were developed by cocaine (5 or 10 mg/kg, i.p.) in the CPP test. Restraint stress‐induced CPP score(s) is measured after the extinction phase. Data are expressed as the mean ± SE (n = 9–18) and are analysed using two‐way repeated measure ANOVA followed by the Holm–Sidak post hoc t test (**p < 0.01 and *p < 0.05 vs. control [at the same day point], ^# p < 0.05 vs. Day 10 for each group [each development phase] and ^@@ p < 0.01 vs. Day 20 of each group [last extinction date for each group]). (C–E) The protein expression in NAcc of CRYAB is detected by Western blotting with specific antibodies and normalized to β‐actin. (C) The CRYAB expression is presented on each phase (n = 1). (D) Data are expressed as the mean ± SE (n = 4) and are analysed using one‐way ANOVA followed by the Holm–Sidak post hoc t test (**p < 0.01 vs. control). (E) Data are expressed as the mean ± SE (n = 9 and 10 for the control and cocaine 10 mg/kg groups, respectively) and are analysed using Student's t test (*p < 0.05 vs. control). NAcc, nucleus accumbens.

FIGURE 3

CRYAB‐expressing cell types in the cocaine‐administered mice NAcc and reduced Extracellular CRYAB levels in oligodendrocytes in stress‐induced relapse. (A) The mice are injected with cocaine (5 mg/kg [i.p.]). The mice brain sections (10‐μm‐thick) are reacted with anti‐CRYAB (green) and antieach brain cell marker (red). MBP, NeuN, GFAP and IBA1 are markers of oligodendrocytes, neurons, astrocytes and microglia, respectively. Closed arrows indicate the colocalization of each marker and CRYAB. Open arrows indicate the non‐colocalization of each marker and CRYAB. Original magnification is 200X. The scale bar is 25 μm. (B) The CRYAB protein levels are measured in culture media of primary human oligodendrocytes by using ELISA kit. Primary human oligodendrocytes are cultured in control or cocaine‐treated media (1 or 10 μM) for 24 h, followed by treatment with corticosterone (200 ng/mL) for 1 h. Data are expressed as the mean ± SE (n = 4 for each cocaine‐treated group without corticosterone, corticosterone control and 1‐μM cocaine‐treated group with corticosterone, respectively; n = 6 for 10 μM cocaine‐treated group with corticosterone) and are analysed using two‐way repeated measure ANOVA followed by the Holm–Sidak post hoc t test (**p < 0.01 vs. each control, ^## p < 0.01 vs. each cocaine at the same treatment dose). NAcc, nucleus accumbens.

FIGURE 4

Dopamine transporter availability and dopamine levels in CRYAB KO mice. (A–C) DAT availability is measured in the striatal brain slices of WT and CRYAB KO mice. DAT availability is visualized using a false fluorescent neurotransmitter (FFN102, 500 μM). Black square is a marker on the area of caudal putamen (CPu) and NAcc in the brain slice for calculating the fluorescence. (B) Data are expressed as the intensity mean ± SE (n = 6 and 4 for the intensity on CPu and NAcc groups, respectively) and are analysed using two‐way repeated measure ANOVA followed by the Holm–Sidak post hoc t test (**p < 0.01 vs. each vehicle control, ^## p < 0.01 vs. each WT‐pair). (C) Data are expressed as the intensity change (% of WT‐vehicle group) mean ± SE (n = 6 and 4 for the intensity on CPu and NAcc groups, respectively) and are analysed using Student's t test (**p < 0.01 vs. each WT group). (D–F) The dopamine levels are measured in the mice NAcc via microdialysis probe. (D) Regions where the microdialysis probe is inserted. (E) The basal levels of dopamine are measured. Data are expressed as the mean ± SE (n = 4) and are analysed using Student's t test (**p < 0.01 vs. WT mice). (F) Cocaine (10 mg/kg, i.p.)‐induced dopamine levels (% of basal level) are measured. Data are expressed as the mean ± SE (n = 4) and are analysed using two‐way repeated measure ANOVA followed by the Holm–Sidak post hoc t test (*p < 0.05 vs. each basal level [at 0 min], ^# p < 0.05 vs. WT group at the same time). NAcc, nucleus accumbens.

FIGURE 5

Schematic diagram and detection of the target protein binding to CRYAB. (A) Each step followed for detecting the target protein is briefly described. (B) The protein expression of EAAT2 is detected using Western blotting with specific antibodies and normalized to GAPDH in the NAcc of mice. Data are expressed as the means ± SE (n = 3 and 6 for the control and cocaine 10 mg/kg groups, respectively) and are analysed using Student's t test (*p < 0.05 vs. control). (C) Co‐IP and Western blotting are performed to reveal whether the binding of EAAT2 to CRYAB in cocaine‐treated mice NAcc. The samples of mice NAcc are used for Co‐IP with CRYAB. After Co‐IP, the elution samples are detected using Western blotting with EAAT2 antibody.

FIGURE 6

Conditioned place preference induced by cocaine was increased in Cryab knockout mice. (A) Diagrams illustrating the timeline of cocaine‐induced CPP procedures in wild‐type and CRYAB KO mice. (B) Cocaine‐induced CPP score are measured using the CPP test. Data are expressed as the mean ± SE (n = 12, 11, 9 and 11 for WT‐control, WT‐cocaine, CRYAB KO‐control and CRYAB KO‐cocaine, respectively) and are analysed using two‐way repeated measure ANOVA followed by the Holm–Sidak post hoc t test (**p < 0.01 vs. CRYAB KO‐control, ^# p < 0.05 vs. WT‐cocaine 2.5 mg/kg). NAcc, nucleus accumbens.