Research ArticleNEUROSCIENCE

Prior alcohol use enhances vulnerability to compulsive cocaine self-administration by promoting degradation of HDAC4 and HDAC5

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Science Advances  01 Nov 2017:
Vol. 3, no. 11, e1701682
DOI: 10.1126/sciadv.1701682
  • Fig. 1 Prior alcohol use enhances persistence, motivation, and compulsivity for cocaine self-administration.

    (A) Drug treatment paradigm to study alcohol and cocaine coadministration. Access to voluntary alcohol (10% alcohol, 2 hours per day) and voluntary cocaine (self-administration, 0.8 mg/kg per injection) was restricted to 3 p.m. to 5 p.m. and 11 a.m. to 2 p.m., respectively, to avoid metabolic interaction between the two drugs. (B) Alcohol self-administration. Animals in the alcohol-primed group began drinking alcohol on day 1 of the paradigm; animals in the alcohol-concurrent group began drinking on day 11. All groups start cocaine self-administration (0.8 mg/kg per infusion) on day 11. The average alcohol intake during the alcohol-priming period was 1 g/kg during the first 5 days, increasing to 1.2 g/kg during the second 5 days. Alcohol intake was similar in the alcohol-primed versus alcohol-concurrent groups from days 11 to 32 [two-way repeated-measures (RM) analysis of variance (ANOVA): Treatment group: F1,12 = 1.398, P = 0.26; Treatment day: F21,252 = 10.71, P < 0.0001; Interaction: F21,252 = 1.14, P = 0.303; n = 6 to 8 per group]. (C) Prior exposure to alcohol does not affect the acquisition of lever pressing on an FR5 schedule of reinforcement. Animals were started on FR1 and gradually increased to FR3 (1 to 2 days) and then to FR5. Animals reached FR5 after 5.9, 6.2, and 6.6 days for water control, alcohol-primed, and alcohol-concurrent groups, respectively (one-way ANOVA: F2,21 = 0.63, P = 0.5425, not significant; n = 6 to 8 per group). (D) Prior alcohol exposure does not enhance lever pressing for cocaine reward (0.8 mg/kg per injection) during the maintenance phase of cocaine self-administration. The analysis showed no main effect for treatment group, treatment day, or an interaction between the two factors (two-way ANOVA: Group: F2,123 = 1.119, P = 0.3298, not significant; Treatment day: F6,123 = 1.44, P = 0.20, not significant; Interaction: F12,123 = 0.16, P = 0.9995, not significant). (E) Alcohol preexposure enhances persistence of cocaine seeking during unrewarded time-out sessions, averaged over the last 3 days of the maintenance phase (B) ( one-way ANOVA: F2,19 = 3.66, P = 0.045; Tukey post hoc: P = 0.047, alcohol primed versus alcohol-naïve; n = 6 to 8 per group). (F) Alcohol preexposure enhances motivation for cocaine self-administration in a progressive ratio schedule of reinforcement (one-way ANOVA: F 2,20 = 6.45, P = 0.007; Tukey post hoc analysis: P < 0.01, control versus alcohol-primed; P < 0.01, concurrent versus alcohol-primed; n = 6 to 8 per group). (G) Alcohol preexposure enhances compulsive cocaine self-administration. Successive increases of the footshock intensity resulted in a decrease in lever pressing for all groups. Alcohol pretreated animals have significant resistance to footshock (two-way RM ANOVA: Group: F2,19 = 4.76, P = 0.02; Footshock: F3,57 = 67.94, P < 0.0001; Footshock × Group interaction: F6,57 = 1.829, P = 0.1095; n = 6 to 8 per group). The baseline number of cocaine infusions (at 0.0 mA) did not differ between groups (AN, 19.67 ± 2.10; AP, 19.7 ± 2.32; AC, 20.00 ± 2.46). (H) Alcohol preexposure does not alter shock-resistant lever pressing for sugar pellets in food-restricted animals (two-way RM ANOVA: Group: F1,9 = 0.003, P = 0.96, not significant; Footshock: F3,27 = 131.3, P < 0.0001; Footshock × Group interaction: F3,27 = 0.64, P = 0.59, not significant; n = 5 to 6 per group). Baseline amount of sugar pellet reward (at 0.0 mA) did not differ between groups (AN, 46.60 ± 3.3; AP, 49.33 ± 2.15). *P < 0.05. Data are means ± SEM.

  • Fig. 2 Alcohol use creates a permissive epigenetic environment for cocaine-induced gene expression.

    (A) Diagram of sequential drug administration paradigm. Animals drank alcohol in a limited access paradigm [alcohol 10% (v/v); 2 hours per day; average intake, 1.11 ± 0.1 g/kg per day; n = 14] for 10 days. An acute cocaine injection (20 mg/kg intraperitoneally) was given 18 hours after the last alcohol ingestion to avoid pharmacokinetic interaction between alcohol and cocaine. (B) A single cocaine injection causes increased expression of the ΔFosB transcript in alcohol-naïve animals (one-way ANOVA: F3,20 = 20.15, P < 0.0001; Sidak post hoc: water/saline-injected versus water/cocaine-injected, P < 0.05), whereas alcohol pretreated animals have significantly enhanced cocaine-induced ΔFosB induction (P < 0.05, alcohol/cocaine-injected versus water/cocaine-injected; P < 0.0001, alcohol/cocaine-injected versus water/saline-injected, alcohol/saline-injected; n = 6 to 7 per group). (C) ChIP experiment for acetylated histone H3K27 at the FosB region of interest shows increased acetylation in animals treated with 10% alcohol, 2 hours per day for 10 days (average intake, 1.1 ± 0.1 g/kg per day, n = 5, P < 0.01 versus water control, n = 3 to 5 per group). (D) Immunoblotting experiment for H3K27 acetylation in animals that drank for 10 consecutive days [10% (v/v) alcohol, 2 hours per day; average intake, 0.98 ± 0.12 g/kg per day; n = 5] and euthanized 18 hours after the last alcohol ingestion shows global increase in H3K27 acetylation in the nucleus accumbens (P < 0.05, 0 hours versus water control; n = 4 to 5 per group). P.O., per oral. *P < 0.05, **P < 0.01, ***P < 0.0001. Data are means ± SEM.

  • Fig. 3 Long-term, but not short-term, alcohol use promotes progressive decrease of nuclear HDAC activity following alcohol cessation.

    (A) Diagram of 10-day voluntary drinking protocol. Alcohol-treated animals were sacrificed at sequential time points after the last alcohol ingestion on day 10: 0 (end of alcohol drinking session), 12, 18, and 22 hours after the last alcohol ingestion (average intake, 0.95 ± 0.05 g/kg per day; n = 20). (B) Short-term (2-day) alcohol exposure paradigm (average alcohol intake, 1.07 ± 0.16 g/kg per day; n = 20). (C) Ten-day exposure to alcohol causes a decrease in nuclear HDAC activity in the nucleus accumbens (one-way ANOVA: F4,19 = 3.669, P = 0.0224; Sidak post hoc: P = 0.026, water versus alcohol 22 hours after treatment). (D) Two-day exposure to alcohol does not result in decreased nuclear HDAC activity (one-way ANOVA: F4,18 = 0.794, P = 0.5442). (E) HDAC activity decreases progressively following cessation of alcohol treatment in animals treated with alcohol for 10 days (two-way ANOVA: Time after treatment cessation: F3,32 = 3.165, P = 0.038; Treatment group: F1,32 = 17.18, P = 0.0002; Interaction: F3,32 = 4.715, P = 0.0078; Sidak post hoc: no significant change at 0 and 12 hours, P = 0.035 at 18 hours, and P = 0.0003 at 22 hours; n = 4 to 5 per group). (F) Immunoblotting experiment for H3K27 acetylation shows enhancement of global H3K27 acetylation at 12, 18, and 22 hours after the last alcohol ingestion (one-way ANOVA: F4,16 = 5.45, P = 0.0058; Sidak post hoc: P = 0.0033, P = 0.006, P = 0.010 at 12, 18, and 22 hours, respectively, versus water control group; n = 4 to 5 per group). (G) Two-day exposure to alcohol does not result in enhanced H3K27 acetylation in the nucleus accumbens (one-way ANOVA: F4,14 = 0.3981, P = 0.8067; n = 4 to 5 per group). *P < 0.05, **P < 0.01. Data are means ± SEM.

  • Fig. 4 Alcohol use promotes degradation of nuclear HDAC4 and HDAC5 in the nucleus accumbens.

    (A) Immunoblotting experiment for HDAC4 in the nuclear lysates of nucleus accumbens cells shows that alcohol cessation following 10 days of alcohol use (Fig. 3A) is associated with significantly decreased levels of HDAC4 in the nucleus accumbens 18 hours after the last alcohol ingestion (one-way ANOVA: F4,17 = 4.37, P = 0.0130; Sidak post hoc: P < 0.05, alcohol/18-hour cessation versus water control; n = 4 to 5 per group). (B) Alcohol use is not associated with changes in HDAC4 levels in the cytoplasmic fractions of nucleus accumbens cells (one-way ANOVA: F4,20 = 0.5071, P = 0.7310; n = 4 to 5 per group). (C) Quantitative real-time PCR (qRT-PCR) analysis for HDAC4 mRNA in nucleus accumbens lysates after 10 days of alcohol use shows no change in HDAC4 mRNA 18 hours after the last alcohol ingestion (P > 0.05, alcohol pretreated versus water control; n = 4 to 5 per group). (D) Intra-nucleus accumbens delivery of the proteasomal inhibitor lactacystin rescues the decrease in HDAC activity observed following alcohol cessation (one-way ANOVA: F2,13 = 7.97, P = 0.0055; Sidak post hoc: P < 0.01, water versus alcohol; P < 0.05, alcohol versus alcohol + lactacystin; n = 6 to 8 per group). (E) Intra-nucleus accumbens delivery of the proteasomal inhibitor lactacystin rescues the decrease in nuclear HDAC4 observed following alcohol cessation (one-way ANOVA: F2,13 = 7.97, P = 0.0055; Sidak post hoc: P < 0.01, water versus alcohol; P < 0.05, alcohol versus alcohol + lactacystin; n = 6 to 8 per group). (F) Alcohol treatment and lactacystin infusion had no significant effects on HDAC4 levels in the cytoplasm (one-way ANOVA: F4,20 = 0.507, P = 0.7310; n = 6 to 8 per group). *P < 0.05, **P < 0.01. Data are means ± SEM.

  • Fig. 5 Selective degradation of HDAC4 and HDAC5 by the class II–specific HDAC inhibitor MC1568 enhances compulsive cocaine self-administration.

    (A) Treatment paradigm to test molecular and behavioral effects of the class IIa selective HDAC inhibitor MC1568. (B) HDAC activity assay of nuclear lysates of nucleus accumbens cells isolated from animals following 10 daily (0.5 mg/kg intraperitoneally) treatments shows 35% decrease in HDAC activity in comparison to control (vehicle-treated) animals (P < 0.01 versus vehicle control; n = 5 per group). (C) Immunoblot for HDAC1, HDAC2, HDAC4, and HDAC5 of nucleus accumbens lysates after 10-day treatment with MC1568 (0.5 mg/kg) shows selective decreases of nuclear accumulation of HDAC4 and HDAC5 (P < 0.05), and no significant change in HDAC1 or HDAC2 (P > 0.5 versus vehicle control; n = 7 to 8 per group). (D) MC1568 did not change cytoplasmic accumulation of HDAC4 or HDAC5. (E) Animals in the MC1568 treatment group had significantly higher motivation in a progressive ratio schedule of reinforcement at a unit cocaine dose of 0.4 mg/kg (two-way RM ANOVA: Treatment group: F1,10 = 7.33, P = 0.022; Unit cocaine dose: F2,20 = 56.51, P < 0.0001; Interaction: F2,20 = 5.08, P = 0.0164; Tukey post hoc: P < 0.01 at 0.4 mg/kg; n = 5 to 6 per group), with no significant difference at a lower unit dose of 0.2 mg/kg. (F) MC1568 enhances persistence of cocaine seeking during unrewarded time-out sessions (P < 0.05, n = 6 per group), averaged across 3 days before progressive ratio testing. (G) MC1568-treated animals are more compulsive, with significantly higher percentage of rewards earned during 0.2 and 0.3 mA of punishment (two-way RM ANOVA: Group: F1,9 = 7.396, P = 0.023; Footshock: F3,27 = 96.94, P < 0.0001; Footshock × Group interaction: F3,27 = 3.98, P = 0.019; P < 0.05 at 0.2 and 0.3 mA; n = 5 to 6 per group). The average cocaine infusions at baseline (0.0 mA) did not differ between groups: vehicle, 19.17 ± 1.77; MC1568, 18.80 ± 2.35. (H) MC1568 does not alter shock-resistant lever pressing for sugar pellets in food-restricted animals (two-way RM ANOVA: Group F1,10 = 0.2159, P = 0.65, not significant; Footshock F3,30 = 137.6, P < 0.0001; Footshock × Group interaction: F3,30 = 0.3396, P = 0.7968; n = 6 per group). Baseline amount of sugar pellet reward (at 0.0 mA) did not differ between groups (AN, 53.40 ± 0.67; AP, 53.20 ± 0.73). TBP, TATA-binding protein; IP, intraperitoneal. *P < 0.05, **P < 0.01. Data are means ± SEM.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/11/e1701682/DC1

    Supplementary Materials and Methods

    table S1. Alcohol and cocaine and cocaethylene levels.

    fig. S1. Prior alcohol use does not enhance daily cocaine intake.

    fig. S2. Prior cocaine use decreases preference for alcohol.

    fig. S3. Initiation of cocaine self-administration on day 11 resulted in decreased alcohol intake in alcohol-primed animals.

    fig. S4. Acetylation at H3 lysine 27 is a key regulatory mark for FosB.

    fig. S5. Alcohol use promotes decreased nuclear accumulation of HDAC4 in nucleus accumbens.

    fig. S6. Chronic alcohol use promotes proteasome-mediated degradation of HDAC5 in the nucleus accumbens.

    fig. S7. MC1568 treatment does not enhance daily cocaine intake.

    fig. S8. Subcellular fractionation.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • table S1. Alcohol and cocaine and cocaethylene levels.
    • fig. S1. Prior alcohol use does not enhance daily cocaine intake.
    • fig. S2. Prior cocaine use decreases preference for alcohol.
    • fig. S3. Initiation of cocaine self-administration on day 11 resulted in decreased alcohol intake in alcohol-primed animals.
    • fig. S4. Acetylation at H3 lysine 27 is a key regulatory mark for FosB.
    • fig. S5. Alcohol use promotes decreased nuclear accumulation of HDAC4 in nucleus accumbens.
    • fig. S6. Chronic alcohol use promotes proteasome-mediated degradation of HDAC5 in the nucleus accumbens.
    • fig. S7. MC1568 treatment does not enhance daily cocaine intake.
    • fig. S8. Subcellular fractionation.

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