Long-Term Excessive Alcohol Consumption Enhances Myelination in the Mouse Nucleus Accumbens

Abstract

Chronic excessive alcohol (ethanol) consumption induces neuroadaptations in the brain's reward system, including biochemical and structural abnormalities in white matter that are implicated in addiction phenotypes. Here, we demonstrate that long-term (12 week) voluntary ethanol consumption enhances myelination in the nucleus accumbens (NAc) of female and male adult mice, as evidenced by molecular, ultrastructural, and cellular alterations. Specifically, transmission electron microscopy analysis showed increased myelin thickness in the NAc following long-term ethanol consumption, while axon diameter remained unaffected. These changes were paralleled by increased mRNA transcript levels of key transcription factors essential for oligodendrocyte (OL) differentiation, along with elevated expression of critical myelination-related genes. In addition, diffusion tensor imaging revealed increased connectivity between the NAc and the prefrontal cortex, reflected by a higher number of tracts connecting these regions. We also observed ethanol-induced effects on OL lineage cells, with a reduction in the number of mature OLs after 3 weeks of ethanol consumption, followed by an increase after 6 weeks. These findings suggest that ethanol alters OL development prior to increasing myelination in the NAc. Finally, chronic administration of the promyelination drug clemastine to mice with a history of heavy ethanol consumption further elevated ethanol intake and preference, suggesting that increased myelination may contribute to escalated drinking behavior. Together, these findings suggest that heavy ethanol consumption disrupts OL development, induces enhanced myelination in the NAc, and may drive further ethanol intake, reinforcing addictive behaviors.

Keywords: addiction; alcohol; animal models; ethanol; myelin; myelin basic protein; nucleus accumbens; oligodendrocytes.

Figure 1.

Voluntary ethanol consumption increases myelin-related gene expression in the NAc in a time-dependent manner. Amount of alcohol (g/kg/24 h) consumed over 84 d (12 weeks). A, Mice consumed ethanol in the intermittent access to 20% ethanol two-bottle choice procedure for 3, 6, or 12 weeks. Control mice consumed only water. Tissues were collected 24 h after the last drinking session (ethanol group). B, C, Ethanol intake (B) and preference (C) over 84 d. Data are expressed as mean ± SEM intermittent daily averages. D–I, mRNA levels of Sox10 (D), Olig2 (E), Myrf (F), Mbp (G), Plp1 (H), and Mag (I) in the NAc, as determined by qRT-PCR and normalized to the level of Gapdh mRNA. Bar graphs represent mean + SEM. n = 5–8 per group; for more details on n per group and sex distribution, see Extended Data Table 1-1; *p < 0.05; **p < 0.01; relative to corresponding water-drinking controls.

Figure 2.

Long-term ethanol consumption increases MBP protein levels in the NAc and connectivity between the NAc and the PFC. Mice consumed ethanol in the intermittent access to 20% ethanol two-bottle choice procedure for 12 weeks. Control mice consumed only water. Tissues were collected 24 h after the last drinking session (ethanol group). A, Schematic representation of the sampling region (Paxinos and Franklin, 2019). Scale bar, 100 µm. B, Quantification of Western blot of MBP levels in the NAc, normalized to GAPDH levels, expressed as a percentage of water-drinking controls. C, Representative blots. See Extended Data Figure 2-1 for the whole blot image. D, Representative immunofluorescence images of MBP staining and DAPI nuclear staining in the NAc. E, Quantification of the number of MBP-positive events in the NAc. F, Mean area of MBP-positive events in the NAc. G, Representative images of tracts between the NAc and the PFC, analyzed from DTI scans. H, Quantification of the number of tracts between the NAc and PFC. Bar graphs represent mean + SEM. n = 8–9 per group for immunofluorescence and Western blot; n = 21–23 per group for DTI; for more details on n per group and sex distribution, see Extended Data Table 2-1. *p < 0.05.

Figure 3.

Long-term ethanol consumption affects myelin ultrastructure in the NAc. A, Representative images of myelin ultrastructural in the NAc of ethanol-drinking mice, as compared with water-drinking controls. Scale bars, 500 nm. B, A scatterplot of individual g-ratio versus axon diameter. Approximately 300 myelinated axons were considered per group. The data were fitted by simple linear regression, and slopes and intercepts are indicated. C, Quantification of g-ratio in the NAc. D, Myelin thickness (half of the difference between the axon + myelin diameter and the axon diameter in micrometer). E, Axon diameter. Data show mean + SEM. n = 4 per group (2 males, 2 females); **p < 0.01.

Figure 4.

Long-term ethanol consumption affects OL lineage cells in the mouse NAc in a time-dependent manner. Mice consumed ethanol in the intermittent access to 20% ethanol two-bottle choice procedure for 3, 6, or 12 weeks. Control mice consumed only water. Tissues were collected 24 h after the last drinking session (ethanol group). A–C, To identify OPC, total OL and mOL immunohistochemistry was performed using antibodies against PDGFRα+ (A), OLIG2+ (B), the mOLs marker APC (CC1+; C) and the OL lineage marker OLIG2+. D–F, Representative immunofluorescence staining image of anti-PDGFRα (blue; D), anti-OLIG2 (green; E), and anti-OLIG2 + CC1 (merge in yellow; F). Scale bar, 100 µm. Bar graphs represent mean + SEM. n = 7–11 per group; for more details on n per group and sex distribution, see Extended Data Table 4-1. *p < 0.05; **p < 0.01; ***p < 0.0001.

Figure 5.

Effects of long-term ethanol consumption on OL proliferation and differentiation properties. Mice consumed ethanol in the intermittent access to 20% ethanol two-bottle choice procedure for 12 weeks. Control mice consumed only water. Tissues were collected 24 h after the last drinking session (ethanol group). Immunohistochemistry was performed to evaluate OPC proliferation and OL differentiation properties. A, Number of Ki67+ PDGFRα+ cells in the NAc, determined by immunohistochemistry, expressed as the percentage of the water-drinking control group's value. B, Representative immunofluorescence images of Ki67+ (green), PDGFRα+ (yellow), and Ki67+ PDGFRα+ merged images, with arrows pointing to PDGFRα+Ki67 + colocalized cells. Scale bar, 50 µm. C, H3K9me3 intensity in CC1 + cells. D, Representative immunofluorescence images of H3K9me3+ (green), CC1+ (yellow), and H3K9me3 intensity in CC1 + cells, arrows pointing to CC1+ H3K9me3 + colocalized cells. Scale bar, 50 µm. Bar graphs represent mean + SEM. n = 6–7 per group; for more details on n per group and sex distribution, see Extended Data Table 5-1.

Figure 6.

Chronic systemic administration of clemastine elevates ethanol consumption and increases MBP expression and mOL number in the NAc of mice. A, Experimental scheme. Following 12 weeks of ethanol drinking in the intermittent access to 20% ethanol two-bottle choice procedure, daily treatment with clemastine (10 mg/kg, i.p.) or vehicle was initiated for 7 weeks. Mouse ethanol consumption and preference were recorded. NAc tissues were collected 24 h after the last drinking session and processed for Western blot or immunohistochemistry. B, C, Ethanol intake (B) and preference (C) per 24 h session were averaged by week. D, MBP levels in the NAc, as determined by Western blot and normalized to GAPDH levels, expressed as the percentage of the levels determined in the vehicle-treated group. E, Representative Western blot images. See Extended Data Figure 6-1 for the whole blot image. F, G, Identification of total OLs (F) and mOL (G) lineage cells in the NAc, as determined by immunohistochemistry performed using antibodies against the mOLs marker APC (CC1+) and the OL lineage marker OLIG2+. H, Representative immunofluorescence image of OLIG2 + CC1+ cells. Scale bar, 100 µm. Bar graphs represent mean + SEM. n = 12 per group for behavioral data; n = 5–6 for biochemical data; for more details on n per group and sex distribution, see Extended Data Table 6-1; ^#p = 0.07; *p < 0.05; ***p < 0.001, relative to vehicle-treated controls.