Adolescent drinking causes a loss of aspartoacylase-expressing oligodendrocytes and hypomyelination of anterior cingulate and corpus callosum axons in male mice, but not females

21 We kindly thank Rithika Senthilkumar and Faye Reagan for their assistance with these studies, 22 and the university animal husbandry and veterinary staff for their dedicated support in 23 maintaining the health and welfare of our mice. We performed confocal imaging and analyses 24 and imaging in the UMass IALS Light Microscopy Facility and Nikon Center of Excellence, and 25 we thank the Director, Dr. James Chambers, for his support and guidance. The work was 26 funded with support from the National Institutes of Health R01AA024774 (HNR) and NIH 27 R21AA031376 (HNR), the National Science Foundation Graduate Fellowship Research 28 Program 1938059 (AFB), and the UMass Spaulding-Smith Fellowship Program (AFB). Graphs 29 were created with GraphPad PRISM and figures were created with BioRender.com. 30 31 Key Words: 32 Adolescence; Alcohol; Sex Difference; Oligodendrocyte; Aspartoacylase 33 34 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 2

35 Adolescent binge drinking is a strong predictor of alcohol use disorder and related mental health 36 outcomes in adulthood, which may be due to disruptions in myelination during this dynamic 37 period of brain development. White matter expansion in frontal regions during adolescence is 38 essential for mature decision-making and stress regulation, yet the cellular mechanisms by 39 which alcohol disrupts this process remain poorly understood. We used multi-label 40 immunofluorescence and confocal microscopy to visualize proteins in oligodendrocyte lineage 41 cells and myelin ensheathment of axons in the anterior cingulate cortex (Cg1) and corpus 42 callosum (CC) following four weeks of episodic voluntary binge drinking using the Drinking-in-43 the-Dark model in adolescent male and female C57BL/6NJ mice beginning on postnatal day 28. 44 Contrary to our initial hypothesis that alcohol targets early-stage oligodendrocyte precursor cells 45 (OPCs), binge drinking selectively depleted mature oligodendrocytes expressing aspartoacylase 46 (ASPA) in the Cg1 and CC of male mice, but not females. This enzyme is essential for lipid 47 biosynthesis and myelin production, and this cell-specific loss was accompanied by significant 48 hypomyelination of axons only in males. These findings identify a later maturational stage of 49 oligodendroglial development as a sex-dependent target of alcohol, advancing our mechanistic 50 understanding of prefrontal myelin deficits in adolescent drinking. Furthermore, ASPA emerges 51 as a potential therapeutic target for alcohol use disorder and demyelinating diseases, with 52 differential vulnerability across sex carrying important implications for adult neurodevelopmental 53 outcomes. 54 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 3

55 Over 134 million teenagers and adults consumed alcohol in the United States in 2024, 56 with 43.1% reporting binge drinking within the past month (Substance Abuse and Mental Health 57 Services Administration, 2025a). While the overall prevalence of binge drinking among adult 58 men and women declined from 2023 to 2024, rates among adolescents aged 12-17 has 59 remained unchanged over the last few years (2.9% in young males and 4.1% in young females, 60 (Substance Abuse and Mental Health Services Administration, 2025b, 2025c)). Binge drinking is 61 the rapid consumption of alcohol that produces blood alcohol concentrations of 0.08 g/dL or 62 higher within two hours, and this is the most common, costly, and deadly type of excessive 63 drinking (National Institute on Alcohol Abuse and Alcoholism, 2004; Stahre et al., 2014; Sacks et 64 al., 2015). Binge drinking during adolescence is strongly associated with the later development 65 of alcohol use disorder (AUD), a chronic relapsing condition characterized by maladaptive 66 alcohol use that persists despite physiological and psychological distress (Chou and Pickering, 67 1992; American Psychiatric Association, 2013; Addolorato et al., 2018). The neurocircuits 68 implicated in AUD and co-occurring mental health conditions are known to differ with sex 69 (Flores-Bonilla and Richardson, 2020; Lees et al., 2020; Radke et al., 2021; Bowen et al., 70 2022). Determining how adolescent drinking affects maturation of these neurocircuits in males 71 and females may help elucidate the underlying causes of cognitive and behavioral health 72 outcomes in adulthood. 73 Brain maturation during adolescence includes the expansion of myelinated white matter 74 fiber tracks in frontal brain regions, which is thought to contribute to improved decision-making, 75 cognition, stress-regulation, and impulse control in adulthood (Gogtay et al., 2004). 76 Oligodendrocytes (OLs) are the glial cells that form insulating myelin sheaths by wrapping 77 concentric layers of lipid-rich processes around axons (Simons and Nave, 2016; Stadelmann et 78 al., 2019) 79 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 4 Myelin sheaths restrict membrane conductance to the nodes of Ranvier that are 80 enriched with voltage-gated ion channels, leading to faster neurotransmission through saltatory 81 conduction (Seidl, 2014). The functional benefit of prefrontal myelination during adolescence 82 has been demonstrated in preclinical animal studies. For example, myelination of axons 83 extending from the corpus callosum into the anterior cingulate cortex early in adolescence 84 (McDougall et al., 2018; Drzewiecki et al., 2020) corresponds with a marked increase in the 85 transmission speed of action potentials along these axons in male rats (McDougall et al., 2018). 86 In addition to the increased risk of AUD, adolescent drinking has also been linked to 87 other negative health outcomes in adulthood including impaired higher executive functions, 88 enhanced reward and sensation-seeking, and a dysregulated response to stress (McCarty et 89 al., 2004; Peters et al., 2015; Squeglia et al., 2015; Elsayed et al., 2018), reviewed in (Crews et 90 al., 2016).These functional deficits observed in adulthood could be due to alcohol-induced 91 disruptions in myelination during adolescent development, as reviewed in (Rice and Gu, 2019). 92 Indeed, lower expression of myelin-associated genes and myelin deficits have been observed in 93 the anterior cingulate following adolescent drinking in humans, rats and mice, with greater 94 effects in males compared to females (Vargas et al., 2014; Wolstenholme et al., 2017; Morris et 95 al., 2019; Tavares et al., 2019). 96 Establishing a causal link between alcohol-induced myelin loss in adolescence and 97 negative health outcomes in adulthood requires a deeper understanding of the cellular 98 mechanisms underlying these changes in myelin, yet these remain elusive. Alcohol may be 99 disrupting axonal myelination in the developing brain of adolescents by targeting OL precursor 100 cells (OPCs) and triggering apoptotic cell death or preventing these cells from differentiating into 101 OLs. In support of this, there is evidence of OPC apoptosis and attenuated differentiation in 102 human fetal brain tissue following alcohol exposure during embryonic development (Darbinian 103 et al., 2021). Similar results have also been reported in rodents exposed to prenatal alcohol 104 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 5 (Newville et al., 2017). Alternatively, adolescent alcohol may be targeting this lineage after 105 differentiation, as has been observed in fetal brains of macaques following alcohol exposure 106 (Creeley et al., 2013). 107 The goal of the current study was to identify cellular mechanisms that could account for 108 myelin changes after repeated binge drinking in adolescence. Using multi-label 109 immunofluorescence and confocal microscopy we tracked and quantified cellular proteins in 110 oligodendroglia lineage cells and myelin ensheathment of axons in the anterior cingulate cortex 111 and corpus callosum following repeated cycles of voluntary binge drinking of alcohol (or water) 112 using the Drinking-in-the-Dark model in adolescent male and female mice. Contrary to our initial 113 hypothesis, we found evidence of oligodendroglia lineage disruption in later stages of cellular 114 development when mature OLs are making the aspartoacylase enzyme necessary for lipid 115 biosynthesis and myelin production. Most notably, these cells were especially sensitive to high 116 levels of alcohol, but the effects were unique to males—explaining why reduced myelin density 117 was not observed in binge drinking females. These findings offer insight into the mechanisms 118 underlying prefrontal myelin deficits associated with adolescent alcohol use, and differential 119 sensitivity across sex may have important implications for adult outcomes. 120 121

122 Animals 123 C57BL/6NJ wildtype male and female mice arrived at 3 weeks of age from Jackson 124 Laboratory (Bar Harbor, ME; stock #005304) and were housed in same sex groups of 3-5 mice 125 per cage. All care of and experimental procedures with animals were performed in strict 126 compliance with the University of Massachusetts Amherst Institutional Animal Care and Use 127 Committee and the NIH Guide for the Care and Use of Laboratory Animals. 128 Drinking-in-the-Dark (DID) 129 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 6 Male and female C57BL/6NJ wild-type mice (n=8/alcohol group/sex) were exposed to 130 voluntary alcohol binge drinking using the well-established limited access Drinking-in-the-Dark 131 (DID) procedure (Rhodes et al., 2005a; Thiele and Navarro, 2014; Thiele et al., 2014; Wilcox et 132 al., 2014). Mice were transferred to a reverse cycled room (11:00 lights OFF/ 23:00 lights ON) 133 for acclimatization for 1 week and were single housed at postnatal day (PD) 28 for DID. DID 134 began 3 hours into the dark cycle on PD28. Mice were given access to either 20% (v/v/) alcohol 135 (or water for controls) for 2 hours on 3 consecutive days, followed by a “binge day” with 4 hours 136 of access. This was followed by a 3-day rest period in the home cage without access to alcohol 137 before the next drinking DID cycle began. Mice completed four DID cycles in total from PD28-138 56. To access drinking levels, bottles were weighed before and after each bout to provide a 139 measure used to calculate daily g/kg intake of alcohol for each animal or ml/kg water intake for 140 control animals. Spillage bottles containing either water or alcohol were handled identically to 141 the experimental bottles to estimate the loss of liquid during the manipulation and due to 142 evaporation. The average spillage volumes were subtracted from the experimental volumes of 143 alcohol or water. Negative values were counted as 0 g/kg intake for alcohol or water. 144 Intracardial perfusions and brain tissue processing 145 Four days after the last drinking bout (PD56), animals were deeply anesthetized with 146 pentobarbital sodium solution (Nembutal, 200 mg/kg ip). They were intracardially perfused with 147 0.9% saline (room temperature) for 5 min, followed by 4% paraformaldehyde / 0.1M sodium 148 tetraborate (chilled to 4°C, pH 9.4) for 5 min at a rate of 6 ml/min. Brains were rapidly extracted 149 and post-fixed overnight in 4% paraformaldehyde at 4°C. Brains were then immersed in a 10% 150 sucrose/phosphate buffered saline solution at 4°C for 1 day and 30% sucrose/phosphate 151 buffered saline solution for another 24 hours. Brains were then snap frozen by submerging 152 briefly in -50°C isopentane (2-methylbutane; Thermo Fisher Scientific, catalogue #03551-4, Fair 153 Lawn, NJ, USA) and stored at -80°C until cryo-sectioning. A freezing sliding microtome was 154 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 7 used to slice 35 µm thick coronal serial sections that were collected in strict anatomical order 155 and stored in cryoprotectant solution in a 1:6 series at -20°C until immunofluorescence 156 experiments were performed. 157 Immunofluorescence 158 In Experiment 1, we used free-floating sections at AP+1.7mm to align anatomically with 159 previous studies showing prefrontal myelin loss after adolescent exposure to alcohol drinking in 160 rats or binge intragastric alcohol administration in mice (Vargas et al., 2014; Papp-Peka et al., 161 2016; Rice et al., 2019; Tavares et al., 2019). In Experiment 2, we expanded the sampling 162 region to include more sections (AP+2.0mm, AP+1.7mm, AP+0.9mm) and test whether 163 oligodendroglial cellular changes extend beyond the medial prefrontal cortex. For both 164 experiments, floating sections were first thoroughly washed with PBS to remove the 165 cryoprotectant storage solution. This was followed by washes in 0.3% Triton-X in PBS (PBS-Tx) 166 to enable primary antibodies to permeate cytoplasmic and nuclear membranes and bind to 167 target proteins. Tissue samples were incubated in 50 mM ammonium chloride solution 168 (NH4Cl/PBS-Tx) for 30 minutes at 25°C to quench autofluorescence followed by rinses in PBS-169 Tx. They were then incubated in a 3% hydrogen peroxide/PBS solution for 30 minutes at 25°C 170 to block endogenous peroxidase. To reduce non-specific binding of secondary antibodies, 171 tissues were incubated in a 5% normal horse serum/PBS-Tx solution for 1 hour at 25°C. 172 Samples were then thoroughly washed in PBS-Tx, followed by incubation with primary 173 antibodies specific to each experiment overnight at 4°C (see Table 1 for dilutions). After several 174 washes in PBS-Tx, sections were incubated in secondary antibodies at room temperature 175 (25°C) for 2 hours. Sections were then washed in PBS and incubated in a Cy3-conjugated 176 streptavidin/PBS-Tx solution for 1 hour at 25°C to amplify and visualize the CC1 signal. 177 Following PBS rinses, nuclei were then fluorescently stained using 0.4 mg/ml 178 diamidinophenolindole (DAPI), which binds to the adenine–thymine-rich regions in DNA 179 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 8 (Kapuscinski, 1995). Sections were washed in PBS and mounted on subbed glass slides and 180 allowed to thoroughly dry in a dark room for a minimum of 12 hours. Slides were next cleared in 181 xylenes and DPX mounting media was used to adhere glass coverslips (Fisher Scientific 182 #50980370). 183 Experiment 1: Test for evidence of reduced oligodendrocyte progenitors and 184 mature cells accompanying myelin loss following adolescent drinking. We used multi-185 label immunofluorescence to detect and visualize oligodendroglia- and myelin-related proteins 186 in medial prefrontal white and gray matter regions at the location where axons of the corpus 187 callosum forceps minor (CCFM) branch out into the cortical layers of the anterior cingulate (Cg1). 188 This region of interest was selected based on previous reports identifying it as a sight of 189 dynamic adolescent myelination (McDougall et al., 2018; Drzewiecki et al., 2020) and sensitivity 190 to alcohol (Vargas et al., 2014; Tavares et al., 2019). Antibodies and reagents are detailed in 191 Table 1. To assess myelinated fiber density, we used a primary antibody that recognizes myelin 192 oligodendrocyte glycoprotein (MOG), which is located on the outer layer of the myelin sheath 193 (Clements et al., 2003; Ambrosius et al., 2020). Primary antibodies that recognize proteins that 194 are expressed at specific oligodendroglia lineage stages were used to delineate between 195 oligodendroglia precursor cells (OPCs) and oligodendroglia (OLs) for cellular density analyses: 196 anti-platelet-derived growth factor receptor alpha (PDGFRɑ) primary antibody for OPCs and 197 anti-CC1 primary antibody for OLs that express RNA-binding protein Quaking Isoform 7 (QKI-7). 198 Fluorophore-conjugated secondary antibodies used to visualize these proteins were donkey 199 anti-rabbit-AF647 for MOG, a donkey anti-goat-AF488 for PDGFRɑ, and a horse anti-mouse 200 biotin antibody followed by incubation with Cy3-streptavidin for QKI-7. 201 Experiment 2: Test for evidence of reduced aspartoacylase production in mature 202 oligodendrocytes following adolescent drinking. To study the late maturational stages of 203 cellular development we used a combination of antibodies to test for the presence or absence of 204 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 9 the aspartoacylase enzyme in mature OLs (antibodies and reagents are summarized in Table 205 1). These analyses expanded the sampling region to include cingulum bundle (CCcing) axons, 206 which extend into the Cg1 more posteriorly after crossing the midline. Mature OLs expressing 207 QKI-7 were immunolabeled using anti-CC1 antibody and co-labeled with antibody against the 208 aspartoacylase (ASPA) protein to determine if these mature OLs were producing the enzyme 209 required to form and maintain myelin sheaths (Madhavarao et al., 2004; Francis et al., 2012, 210 2016; Grønbæk-Thygesen and Hartmann-Petersen, 2024; Takeda et al., 2024). This allowed us 211 to delineate the specific maturational stage at which OLs were impacted by alcohol. In a subset 212 of mice, sections were also immunolabeled for myelin basic protein to confirm ASPA-expressing 213 cells were producing myelin sheath proteins. We therefore included incubations in chicken anti-214 MBP primary antibody and Alexa 488-conjugated goat anti-chicken antibody steps for those 215 sections. Fluorophore-conjugated secondary antibodies used for detection were a horse anti-216 mouse biotin antibody followed by incubation with Cy3-streptavidin for QKI-7 and a donkey anti-217 rabbit-AF647 for ASPA. 218 Confocal microscopic imaging 219 All images in Experiment 1 were acquired on the A1R-TIRF confocal microscope and all 220 images in Experiment 2 were acquired on the CREST-V2 confocal microscope at the UMass 221 IALS Nikon Center for Excellence Light Microscopy Facility Core. Both microscopes are 222 connected to the Nikon NIS-Elements platform to process and analyze images. To assess white 223 matter, we sampled from the location where corpus callosum axons extend medially out into the 224 layer VI of the anterior cingulate cortex. At the level of the medial prefrontal cortex this is called 225 the forceps minor or CCFM and at the level of the bed nucleus of the stria terminalis this is called 226 the cingulum or CCcing. To assess prefrontal gray matter, we sampled from layers II/III within the 227 anterior cingulate cortex (Cg1). In the first study, sections containing the medial prefrontal cortex 228 (AP+1.7 mm distance from bregma) were triple immunofluorescent labeled for MOG, PDGFRɑ, 229 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 10 and QKI-7, and counterstained with DAPI fluorescent nuclear stain. In the second study, 230 sections were selected from the following anatomical locations: AP+2.0mm, AP+1.7mm, and 231 AP+0.9mm distance from bregma. These sections were double immunofluorescent labeled for 232 QKI-7 and ASPA, and counterstained with DAPI nuclear stain. Within each A/P distance from 233 bregma and each hemisphere, one white matter (CCFM) and one gray matter (Cg1) region of 234 interest (ROI) were selected for imaging. A 20X objective was used for acquiring z-stacks 235 images for both the A1R-TIRF (Experiment 1) and CREST-V2 (Experiment 2) confocal 236 microscopes. 237 Quantification of myelinated axons 238 Sections that were AP+1.7mm were used for analysis of myelin density in the ROIs. 239 General Analysis 3 with NIS-AR was used to quantify MOG positive fiber density by thresholding 240 the images in the infra-red channel (647nm). The percentage of area covered by myelinated 241 fibers over the total area was calculated to quantify myelinated fiber density. 242 Quantification of oligodendroglial lineage cell populations 243 An optical configuration was designed within the NIS-Advanced Research (NIS-AR) 244 software, allowing all imaging to occur with identical laser power and gain parameters. First, a 245 10x-stitched large image of the entire tissue section was acquired on the 405 nm channel for 246 DAPI visualization of neural architecture. This image was used to digitally select our ROIs in 247 each hemisphere. For each ROI, a 20x z-stack with a depth of 10µm and a step size of 1µm 248 was acquired. A custom-designed semi-automatic cell counting program in General Analysis 3 249 within NIS-AR quantified multiple cell types in three-dimensions simultaneously. 250 Statistics 251 Statistical analyses were conducted using GraphPad Prism and confirmed using IBM 252 SPSS Statistics 28.0.1 for Mac. Graphs were generated using GraphPad Prism version 9.4.1 for 253 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 11 Mac (GraphPad Software, San Diego, CA). To examine if overall alcohol consumption was 254 comparable in groups of males and females in this study, we first ran a three-way mixed model 255 analysis of variance (MM-ANOVA) with one between-subjects factor and two within-subjects 256 factors. Specifically, sex (male vs female) was the between-subject factor and DID cycle (weeks 257 1, 2, 3, and 4) and access length (average of three 2-hour “baseline” days vs one 4-hour “binge” 258 day) were the within-subjects factors. There were no sex differences in alcohol intake (F (1, 42) = 259 1.288, p = 0.2629) and no significant interactions (sex X DID cycle, F (3, 42) = 1.045, p = 0.3828; 260 sex X access length, F (1, 42) = 2.132, p = 0.1517. To maximize statistical power for testing 261 specific hypotheses, subsequent analyses were conducted separately in males and females. 262 Data were analyzed using one-way and two-way repeated measures analysis of variance (RM-263 ANOVA). Bonferroni’s or Tukey’s multiple comparisons test were used for post-hoc analyses 264 following significant main effects or interactions, as appropriate. Bonferroni post-hoc analyses 265 were used for comparing two groups, e.g., baseline vs binge. Tukey post-hoc analysis were 266 used for comparing each group with every other group (e.g., compare each DID cycle). In 267 Experiment 1, myelin fiber density (% of area covered by MOG), density of OPCs (PDGFRɑ+ 268 cells/mm2), and density of mature OLs (QKI-7+ cells/mm2) were analyzed using unpaired two-269 tailed t-tests with treatment (alcohol vs control) as the between-subject factor. Pearson’s 270 correlations were used for simple linear regression analyses of relationships between alcohol 271 intake in the last week and the density of myelinated axons, OPCs, and OLs. In Experiment 2, 272 the density of pre-myelinating (QKI-7+/ASPA- cells/mm2), myelinating (QKI-7+/ASPA+ 273 cells/mm2), and post-myelinating (QKI-7-/ASPA+ cells/mm2) OLs were analyzed in three 274 anterior-to-posterior locations using two-way RM-ANOVA with treatment (alcohol vs control) as 275 the between-subject factor with distance from bregma (AP+2.0mm vs AP+1.7mm vs 276 AP+0.9mm) as the within-subject factor. Pearson’s correlations were used for simple linear 277 regression analyses of relationships between alcohol intake in the last week and the density of 278 pre-myelinating, myelinating, and post-myelinating cells. Alcohol consumption in on the last DID 279 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 12 cycle was also used to rank alcohol drinking mice as “high” vs “low” alcohol intake groups based 280 on a median split. Two-tailed t-tests were used to test if there were fewer QKI-7+/ASPA+ 281 cells/mm2 in high drinkers compared to low drinkers. Data are presented as mean ± standard 282 error of the mean (SEM) unless otherwise indicated. The criterion for statistical significance was 283 p ≤ 0.05. 284 285

286 Alcohol drinking was comparable in adolescent male and female mice. 287 An overview of the experimental design and drinking data is shown in Fig 1. Beginning 288 on PD28, the DID model was used to expose mice to two weeks of binge drinking of 20% v/v 289 alcohol (or water for controls, Fig. 1A). Three days after the last drinking session, mice were 290 perfused and brains processed for immunofluorescence experiments, followed by confocal 291 imaging and cellular analyses. Voluntary alcohol intake was greater in the four-hour “binge” day 292 compared to the average daily drinking during baseline days in males (Fig. 1B; main effect of 293 DID cycle (F (3, 42) = 23.87, p < 0.0001), access length (F (1, 14) = 14.98, p = 0.0017) and cycle x 294 length interaction (F (3, 42) = 4.332, p = 0.0016)). Post-hoc analyses showed increased drinking 295 on the binge day compared to the average intake on the baseline days on DID cycles 2, 3 and 4 296 in males (Bonferroni’s multiple comparisons test, ps = 0.0003 on week 2 and 3, and p = 0.030 297 on week 4). In females, alcohol consumption was greater on the binge day compared to the 298 average baseline drinking (Fig. 1C), with analyses showing a main effect of access length (F (1, 299 7) = 37.08, p = 0.0005) and cycle x length interaction (F (3, 21) = 3.99, p = 0.02). Post-hoc 300 analyses showed that binge day alcohol consumption was greater than the average intake on 301 the baseline days in females on DID cycles 1, 2 and 3 (Bonferroni’s multiple comparisons test, p 302 = 0.0007 on cycle 1, and p < 0.0001 on cycle 2 and 3). 303 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 13 Total alcohol intake in adolescent males per DID cycle showed a significant difference (F 304 (2.723, 19.06) = 11.72, p = 0.0002) and post-hoc analyses found a significant increase in total alcohol 305 intake between cycle 1 and every following DID cycle (Fig. 1D, Tukey’s multiple comparisons 306 test, p = 0.0093 for cycle 1 vs 2, p = 0.0031 for cycle 1 vs 3, and p = 0.0249 for cycle 1 vs 4). No 307 significant differences were found between DID cycle 2 vs 3, cycle 2 vs 4, or cycle 3 vs 4 (all ps 308 > 0.05). Adolescent female mice also showed a significant difference in total alcohol intake per 309 DID cycle (F (1.555, 10.89) = 4.539, p = 0.0440), and post-hoc analyses showed an increase in total 310 alcohol intake only between DID cycle 2 vs 3 (Fig. 1E, Tukey’s multiple comparisons test, p = 311 0.0038). On binge days, mice surpassed alcohol consumption greater than 3 g/kg: in cycles 2 312 and 3 in males and in cycles 2, 3, and 4 in females. Alcohol consumption at 3 g/kg and greater 313 is predicted to correlate with BAC over 80 mg/dl, a value that fits the criteria of alcohol binge 314 drinking in humans (Rhodes et al., 2005b; Crabbe et al., 2009). In males, the average intake 315 across DID cycles 3-4 is significantly greater in both baseline and binge access compared to 316 cycles 1-2 (Supplemental Fig. 1A, F (1, 7) = 17.28, p = 0.0005, main effect of cycle). This 317 suggests that males increase alcohol intake across adolescent development. In contrast, 318 females do not show a significant difference in the average intake between DID cycles 1-2 and 319 3-4 (Supplemental Fig. 1B, F (1, 7) = 4.36, p = 0.07, main effect of cycle), suggesting that 320 females consume higher amounts of alcohol from the onset of adolescence. 321 Water intake in control male mice was not different across the DID cycle or between 322 baseline and binge sessions (Supplemental Fig. 2A, repeated measures two-way ANOVA, p > 323 0.05). Water intake in control female mice was significantly different on the baseline and binge 324 days (Supplemental Fig. 2B, main effect of access, repeated measures two-way ANOVA, p < 325 0.05). No differences in the total intake per DID cycle was found in the control groups 326 (Supplemental Fig. 2C, F (1.269, 8.884) = 3.192, p = 0.1030 in males and Supplemental Fig. 2D, F 327 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 14 (2.214, 15.50) = 2.568, p = 0.1048 in females). These data support the interpretation that increased 328 drinking on binge days is not due to longer time of access. 329 Alcohol reduces myelination of anterior cingulate and corpus callosum axons in male mice, but 330 not females. 331 Based on our previous findings showing sex differences in prefrontal myelin loss 332 following adolescent alcohol drinking in rats (Vargas et al., 2014; Tavares et al., 2019), the 333 current study tested if differential sensitivity extended to male and female mice as well. We 334 fluorescently labeled MOG—a protein that is enriched in myelin sheaths—to visualize segments 335 of myelin ensheathing prefrontal axons extending from the front branches of the corpus 336 callosum (forceps minor of the corpus callosum/cingulate cortex-layer VI or CCFM) out into the 337 superficial layers of the cingulate cortex (Cg1; Fig. 2A-C). Representative images of each ROI 338 of each treatment and sex group with MOG+ immunolabeling are shown in Fig. 2D-E. There 339 was a significant reduction of myelin density in the CCFM found between alcohol males 340 compared to controls (Fig. 2F, unpaired two-tailed t-test, t (14) = 3.975, p = 0.0014). In the 341 anterior cingulate cortex (Cg1) there was also a significant reduction of myelin density after 342 alcohol drinking in males (Fig. 2F, unpaired two-tailed t-test, t (14) = 2.162, p = 0.0484). In 343 females, there were no differences in the myelin density between the alcohol group and controls 344 in the CCFM region (Fig. 2G, unpaired two-tailed t-test, t (13) = 1.140, p = 0.2747) nor the Cg1 345 region (Fig. 2G, unpaired two-tailed t-test, t (13) = 0.5317, p = 0.6067). Given that males showed 346 increased intake in DID cycle 4 compared to cycle 1, we tested the relationship between alcohol 347 intake and myelin density. No significant relationships were found between the total alcohol 348 intake of DID cycle 4 and the density of myelin sheaths in either CCFM or Cg1 region of male 349 (Fig 2H, simple linear regression, r2 = 0.06, p = 0.57 in the CCFM region and r2 = 0.03, p = 0.66 350 in the Cg1 region) or female (Fig. 2I, r2 = 0.35, p = 0.16 in the CCFM region and r2 = 0.24, p = 351 0.22 in the Cg1 region) mice. 352 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 15 The pool of oligodendroglial precursor cells is not reduced by alcohol in male mice 353 To test the hypothesis that alcohol disrupts oligodendroglial in the early phases of cell 354 development, thus reducing the entire OL lineage thereafter, we quantified the density of 355 PDGFRɑ+ OPCs in the Cg1 and CCFM (Fig. 3A-E). Contrary to our initial hypothesis, myelin 356 deficits following alcohol drinking in males were not explained by a significant reduction in the 357 OPC pool in these regions (CCFM, Fig. 3F, unpaired two-tailed t-test, t (14) = 0.2976, p = 0.7704; 358 Cg1, Fig. 3F, paired two-tailed t-test, t (14) = 1.732, p = 0.1052). Likewise, alcohol did not 359 significantly alter the OPC pool in the CCFM (Fig. 3G, unpaired two-tailed t-test, t (11) = 0.3358, p 360 = 0.7433) or the Cg1 region (Fig. 3G, unpaired two-tailed t-test, t (11) = 0.6082, p = 0.5554) of 361 females. Despite a lack of significant differences in the density of OPCs in either region 362 following alcohol, there were correlations between the level of alcohol intake in the last week of 363 DID and OPC density in males. Using simple linear regression analyses, we detected a modest 364 but significant negative relationship between alcohol intake and OPC density in the Cg1 region 365 (Fig. 3H, r2 = 0.60, p = 0.03) and a modest positive significant relationship between these 366 variables in the CCFM region (Fig. 3H, r2 = 0.56, p = 0.03) and of male mice. Thus, higher intake 367 in male mice correlated with lower OPC density in gray matter and higher OPC density in white 368 matter, possibly indicating regional differences in the rate and timing of oligodendrogenesis 369 changes that were captured four days after drinking ended. No significant correlations between 370 these two variables were found in females (Fig. 3I, simple linear regression, r2 = 0.10, p = 0.53 371 in the CCFM region and r2 = 0.08, p = 0.59 in the Cg1 region). 372 The density of mature oligodendrocytes is increased by alcohol in the anterior cingulate. 373 Using the well-established CC1 antibody to detect the RNA-binding QKI-7 protein as an 374 marker for mature OLs, we tested for a decrease in OL maturation in binge drinking males (Fig. 375 4A-E). We first confirmed the QKI-7+ cells had fully differentiated by the absence of PDGFRɑ+ 376 signal in the cells (data not shown). Contrary to our prediction, mature QKI-7+ cell density was 377 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 16 not significantly reduced by alcohol in the CCFM region in males (Fig. 4F, unpaired two-tailed t-378 test, t (14) = 0.8772, p = 0.3952). Instead, we found a trend of an increase in the number of 379 mature oligodendroglia in the Cg1 after drinking in males (Fig. 4F, unpaired two-tailed t-test, t (14) 380 = 2.014, p = 0.0636), and this alcohol-induced increase was significant in females (Fig. 4G, 381 unpaired two-tailed t-test, t (11) = 2.929, p = 0.0137). Similar to males, the density of mature OLs 382 was not impacted by alcohol in the CCFM region (Fig. 4G, paired two-tailed t-test, t (11) = 0.1475, 383 p = 0.8854). The increase in QKI-7+ cell density in the Cg1 region in females and a trend of an 384 increase in males four days after alcohol could be reflecting increases in differentiation and 385 maturation of OLs in response to myelin loss, albeit an unsuccessful attempt at rescuing myelin 386 deficits in males. Given the differential levels of alcohol consumption, we investigated whether 387 total alcohol intake during the last DID cycle predicted the density of mature OLs. No significant 388 correlations were found in males (Fig. 4H, simple linear regression, r2 = 0.16, p = 0.32 in the 389 CCFM region and r2 = 0.05, p = 0.60 in the Cg1 region) or in females (Fig. 4I, simple linear 390 regression, r2 = 0.15, p = 0.44 in the CCFM region and r2 = 0.32, p = 0.44 in the Cg1 region). 391 Alcohol decreased the density mature OLs expressing aspartoacylase in male mice. 392 While the pool of QKI-7 expressing OLs appeared normal or even elevated in binge 393 drinking males, we reasoned that there may be differences among this population that could 394 explain hypomyelination of prefrontal axons in these animals. One possibility is that alcohol may 395 disrupt the ability of mature OLs to myelinate. To test this possibility, we co-immunolabeled 396 sections with both QKI-7+ and aspartoacylase (ASPA) to distinguish between mature OLs that 397 were in a pre-myelinating stage from those that were forming myelin sheaths (Fig. 5). ASPA 398 catalyzes deacetylation of N-acetyl aspartate (NAA) into free acetate, a precursor necessary for 399 the synthesis of lipids used for myelin sheaths (Madhavarao et al., 2002). Thus, if alcohol was 400 interfering with late cellular maturational stages in OLs they may be kept at the pre-myelinating 401 stage, preventing the lipid synthesis necessary for the formation of myelin resulting in 402 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 17 hypomyelination. Using ASPA as a proven specific marker for actively myelinating OLs (Pan et 403 al., 2020), we phenotyped mature OLs into ASPA+ vs ASPA- to examine their capacity to 404 synthesize myelin sheaths. We tested the following predictions: 1) alcohol would decrease in 405 density of myelinating OLs (QKI-7+/ASPA+ cells) in male mice, and 2) this would be 406 accompanied by an increase in the density of pre-myelinating OLs (QKI-7+/ASPA- cells) 407 indicating that mature OLs were stuck at this late maturational stage of oligodendrogenesis. 408 As myelin deficits were observed in the medial prefrontal cortical regions (bregma 409 AP+1.7mm), but a single myelinating OL can generate between 20 and 60 myelinating 410 processes with intermodal lengths of about 20 mm–200 mm (Simons and Nave, 2016). Thus, 411 we expanded our sampling area at distance range from bregma AP+2.0mm to AP+0.9mm to 412 capture all myelinating OLs within reach of AP+1.7mm (Fig. 5A). There were three distinct 413 populations of mature OLs (Fig. 5B): pre-myelinating OLs (QKI-7+/ASPA-, red), myelinating 414 OLs (QKI-7+/ASPA+, pink) and a third –albeit smaller– population of QKI-7-/ASPA+ OLs we call 415 “post-myelinating” (white). Representative images of AP+1.7mm for mice in the control and 416 alcohol group are shown in Fig. 6A. 417 In the CCFM region of males, no significant difference in the density of pre-myelinating 418 OLs was found between the control and alcohol groups (Fig. 6B, two-way MM-ANOVA, F (1, 14) = 419 0.01331, p = 0.9098) and no significant differences were found between AP distances from 420 bregma (Fig. 6B, two-way MM-ANOVA, F (2, 25) = 0.7114, p = 0.4992). Similarly to males, the 421 population density of mature pre-myelinating OLs in the CCFM region in female mice showed no 422 significant change with alcohol drinking during adolescence (Fig. 6B, two-way MM-ANOVA, F (1, 423 14) = 0.1648, p = 0.6969) nor any differences between AP distances from bregma were found 424 (Fig. 6B, two-way MM-ANOVA, F (2, 28) = 0.3294, p = 0.7221). 425 The density of mature myelinating OLs in the CCFM region of males was significantly 426 lower in mice that consumed alcohol during adolescence compared to controls (Fig. 6C, two-427 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 18 way MM-ANOVA, main effect of treatment, F (1, 14) = 6.174, p = 0.0262). The population of 428 mature myelinating OLs (QKI-7+/ASPA+) in the CCFM region of males was found to be 429 significantly different between AP distances from bregma (Fig. 6C, two-way MM-ANOVA, F (2, 25) 430 = 5.648, p = 0.0095). Follow-up analysis indicated that the average myelinating OL density in 431 the AP+2.0mm was significantly lower when compared to AP+0.9mm (Tukey’s post-hoc, p = 432 0.0069). No differences were found between AP+2.0mm and AP+1.7mm (Tukey’s post-hoc, p = 433 0.2298) nor between AP+1.7mm and AP+0.9mm (Tukey’s post-hoc, p = 0.2971). In contrast to 434 males, we found no significant differences between alcohol and control groups in the density of 435 mature myelinating OLs in the CCFM region of female mice (Fig. 6C, two-way MM-ANOVA, F (1, 436 14) = 03654. p = 0.5552). No significant differences were found in the cell density of myelinating 437 OLs between AP distances from bregma (Fig. 6C, two-way MM-ANOVA, F (2, 28) = 1.375, p = 438 0.2694) in the CCFM region of females. 439 Alcohol consumption during adolescence significantly decreased the density of mature 440 post-myelinating OLs in the CCFM region of males (Fig. 6D, two-way MM-ANOVA, main effect of 441 treatment, F (1, 14) = 6.415, p = 0.0239). The density of post-myelinating OLs did not differ across 442 the anterior-to-posterior locations sampled (Fig. 6D, two-way MM-ANOVA, F (2, 25) = 2.489, p = 443 0.1033). In females, we found no differences in the population of post-myelinating OLs in the 444 CCFM between alcohol and control groups (Fig. 6D, two-way MM-ANOVA, F (1, 14) = 0.2725, p = 445 0.6098) or across the anterior-to-posterior locations (Fig. 6D, two-way MM-ANOVA, F (2, 28) = 446 0.5079, p = 0.6072). Adolescent alcohol drinking increased the proportion cells that remained in 447 the pre-myelinating state in the CCFM region of males (44% vs 72% in pre-myelinating OLs, Fig. 448 6E, unpaired t-test, p = 0.02), whereas the shift in females was marginal (68% vs 73% in pre-449 myelinating OLs, Fig. 6F, unpaired t-test, p > 0.05). 450 Alcohol decreased the density of post-myelinating OLs in the Cg1 region in male mice. 451 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 19 The population density of pre-myelinating, myelinating, and post-myelinating OLs was 452 assessed in the anterior cingulate cortex (Cg1) at the same three anatomical locations as 453 described above for the corpus callosum white matter analyses (AP+2.0mm, AP+1.7mm, and 454 AP+0.9mm, Fig. 5A, outlined by a white box). Representative images of cells at AP+1.7mm for 455 male and female mice in the control and alcohol groups are shown in Fig. 7A. In the Cg1 region 456 of males, no significant differences in the average density of pre-myelinating OLs were found 457 between alcohol and control groups (Fig. 7B, two-way MM-ANOVA, F (1, 14) = 0.03337, p = 458 0.8577) or across the three AP distances from bregma (Fig. 7B, two-way MM-ANOVA, F (2, 25) = 459 0.5619, p = 0.5772). In female mice, we found no significant effect of alcohol in the population 460 density of mature pre-myelinating OLs (Fig. 7B, two-way MM-ANOVA, F (1, 14) = 0.030, p = 461 0.8647). There were also no significant differences across the AP distances from bregma (Fig. 462 7B, two-way MM-ANOVA, F (2, 27) = 1.078, p = 0.3544). 463 No significant differences were found between alcohol and control groups in the average 464 density of mature myelinating OLs in the Cg1 region (Fig. 7C, two-way MM-ANOVA, F (1, 14) = 465 2.647, p = 0.1261); however, there was a significant main effect of AP distance from bregma 466 (Fig. 7C, two-way MM-ANOVA, F (2, 25) = 9.953, p = 0.0007). The average mature myelinating 467 OL density in AP+0.9mm was significantly higher compared to AP+2.0mm (Fig. 7C, Tukey’s 468 post-hoc, p = 0.0010) and compared to AP+1.7mm (Fig. 7C, Tukey’s post-hoc, p = 0.0049). In 469 females, there was no effect of alcohol on the population density of mature myelinating OLs in 470 the Cg1 region (Fig. 7C, two-way MM-ANOVA, F (1, 14) = 0.008, p = 0.9295) and no significant 471 differences across the three AP distances from bregma (Fig. 7C, two-way MM-ANOVA, F (2, 27) = 472 1.136, p = 0.3359). 473 In male mice, adolescent drinking decreased the average density of post-myelinating 474 OLs in the Cg1 region, similar to what was observed in the CCFM (Fig. 7D, two-way MM-475 ANOVA, main effect of treatment, F (1, 14) = 6.247, p = 0.0255). There was also a significant 476 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 20 interaction between treatment and anatomical location (mm distance from bregma) (Fig. 7D, 477 two-way MM-ANOVA, F (2, 25) = 4.495, p = 0.0215), with follow-up analyses showing a significant 478 decrease in the average density of post-myelinating OLs in the alcohol group compared to 479 controls at AP+0.9mm distance from bregma (Fig. 7D, Bonferroni’s post-hoc, p = 0.0015) in the 480 Cg1 region of males. No significant differences between alcohol and control groups were found 481 at AP+2.0mm (Bonferroni’s post-hoc, p > 0.9999) nor AP+1.7mm (Bonferroni’s post-hoc, p = 482 0.7311). We found no significant difference in the population density of mature post-myelinating 483 OLs in the Cg1 region in females that consumed alcohol during adolescence compared to 484 controls (Fig. 7D, two-way MM-ANOVA, F (1, 14) = 0.049, p = 0.8280) and no differences were 485 found between distances from bregma (Fig. 7D, two-way MM-ANOVA, F (2, 27) = 1.345, p = 486 0.2774). The proportion of the population of pre-myelinating OLs vs myelinating OLs in the Cg1 487 region did not significantly change with alcohol in males (Fig. 7E, 57% vs 70% in pre-488 myelinating OLs, unpaired t-test, p > 0.05) or in females (Fig. 7F, 60% vs 71% in pre-489 myelinating OLs, unpaired t-test, p > 0.05). 490 There were no changes in the density of mature OLs in gray or white matter regions of 491 males or females (Fig. 6B, 7B, and Supplemental Fig. 4A). While there was a significant loss 492 of myelinating OLs in the CCFM region and of post-myelinating OLs in both CCFM and Cg1 493 regions of males (Fig. 6C, 6D, 7D, and Supplemental Figure. 4B), there was no change in the 494 total population of mature OLs (all QKI-7+ and ASPA+ cells, Supplementary Fig. 4C). This 495 suggests that there may be a cellular compensation mechanism occurring in these regions that 496 allow the total population of mature OLs to remain stable following alcohol. 497 Alcohol intake during the last week of drinking predicts myelinating OL density in male mice. 498 We noted a bimodal distribution of total intake consumed during DID cycle 4 in males 499 and females (Fig. 1D and E). Accordingly, we used a median split to sub-divide mice into two 500 alcohol intake groups: high drinkers (total intake > 7.4 g/kg in males and 12.6 g/kg in females; n 501 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 21 = 4 / sex) and low drinkers (total intake < 7.4 g/kg in males and 12.6 g/kg in females; n = 4 / 502 sex). The high intake group drank significantly more alcohol during DID cycle 4 compared to the 503 low intake group in both males and females (Fig. 8A, unpaired t-tests, p = 0.02 in males and p = 504 0.005 in females). We next assessed whether there were significant differences in OL density 505 between high and low drinking groups. High drinking male mice had a lower density of 506 myelinating OLs in the Cg1 region at the distance from bregma AP+1.7mm compared to low 507 drinking male mice (Fig. 8B, unpaired t-test, p = 0.0011). In contrast, the density of myelinating 508 OLs in the Cg1 region was similar in the low and high drinking groups in female mice (Fig. 8B, 509 unpaired t-test, p = 0.5722). There was also a significant negative relationship between the total 510 alcohol intake in DID cycle 4 and the density of myelinating OLs in the Cg1 region of male mice 511 (Fig. 8C, simple linear regression, r2 = 0.81, p = 0.0025). There was no correlation between total 512 alcohol consumed in DID cycle 4 and the density of myelinating OLs in the Cg1 region in female 513 mice (Fig. 8C, bottom scatter plot, simple linear regression, r2 = 0.08, p = 0.7819). When both 514 high and low alcohol intake groups were compared to controls, the density of myelinating OLs in 515 the Cg1 region of males was significantly decreased in the high intake group (Dunnett’s post-516 hoc following a significant one-way ANOVA, F (2, 11) = 2.984, p = 0.04, data not shown). No 517 differences were found in the density of myelinating OLs between the three groups in females 518 (one-way ANOVA, F (2, 13) = 0.1471, p = 0.5482, data not shown). These data suggest 519 myelinating Cg1 myelinating OLs are sensitive to increasing alcohol levels in males only. This 520 may reflect sex differences in the population of myelinating OLs during adolescent development. 521 522

523 The present study showed that adolescent drinking disrupts myelination of axons in the 524 anterior cingulate cortex and adjacent white matter of the corpus callosum in male mice. Our 525

indicate that a significant loss in mature OLs expressing ASPA may have caused 526 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 22 hypomyelination of axons in males given the importance of this enzyme for lipid biosynthesis 527 during myelin sheath formation (Hershfield et al., 2006; Mattan et al., 2010; Francis et al., 2016). 528 Most notably while the magnitude of cell loss was even more pronounced in higher drinking 529 males, females appeared resistant to the negative effects of alcohol regardless of how much 530 they drank. Binge drinking females had a higher density of mature OLs (QKI-7+ cells) in the 531 Cg1, and there was a similar trend in male mice. The subtle changes in OPCs and mature OL 532 populations may signify an upregulation in oligodendrogenesis that could have replenished the 533 ASPA+ OL pool in females, protecting them against the hypomyelinating effects of alcohol. 534 Nevertheless, this was clearly insufficient to fill the ASPA+ OLs pool and rescue myelin loss in 535 alcohol males. Our results replicate previous reports of myelin deficits with alcohol in rodents 536 and humans (Jacobus et al., 2009; Vargas et al., 2014; Papp-Peka et al., 2016; Wolstenholme 537 et al., 2017; Rice et al., 2019; Tavares et al., 2019) and fill a significant knowledge gap by 538 providing evidence that alcohol impacts oligodendroglial lineage cells at a later maturational 539 stage of cellular development. By identifying ASPA as a direct or indirect target of alcohol, we 540 highlight the need for further investigation of this enzyme, which is a promising new target for 541 therapeutic intervention in alcohol use disorder and demyelinating diseases. 542 Alcohol induces myelin sheath density loss in male mice 543 We previously reported that the anterior branches of the corpus callosum (CCFM) which 544 project to the Cg1 region undergo substantial increases in myelin density during adolescent 545 development, which significantly speeds up the conduction velocity in these axons (McDougall 546 et al., 2018). These myelinated axons are vulnerable to alcohol consumption during 547 adolescence, as alcohol reduced the density of myelinated fibers in adolescent male, but not in 548 female, rats (Vargas et al., 2014; Tavares et al., 2019). The data from the study herein 549 recapitulated these findings in adolescent male mice. This is consistent with evidence that 550 alcohol is a demyelinating agent in the CNS. Previous studies measuring the gene expression 551 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 23 of myelin-associated glycoprotein (Mag), myelin basic protein (Mbp), myelin-associated 552 oligodendrocytic basic protein (Mobp), and proteolipid protein 1 (Plp) showed reductions in the 553 prefrontal cortex after a bolus high dose of alcohol via gavage in adolescent mice 554 (Wolstenholme et al., 2017). A similar high dose administration of alcohol during adolescence 555 decreased myelin density in the prefrontal cortex, primarily in the axons of parvalbumin-negative 556 neurons (Rice et al., 2019). These deficits in myelination during adolescence have been 557 associated with impairments in working memory and social interaction in young adult mice 558 (Makinodan et al., 2009). 559 Myelin oligodendrocyte glycoprotein (MOG) is a 28 kDa protein located in the outermost 560 layer of the myelin sheath exclusively in the CNS and is a relatively very minor (0.05%) 561 component of myelin (Johns and Bernard, 1999). MOG may play a role as an adhesion protein 562 supporting myelin compaction (Clements et al., 2003). The current study shows evidence that 563 four weeks of voluntary alcohol intake sufficiently perturbs MOG+ myelin density in male mice. 564 Both the density of myelin sheaths and density of OLs expressing Mog mRNA is decreased 565 throughout the CC and the prefrontal cortex in male mice following chronic social defeat stress 566 (Lehmann et al., 2017). Similarly, a bolus dose of alcohol (3g/kg, i.p.) in female rats or chronic 567 exposure to alcohol through continuous home-cage access for 5 months in female mice reduces 568 Mog mRNA and MOG protein levels in the prefrontal cortex (Alfonso‐Loeches et al., 2012; Pascual 569 et al., 2014). Possibly longer exposure to alcohol may be necessary to induce similar deficits in 570 females as we see in males. 571 ASPA-expressing OLs are sensitive to adolescent drinking in male mice 572 ASPA generates the free acetate needed for lipid synthesis in myelin formation through 573 the hydrolyzation of N-acetylaspartate (NAA) released by neurons (Madhavarao et al., 2002; 574 Hershfield et al., 2006; Francis et al., 2012, 2016; Grønbæk-Thygesen and Hartmann-Petersen, 575 2024). ASPA is highly expressed in OLs, located predominantly in the soma in both the nucleus 576 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 24 and cytoplasm, and with 92-98% co-localization with QKI-7+ mature OLs (Baslow et al., 1999; 577 Madhavarao et al., 2004; Hershfield et al., 2006). Expression of ASPA also follows the 578 developmental trajectory of myelination in the CNS, supporting the role of ASPA in myelin 579 synthesis (Kirmani et al., 2003). Deficiency of ASPA enzyme, a characteristic of Canavan 580 disease, disrupts the production of myelin-associated lipids which leads to vacuolation and 581 myelin deficiency (Takeda et al., 2024). 582 High alcohol exposure has been reported to reduce brain levels of ASPA’s substrate 583 NAA. Administration of 3 g/kg of alcohol for 4 days via i.p. injections decreased brain NAA levels 584 in adolescent Swiss-Webster male mice (Baslow et al., 2000). Similarly, in a recent study on 585 adult patients with AUD seeking treatment, NAA levels within the frontal gray and white matter 586 were significantly lower in the high-risk to relapse group compared to light- and non-drinking 587 controls (May et al., 2025). This may reflect a reduction in NAA synthesis in neurons after 588 alcohol. If similar effects happen with alcohol drinking in adolescent male mice, the combination 589 of a limited supply of the NAA substrate from neurons and the lower number of ASPA-590 expressing OLs to catabolize the deacetylation of NAA could conceivably cause a major 591 deficiency in available free acetate, further exacerbating myelin loss. 592 It is unclear whether alcohol drinking prevented mature OLs from starting to express 593 ASPA. Alcohol may be holding mature cells in a pre-myelinating state or may disrupt ASPA 594 expression in actively myelinating OLs preventing them from continuing to form myelin sheaths. 595 While the fate of these “lost” ASPA cells is unknown, the consequences of alcohol could be 596 significant and long-lasting. Considering the possibility that the NAA substrate supplied by axons 597 may already be lowered by alcohol as other studies have shown (Baslow et al., 2000; May et 598 al., 2025; Sommer and Canals, 2025), and if there are also not enough mature OLs expressing 599 ASPA to catalyze deacetylation of the NAA that is available to synthesize myelin sheaths, then 600 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 25 drinking during this time could have lasting effects on prefrontal circuits that impact functions in 601 adulthood. 602 Differential correlations between alcohol and PDGFR+ OL progenitor cells in white and 603 gray matter. 604 In the present study, there were no group differences detected in OPC density in the 605 CCFM and Cg1, despite decreased density of myelinated axons and myelinating OLs in alcohol 606 males compared to controls. This was somewhat surprising because acute cellular injury can 607 trigger apoptotic cell death OPCs in 24 hours (Hill et al., 2017; Chapman et al., 2024) and 608 myelin damage and OL loss accelerates the differentiation of OPC into OLs (Hill et al., 2014; 609 Baxi et al., 2017; Chapman et al., 2023). These two events would be expected to reduce the 610 number of OPCs, but there is a dynamic interplay between cellular division and differentiation 611 that serves to stabilize the OPC pool. In response to an acute demyelinating event and OPC cell 612 death, OPCs divide and a portion of daughter cells differentiate into OLs within a few days (Hill 613 et al., 2014; Baxi et al., 2017). As brains were collected three days after the last alcohol binge 614 day in our study, it is possible that there was enough time for the OPC pool to replenish itself 615 through cell division after alcohol exposure ended. 616 Delving deeper into the OPC population, we found region-specific relationships between 617 OPC density and the total alcohol intake on the last DID cycle. There was a modest negative 618 correlation in gray matter, with higher drinking levels being associated with lower OPC density in 619 the Cg1. Conversely, there was a modest positive correlation in white matter, with higher 620 drinking levels being associated with higher OPC density in the CCFM. Differential dynamics of 621 oligodendrogenesis may explain this, as OPCs proliferate and differentiate faster into OLs in 622 white matter regions compared to gray matter following myelin injury (Baxi et al., 2017). Thus, 623 OPC density could be elevated in the CCFM of high drinking mice because proliferation was 624 initiated earlier in the corpus callosum OPC population, as has been observed after social 625 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 26 chronic stress (Poggi et al., 2022). In the Cg1, OPC populations may be lower due to increased 626 differentiation in response to demyelination (Hill et al., 2014). A recently published study showed 627 that primary culture of OLs from mice cortices have distinct gene expression changes in high 628 (30mM, 138.3 mg/dl BAC) compared to a moderate (10mM, 46.1 mg/dl BAC) concentration of 629 alcohol (Bazzi et al., 2025). At the moderate concentration, alcohol increased genes associated 630 with cell cycle progression through G1 and mitosis (increase in Cyclin B and D) and decreased 631 genes associated with progression though S and G2 (decrease in Cyclin A and E), suggesting 632 disrupted cell division at specific steps. On the other hand, all cyclin genes were downregulated 633 at the high concentration, suggesting a decrease in proliferation at that dose (Bazzi et al., 2025). 634 A modest increase in OPC density with greater alcohol intake in the corpus callosum 635 may be due to several possible events: 1) an increase in proliferation with a failure to initiate 636 differentiation, 2) a failure in OPC migration from white matter to the adjacent gray matter 637 region, or 3) a white vs gray matter difference in the rate of OPC proliferation and differentiation. 638 In support of these possible explanations, other studies have shown that OPC proliferation and 639 differentiation into OLs occur at a greater rate in white matter compared to gray matter (Dimou 640 et al., 2008; Rivers et al., 2008; Viganò et al., 2013; Young et al., 2013) and both proliferation 641 and differentiation are increased following demyelination, at a faster rate in the corpus callosum 642 compared to the cingulate cortex (Hill et al., 2014; Baxi et al., 2017). During a demyelinating 643 insult such as cuprizone, OLs regenerate by OPC proliferation and migration during active 644 demyelination (Mason et al., 2000). Alcohol consumption can specifically inhibit OL 645 differentiation without changing the OPC population density (Guo et al., 2021). Furthermore, the 646 OPCs that migrated the longest distance in response to injury were the subpopulation of 647 progenitor cells that did not differentiate into OLs (Chapman et al., 2023). If alcohol disrupts 648 OPC migration, this may have contributed to the region-specific changes in OPC density we 649 observed in the present study. Therefore, these events may, in combination, compound and 650 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 27

in the modest increase in OPC density of the CCFM region in males and a modest 651 decrease in the Cg1 region. Our findings provide additional support for the notion that OPCs 652 respond differently to demyelination and injury in gray versus white matter. 653 Evidence for elevations in the density of QKI-7 expressing OLs after alcohol drinking 654 During OL maturation, these cells start producing specific proteins that are necessary for 655 the production and maintenance of myelin sheaths (Huang et al., 2023). Both pre-myelinating 656 and myelinating OLs express Quaking Protein-Isoform 7 (QKI-7, labeled by the CC1 antibody), 657 a protein that binds and stabilizes the mRNA of myelin structural proteins including myelin basic 658 protein (Bin et al., 2016). The average density of pre-myelinating OLs was not affected by 659 alcohol in adolescent mice; however, there is a hint of change with alcohol in males. There was 660 a trend of an increase in the density of QKI-7 OLs in the cingulate cortex. This pooling of mature 661 OLs may indicate 1) an inability to express ASPA and generate myelin sheaths following 662 alcohol, or 2) an enhanced OPC differentiation. Recent findings in the nucleus accumbens 663 indicate increases in differentiation (measured as an increased number of CC1+ QKI-7 mature 664 OLs) at 6 weeks of alcohol consumption in adult mice (Liran et al., 2025) and our findings 665 showing a negative correlation between alcohol intake and OPC density may suggest a possible 666 upregulation of OPC differentiation in the Cg1 of males. In mice treated with cuprizone to induce 667 demyelination, the density of CC1+ QKI-7 OLs showed long-term (6 weeks post-treatment) 668 increase in the corpus callosum but decrease in the cingulate cortex (Baxi et al., 2017), 669 suggesting timing-dependent differences between the white matter and gray matter regions in 670 the cellular response to a demyelinating event. 671 Alcohol consumption was comparable between adolescent male and female mice 672 The drinking-in-the-dark (DID) protocol was used for alcohol administration because it 673 reliably elicits binge-like drinking behavior and achieve biologically relevant blood alcohol 674 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 28 concentrations (Crabbe et al., 2009; Barkley‐Levenson and Crabbe, 2012; Thiele and Navarro, 2014; 675 Thiele et al., 2014). This voluntary, limited access to alcohol allowed the mice to reach alcohol 676 intake over 3 g/kg for both sexes. This alcohol consumption has been shown to correlate with 677 BAC over 80 mg/dl, a value that fits the criteria of binge drinking in humans (Rhodes et al., 678 2005b; Crabbe et al., 2009; Thiele and Navarro, 2014; Wilcox et al., 2014). We also found 679 higher alcohol consumption on bassline and binge days in the later DID cycles in male mice, 680 similar to the escalated drinking patterns that have been previously reported with the DID model 681 (Wilcox et al., 2014). Female mice showed comparable drinking to male mice, consistent with 682 previous studies that showed similar levels of alcohol consumption between adolescent male 683 and female mice and rats (Schramm-Sapyta et al., 2014; Tavares et al., 2019; Silva-Gotay et al., 684 2021; Edwards et al., 2025). Others have shown that female rodents drink more alcohol than 685 males during adolescence (Walker et al., 2008; Strong et al., 2010) and/or in adulthood (Rhodes 686 et al., 2005b; Walker et al., 2008; Strong et al., 2010; Flores-Bonilla et al., 2021). One study 687 found that alcohol intake from PD30 to PD51 was comparable between male and female rats; 688 however, a shift in increase of alcohol drinking was found in female rats compared to males 689 from PD52 onwards (Lancaster et al., 1996). Sex differences in alcohol drinking in adulthood is 690 driven primarily the magnitude of front-loading these animals exhibit (Flores-Bonilla et al., 2021). 691 When adolescent rodents first start drinking, both males and females consume more alcohol 692 than adults (Bell et al., 2006; Walker et al., 2008; Strong et al., 2010; Schramm-Sapyta et al., 693 2014; Lee et al., 2017). A history of adolescent drinking can lead to higher alcohol drinking later 694 in adulthood (Gilpin et al., 2012; Pandey et al., 2015; Younis et al., 2019), and greater effects 695 have been reported in females (Strong et al., 2010). High alcohol drinking (HAD) rat strains also 696 show higher intake during adolescence compared to adulthood, particularly adolescent males 697 show the highest drinking–while adolescent females show the lowest drinking– of all four age 698 and sex groups tested (Dhaher et al., 2012). There were strain-dependent effects on alcohol 699 drinking modulated by sex in adults, as adult HAD-1 rats did not show sex differences while 700 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 29 adult HAD-2 female rats showed greater intake than males (Dhaher et al., 2012). Similarly, adult 701 male alcohol-preferring rats consumed more alcohol compared to adult females (Bell et al., 702 2006). These differences compared to outbred strains of rats and mice may be a result of 703 selectively breeding (for 75+ generations) for higher intake and introducing a history of 704 excessive alcohol drinking for multiple generations that leads to blunted neuronal activity 705 associated with decision-making related to alcohol drinking behavior (Linsenbardt et al., 2019). 706 Female mice are resilient to alcohol-induced myelin loss 707 We did not detect measurable changes in myelinated fiber density after alcohol drinking 708 in female mice despite exhibiting similar levels of alcohol intake using the DID alcohol binge 709 drinking model. This is consistent with our previous report in adolescent female rats (Tavares et 710 al., 2019). Despite studies showing reduced gene expression and protein levels of myelin-711 associated genes in female rodents with high doses of alcohol (Alfonso‐Loeches et al., 2012; 712 Pascual et al., 2014), these changes may be due to the methods used to expose animals to 713 alcohol. We have found that in female rats, alcohol reduces the length of the nodes of Ranvier 714 located between the contactin-associated protein (Caspr) pairs (Tavares et al., 2019). This has 715 implications for action potential conduction velocity and amplitude, as a reduced nodal length 716 would lead to a decrease in sodium channels available at the nodes (Babbs and Shi, 2013; 717 Arancibia-Cárcamo et al., 2017; Scurfield and Latimer, 2018). 718 Loss of myelin density in the CCFM and Cg1 regions of adolescent males but not females 719 may reflect differences in the pubertal timing of myelination between sexes. Axons that have 720 partial or complete myelin sheaths at the time when demyelination occurs have faster 721 remyelination and are more selectively targeted for remyelination by OLs compared to isolated 722 myelin sheaths (Chapman et al., 2023). Adult males have a higher density of myelin sheaths 723 and OLs in the corpus callosum compared to females of the same age (Cerghet et al., 2006). 724 However, this is mediated by gonadal hormones, as castrated males show lower myelin sheath 725 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 30 density in the corpus callosum and fimbria at the anterior hippocampus compared to intact 726 males (Cerghet et al., 2006). Since pubertal maturation occurs at an earlier age in females 727 compared to males (Tavares et al., 2019), de novo myelin sheaths may be added before the 728 start of alcohol intake in females but not males. By PD28, females may have a greater percent 729 of axons that are partially or completely myelinated while males may have a higher percentage 730 of isolated myelin sheaths. Since brains were collected four days post-alcohol (PD56), this 731 allows enough time for partial and completed myelin sheaths to remyelinate while isolated 732 myelin sheaths take approximately eight days or more for remyelination to occur (Chapman et 733 al., 2023). Ongoing studies are dissecting the sex differences in the rate of myelination during 734 early adolescent development. 735 No change in the population of OPCs, pre-myelinating, myelinating, and post-736 myelinating were found in the cingulate cortex or corpus callosum of female mice in this study. 737 However, alcohol increased the density of mature (QKI-7+ OLs) in the cingulate cortex of female 738 mice, indicating a potential mechanism that ultimately results in the preservation of myelin 739 sheaths. Adult females rodents have a lower myelin and OL density and a higher turnover 740 (increased proliferation and cell death) of OLs in the corpus callosum, fornix, and spinal cord 741 compared to males that is mediated by gonadal hormones (Cerghet et al., 2006). In support of 742 this, administration of 17-b estradiol (alone or combined with progesterone) in male mice 743 partially blunts the effects of cuprizone on OL density loss and demyelination in the corpus 744 callosum (Acs et al., 2009; Taylor et al., 2010). This hormone-mediated ability for greater rate of 745 OL replacement suggests that females may have a mechanism for faster renewal of myelin in 746 response to insults like alcohol. This is the case for 12-month-old female rats following a 747 demyelinating lesion induced by ethidium bromide injection (Li et al., 2006). However, they also 748 found no differences in the remyelination rate in young adult (2-month-old) between male and 749 female rats (Li et al., 2006). Whether sex-specific differences in alcohol-induced myelin loss is 750 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 31 due to 1) timing of exposure relative to the surge of gonadal hormones, 2) sex differences in OL 751 renewal and/or remyelination rate, or 3) hormonal neuroprotection conferring resiliency to OLs 752 in females is still a subject of active research. 753

754 We have demonstrated that myelinating (ASPA+) OLs are particularly vulnerable to 755 alcohol in adolescent male mice, shifting the dynamics of differentiation and maturation of the 756 oligodendrocyte lineage, resulting in loss of myelin sheaths. These results contribute to the 757 growing body of evidence that alcohol disrupts the maturation of frontotemporal circuits, leading 758 to delayed processing and both functional and behavioral consequences, increasing the risk of 759 developing AUD later in life. 760 761 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 32

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The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 40 Walker BM, Walker JL, Ehlers CL (2008) Dissociable effects of ethanol consumpPon during the 1029 light and dark phase in adolescent and adult Wistar rats. Alcohol 42:83–89. 1030 Wilcox M V., Carlson VCC, Sherazee N, Sprow GM, Bock R, Thiele TE, Lovinger DM, Alvarez VA 1031 (2014) Repeated Binge-like ethanol drinking alters ethanol drinking paderns and 1032 depresses striatal GABAergic transmission. Neuropsychopharmacology 39:579–594. 1033 Wolstenholme JT, Mahmood T, Harris GM, Abbas S, Miles MF (2017) Intermident Ethanol during 1034 Adolescence Leads to LasPng Behavioral Changes in Adulthood and Alters Gene 1035 Expression and Histone MethylaPon in the PFC. Front Mol Neurosci 10:307. 1036 Young KM, Psachoulia K, Tripathi RB, Dunn S-J, Cossell L, Adwell D, Tohyama K, Richardson WD 1037 (2013) Oligodendrocyte Dynamics in the Healthy Adult CNS: Evidence for Myelin 1038 Remodeling. Neuron 77:873–885. 1039 Younis RM, Wolstenholme JT, Bagdas D, Benger JC, Miles MF, Damaj MI (2019) Adolescent but 1040 not adult ethanol binge drinking modulates ethanol behavioral effects in mice later in 1041 life. Pharmacol Biochem Behav 184:172740. 1042 1043 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 41 Figure legends: 1044 Figure 1. Overview of study design and alcohol intake in adolescent male and female 1045 mice. A. Schematic diagram of the drinking-in-the-dark (DID) alcohol binge drinking protocol. 1046 One DID cycle is three days of 2-hour “baseline” access (small red circles) to a single bottle of 1047 20% v/v alcohol, one day of 4-hour “binge” access (big red circles) and three days with no 1048 access to alcohol (black circles). Adolescent male and female C57BL6/J mice were allowed 1049 access to alcohol for four cycles beginning on postnatal day (PD) 28. At PD56 mice were 1050 intracardially perfused and brains were processed for immunofluorescence experiments with 1051 antibodies against the indicated proteins. Sections were then imaged using confocal microscopy 1052 and analyzed. B, C. Daily alcohol intake (g/kg) on binge days was higher than the average of 1053 daily drinking during “baseline” days in the same DID cycle (*, all ps < 0.05, repeated measures 1054 two-way ANOVA, Bonferroni’s post hoc) on cycles 2, 3 and 4 in males (B) and cycles 1, 2, and 3 1055 in females (C). D, E. Total weekly alcohol intake was significantly greater (*, all ps < 0.05, 1056 repeated measures one-way ANOVA, Tukey post hoc) in DID cycles 2, 3, and 4 compared to 1057 DID cycle 1 in males (D) and in DID cycle 3 compared to DID cycle 2 in females (E). Data are 1058 presented as mean values ± SEM; *p £ 0.05 = significance criterion; ns = non-significance. 1059 1060 Figure 2. Adolescent drinking decreases the density of myelinated axons the corpus 1061 callosum and anterior cingulate in male mice. A. Composite image of a representative brain 1062 section with DAPI nuclear staining that was produced by stitching together frames of 10x confocal 1063 images. Sections were AP+1.7mm distance from bregma and the CCFM and Cg1 sampling regions 1064 are denoted by the white boxes. Mag bar, 1000 µm. B, C. Representative image of the CCFM (B) and 1065 Cg1 (C) acquired using the 20X objective with immunohistochemical labeling of myelin basic protein 1066 (MOG)+ myelin sheaths in white. D, E. Representative images showing NIS Elements thresholding 1067 of MOG+ myelin sheaths (salmon color) in the CCFM and Cg1 of male (D) and female (E) control and 1068 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 42 alcohol mice. Tissue tears (dashed-line outlined area in E) were excluded from the analysis. Mag 1069 bar, 100 µm. F, G. Alcohol decreases myelin fiber density (% area covered by MOG) in the CCFM and 1070 Cg1 regions of males (F, *, all ps ≤ 0.05, compared to controls, unpaired t-tests) but not females (G, 1071 unpaired t-tests, ps > 0.05). H, I. No relationship was found between the total alcohol intake in cycle 1072 4 and myelin density in males (H) or females (I). AP, anterior-posterior position; CCFM, corpus 1073 callosum forceps minor; Cg1, anterior cingulate cortex; DAPI, 4',6-diamidino-2-phenylindole; OL, 1074 oligodendrocyte, MOG, myelin oligodendrocyte glycoprotein, QKI-7, Quaking protein isoform 7. Data 1075 are presented as mean values ± SEM; *p £ 0.05 = significance criterion; ns = non-significance. 1076 1077 Figure 3. Adolescent drinking does not reduce the density of oligodendrocyte precursor cells 1078 in the corpus callosum and anterior cingulate in mice. A. Composite image of a representative 1079 brain section with DAPI nuclear staining, which was produced by stitching together frames of 10x 1080 confocal images. Sections were AP+1.7mm distance from bregma and the CCFM and Cg1 sampling 1081 regions are denoted by the white boxes. B, C. Representative images of the CCFM (B) and Cg1 (C) 1082 acquired using the 20X objective with immunohistochemical labeling of PDGFRɑ-expressing OPCs 1083 in green. D, E. Representative images of PDGFRɑ cells in the CCFM and Cg1 of a control male, 1084 alcohol male, control female, and alcohol female. F, G. Alcohol did not affect the density of 1085 PDGFRɑ+ cells/mm2 in the CCFM or Cg1 of males and females. H, I. In males, there was a 1086 significant positive relationship between the total alcohol intake in cycle 4 and OPC density in the 1087 CCFM region (H, r2 = 0.56, p < 0.05) and in the Cg1 region there was a significant negative 1088 relationship (I, r2 = 0.60, p < 0.05). No significant correlations were found in females. AP, anterior-1089 posterior position; CCFM, corpus callosum forceps minor; Cg1, anterior cingulate cortex; DAPI, 4',6-1090 diamidino-2-phenylindole; OL, oligodendrocyte, MOG, myelin oligodendrocyte glycoprotein, QKI-7, 1091 Quaking protein isoform 7. Data are presented as mean values ± SEM; *p £ 0.05 = significance 1092 criterion; ns = non-significance. Scale bars = 1000 µm (A), 100 µm (B-E), 20 µm (inset images in B 1093 and C). 1094 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 43 1095 Figure 4. Adolescent drinking increased the density of mature oligodendroglia in the anterior 1096 cingulate in female mice. A. Composite image of a representative brain section with nuclear 1097 staining (DAPI) that was produced by stitching together frames of 10x confocal images. Sections 1098 were AP+1.7mm distance from bregma and the CCFM and Cg1 sampling regions are denoted by the 1099 white boxes. B, C. Representative image of the CCFM and Cg1 acquired using the 20x objective with 1100 immunohistochemical labeling of mature oligodendrocytes with the QKI-7 clone-CC1 antibody in red. 1101 D, E. Representative images of QKI-7 immunohistochemical labeling in the CCFM and Cg1 of control 1102 and alcohol groups in males (D) and females (E). Mag bar, 100 µm. F. No differences were found in 1103 the density of mature oligodendroglia between alcohol drinking group and controls in males in the 1104 CCFM region (unpaired t-test, p>0.05) and a trend of increase in the Cg1 region (unpaired t-test, p = 1105 0.06). G. No changes in the density of differentiated oligodendrocytes were found in the CCFM region 1106 in females (unpaired t-test, p>0.05); in contrast, alcohol drinking during adolescence increased the 1107 density of differentiated oligodendroglial cells in the Cg1 region in female mice (*p < 0.05, compared 1108 to controls, unpaired t-test). H, I. No relationship between the total alcohol intake in cycle 4 and the 1109 density of mature oligodendrocytes was found in males (H) or females (I). Data are presented as 1110 mean values ± SEM; *p £ 0.05 = significance criterion; ns = non-significance. Scale bars = 1000 1111 µm (A), 100 µm (B-E), 20 µm (inset images in B and C). 1112 1113 Figure 5. Phenotyping mature oligodendrocytes. A. Composite images of representative brain 1114 sections were produced by stitching together frames of 10x confocal images of a control female 1115 mouse. Sections were analyzed at AP+2.0 mm, AP+1.7 mm, and AP+0.9 mm distances from 1116 bregma, with sampled regions of the CCFM and Cg1 denoted by white boxes. B. Representative 20x 1117 magnification images showing QKI-7 (red) and ASPA (white) immunofluorescent labeling and DAPI 1118 nucleic acid dye counterstain (blue) in the CCFM and Cg1 of a control female mouse (AP+1.7mm). 1119 Three distinct OL populations were identified: pre-myelinating OLs (QKI-7+/ASPA-, red arrow), 1120 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 44 myelinating OLs (QKI-7+/ASPA+, pink arrow), and post-myelinating OLs (QKI-7-/ASPA+, white 1121 arrow). AP, anterior-posterior position; ASPA, aspartoacylase, DAPI, 4',6-diamidino-2-phenylindole; 1122 OL, oligodendrocyte, QKI-7, Quaking protein isoform 7. Scale bars = 1000 µm (A), 50 µm (B). 1123 1124 Figure 6. Alcohol decreases the population density of myelinating oligodendrocytes in 1125 the CCFM of male mice. A. Representative 20x confocal images of the CCFM region at 1126 AP+1.7mm from bregma in male and female mice following adolescent drinking of alcohol or 1127 water. Insets (white boxes) show higher magnification views; red arrows indicate cells 1128 expressing QKI-7 (pre-myelinating OLs), white arrows indicate cells expressing ASPA 1129 (myelinating OLs), and pink arrows indicate cells expressing both QKI-7 and ASPA (post-1130 myelinating OLs). B. Pre-myelinating OL density was not affected by alcohol in either males or 1131 females (two-way ANOVAs, ns). C, D. Alcohol decreased the density of myelinating (C) and 1132 post-myelinating (D) OLs in males only (two-way ANOVAs, *p < 0.05, main effect of treatment in 1133 males; ns in females). E, F. Alcohol increased the proportion of pre-myelinating OLs in the CCFM 1134 of males (E, unpaired t-test, p < 0.05), but not females (F, unpaired t-test, ns) at AP+1.7mm. 1135 Data are presented as mean values ± SEM with individual values shown in circles (males) or 1136 squares (females); *p £ 0.05 = significant; ns = not significant. Scale bars = 100 µm (A), 50 µm 1137 (A, close-up images). 1138 1139 Figure 7. Alcohol decreases the population density of myelinating oligodendrocytes in 1140 the Cg1 region. A. Representative 20x confocal images of the Cg1 region at AP+1.7mm from 1141 bregma in male and female mice following adolescent drinking of alcohol or water. Zoomed in 1142 images are outlined in white boxes. Red arrows indicate QKI-7 cell, white arrows indicate ASPA 1143 cells, and pink arrows indicate both QKI-7 and ASPA cells. B, C. No differences found in the 1144 density of pre-myelinating and myelinating OLs in males or females (two-way ANOVAs, ns). D. 1145 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 45 Alcohol decreased the density of post-myelinating OLs in males, but not females (*, p 0.05) or 1148 females (F, unpaired t-test, p > 0.05). Mag bar, 100 µm. Mag bars of the close-up images are 50 1149 µm. Data are presented as mean values ± SEM with individual values shown in circles (males) 1150 or squares (females); *p £ 0.05 = significant; ns = not significant. 1151 1152 Figure 8. High alcohol intake predicts low myelinating OL density in the Cg1 region in 1153 males. A. Mice were categorized as “high” or “low” drinking groups based on a median split of 1154 the total amount of alcohol consumed during the last week of drinking (DID cycle 4, Fig 1D and 1155 E). High drinking male mice consumed twice as much alcohol as low drinking males (top bar 1156 graph, *, p < 0.05, unpaired t-test) and high drinking female mice consumed three times as 1157 much alcohol as low drinking females (bottom bar graph, **, p < 0.01, unpaired t-tests). B. 1158 Myelinating OL density in the Cg1 was lower in high-drinking males compared to low-drinking 1159 males in the Cg1 region at (top bar graphs, **p 0.05, unpaired t-test). C. 1161 There was a tight negative correlation between the total amount of alcohol consumed and 1162 myelinating OL density in the Cg1 in males (top scatter plots, (r2 = 0.81, p < 0.01) but not 1163 females (bottom scatter plots, r2 = 0.08, ns). Sections used for analyses were AP+1.7mm 1164 distance from bregma. Data are presented as mean values ± SEM with individual values shown 1165 in circles (males) or squares (females); *p £ 0.05 = significant; ns = not significant. 1166 1167 Figure 9. Working model illustrating how binge drinking impairs myelination of prefrontal 1168 axons during adolescent development. We propose that alcohol targets the OL lineage in the 1169 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 46 late phase of cellular development, resulting in a low number of cells expressing the ASPA 1170 enzyme that is necessary for lipid synthesis during myelination. Even if the loss of ASPA+ OLs 1171 leads to a compensatory increase in OPC differentiation, this response is insufficient to rescue 1172 the myelinating OL population in males, resulting in myelin deficits. Arrows indicate the 1173 hypothesized direct (solid lines) and indirect (dashed lines) effects of alcohol on oligodendroglia. 1174 OPC, oligodendroglia precursor cell; OL, oligodendroglial cell; PDGFR⍺, platelet-derived growth 1175 factor receptor alpha; QKI-7, quaking protein isoform-7; ASPA, aspartoacylase. 1176 1177 Supplemental Figure 1. Alcohol intake across adolescent development. A, B. Male (A) and 1178 female (B) mice showed a significant increase in the average alcohol intake on binge days 1179 compared to baseline days (**, p < 0.01, ***, p < 0.001, two-way ANOVA, main effect of access). 1180 Male mice showed an increased average intake during the DID cycles 3 and 4 compared to the 1181 average intake in cycles 1 and 2 (**, p < 0.01, two-way ANOVA, main effect of DID cycle). Data 1182 are presented as mean values ± SEM with individual values shown in circles (males) or squares 1183 (females); *p £ 0.05 = significant; ns = not significant. 1184 1185 Supplemental Figure 2. Water intake of adolescent male and female mice. A. Male mice did 1186 not show consumption difference between baseline and binge days (two-way ANOVA, p > 0.05). 1187 B. Female mice showed a significant decrease in water intake in the binge session compared to 1188 the average of the baseline intake (*, p 0.05). Data are presented as mean values ± SEM with 1191 individual values shown in circles (males) or squares (females); *p £ 0.05 = significant; ns = not 1192 significant. 1193 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 47 1194 Supplementary Fig 3. Alcohol decreased the total population density of myelinating OLs 1195 with no change in the total population of mature OLs in the CCFM region of male mice. A. 1196 No differences in the total density of mature (QKI-7+) OLs were found in the CCFM or Cg1 1197 regions of males or females (two-way ANOVA, p > 0.05). B. Alcohol decreased the density of 1198 total myelinating OLs in the CCFM region of males (*, ps 0.05). C. No change with alcohol found in the total population of mature 1201 OLs in the CCFM or Cg1 regions of males or females (two-way ANOVA, p > 0.05). Data are 1202 presented as mean values ± SEM with individual values shown in circles (males) or squares 1203 (females); *p £ 0.05 = significant; ns = not significant. 1204 1205 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 48 1206 1207 Table 1. Antibodies and amplification reagents used for immunohistochemical experiments. Reagent Species Supplier Cat # Dilution Primary antibody MOG Rabbit Abcam Ab32760 1:1000 PDGFRɑ+ Goat R&D System AF1062 1:100 QKI-7 (clone-CC1) Mouse Millipore MABC200 1:400 ASPA Rabbit Millipore ABN1698 1:1000 Secondary antibody Anti-Rabbit (AlexaFluor 647) Donkey Jackson ImmunoResearch 1:500 Anti-Goat (AlexaFluor 588) Donkey Jackson ImmunoResearch 1:500 Biotinylated a Mouse (IgG) Horse Vector Laboratories BA-2000 1:200 Cy3-Streptavidin Jackson ImmunoResearch 016-160-084 1:2000 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 49 1208 1209 1210 1211 Figure 1. Overview of study design and alcohol intake of adolescent male and female mice. 0 5 10 15 20 25 0 2 4 6 Time (days) Alcohol intake (g/kg) Males ✱ ✱ ✱ Baseline Binge 0 5 10 15 20 25 0 2 4 6 Time (days) Alcohol intake (g/kg) Females ✱ ✱ ✱Baseline Binge 1 2 3 4 0 5 10 15 20 DID cycle Weekly alcohol intake (g/kg) Males 1 2 3 4 0 5 10 15 20 ✱ ✱ ✱ 1 2 3 4 0 5 10 15 20 DID cycle Weekly alcohol intake (g/kg) Females 1 2 3 4 0 5 10 15 20 a b 2h 4h2h 2h 24h 24h 24h DID cycle (7 days) 105 15 20 25 PD28 PD56 2h 4h2h 2h 24h 24h 24h 2h 4h2h 2h 24h 24h 24h2h 4h2h 2h 24h 24h 24h2h 4h2h 2h 24h 24h 24h 1 Perfusions Myelin density: MOG OL lineage proteins: PDGFRα, QKI-7, ASPA A B D C E was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 5, 2026. ; https://doi.org/10.64898/2026.04.01.715654doi: bioRxiv preprint 50 1212 Figure 2. Adolescent binge drinking decreases myelin fiber density the corpus callosum and cingulate cortex in males. A CCFM MOG Cg1 ControlAlcohol MalesD E CCFM Cg1 FemalesCCFM Cg1 MOG CCFM Cg1 ControlAlcohol B CDAPI 0 20 40 60 80 100MOG+ (% area) CCFM Control Alcohol ✱ 0 20 40 60 80 100 Cg1 ✱ 0 20 40 60 80 100MOG+ (% area) CCFM Control Alcohol 0 20 40 60 80 100 Cg1 Myelin fiber in MalesF Myelin fiber in FemalesG 036912020406080100 Alcohol Intake (g/kg) MOG+ (% area) CCFM Males 036912020406080100 Alcohol Intake (g/kg) Cg1Males 05101520020406080100 Alcohol Intake (g/kg) Cg1Females 05101520020406080100 Alcohol Intake (g/kg) MOG+ (% area) CCFM FemalesH I 51 1213 Figure 3. Adolescent drinking does not change the density of oligodendroglial precursor cells in the corpus callosum and cingulate cortex. PDGFRɑ CCFM PDGFRɑ Cg1 A Cg1 CCFM Cg1 Control D Alcohol Males Control CCFM Cg1 Alcohol E Females CCFMCg1 DAPI PDGFRɑ PDGFRɑ PDGFRɑ PDGFRɑ PDGFRɑ PDGFRɑ PDGFRɑ PDGFRɑ B C 0 100 200 300PDGFRα+ (cells/mm2 ) CCFM Control Alcohol 0 100 200 300 Cg1 0 100 200 300PDGFRα+ (cells/mm2 ) CCFM 0 100 200 300 Cg1 ControlAlcohol OPCs in MalesF OPCs in FemalesG 0369120 100 200 300 Alcohol Intake (g/kg) PDGFRα+ (cells/mm2 ) CCFM Males r2 = 0.56, p = 0.030369120 100 200 300 Alcohol Intake (g/kg) Cg1Males r2 = 0.60, p = 0.02 051015200 100 200 300 Alcohol Intake (g/kg) PDGFRα+ (cells/mm2 ) CCFM Females 051015200 100 200 300 Alcohol Intake (g/kg) Cg1FemalesH I 52 1214 Figure 4. Adolescent drinking increases the density of mature oligodendrocytes in the cingulate cortex in females. QKI - 7 Cg1 QKI - 7 CC FM A DControl CCFM Cg1 Alcohol Males CCFMCg1 DAPI QKI-7 QKI-7 QKI-7 QKI-7 EControl Alcohol CCFM Cg1Females QKI-7 QKI-7 QKI-7 QKI-7 B C 0 500 1000 1500 2000QKI7+ (cells/mm2 ) CCFM Control Alcohol 0 200 400 600 Cg1 p = 0.06 0 500 1000 1500 2000QKI7+ (cells/mm2 ) CCFM Control Alcohol 0 200 400 600 Cg1 ✱ OLs in MalesF OLs in FemalesG 0369120500100015002000 Alcohol Intake (g/kg) QKI7+ (cells/mm2 ) CCFMMales 0369120100200300400 Alcohol Intake (g/kg) Cg1Males 051015200100200300400 Alcohol Intake (g/kg) Cg1Females 051015200500100015002000 Alcohol Intake (g/kg) QKI7+ (cells/mm2 ) CCFMFemales H I 53 1215 1216 1217 Figure 5. Phenotyping mature oligodendrocytes (OLs). A B QKI - 7 DAPI CCFM Myelinating OL Post - Myelinating OL (sheaths completed) Pre - myelinating OL 50 µm 1000 µm CCcing Cg1 CCFM Cg1 CCFM Cg1 Cg1 Post - Myelinating OL (sheaths completed) Myelinating OL Pre - myelinating OL 50 µm 54 1218 1219 1220 Figure 6. Adolescent drinking decreases the density of myelinating oligodendrocytes in the cingulate cortex in males. 2.01.70.90 1000 2000 3000QKI7+/ASPA- (cells/mm2 ) 2.01.70.90 500 1000 1500QKI7+/ASPA+ (cells/mm2 ) ✱ ✱ ✱ 2.01.70.90 20 40 60 80 100 Anterior → Posterior (mm from bregma) QKI7-/ASPA+ (cells/mm2 ) ✱ ✱ ✱ 2.01.70.9 Control Alcohol 2.01.70.9 ControlAlcohol 2.01.70.9Anterior → Posterior (mm from bregma) ControlAlcohol Pre-myelinating OLsBMales Females Myelinating OLs Males Females C Post-myelinating OLsDMales Females ASPA ASPA QKI - 7 Control Male Alcohol Male ASPA QKI - 7 ASPAQKI-7 Control Female ASPAQKI-7 Alcohol Female A 50 µm 50 µm 50 µm 50 µm 100 µm 100 µm 100 µm Control 43.59% Pre-myelinating 56.41% Myelinating 32.32% Myelinating 67.68% Pre-myelinating Control 27.72% Myelinating 72.28% Pre-myelinating Alcohol Alcohol 27.53% Myelinating 72.47% Pre-myelinating E MalesCCFM F FemalesCCFM 100 µm 100 µm 55 1221 1222 Figure 7. Adolescent drinking reduces the density of myelinating oligodendrocytes in the corpus callosum in males. 2.01.70.90 1000 2000 3000QKI7+/ASPA- (cells/mm2 ) 2.01.70.90 500 1000 1500QKI7+/ASPA+ (cells/mm2 ) 2.01.70.90 20 40 60 80 100 Anterior → Posterior (mm from bregma) QKI7-/ASPA+ (cells/mm2 ) ✱ ✱✱ 2.01.70.9 Control Alcohol 2.01.70.9 ControlAlcohol 2.01.70.9Anterior → Posterior (mm from bregma) ControlAlcohol Pre-myelinating OLsBMales Females Myelinating OLs Males Females C Post-myelinating OLsDMales Females A ASPA QKI - 7 ASPA QKI - 7 Control Female Alcohol Female ASPA QKI - 7 ASPA QKI - 7 Control Male Alcohol Male 50 µm 50 µm 50 µm 50 µm 100 µm 100 µm 100 µm 100 µm 100 µm Control 56.70% Pre-myelinating 43.30% Myelinating 39.60% Myelinating 60.40% Pre-myelinating Control 29.90% Myelinating 70.10% Pre-myelinating Alcohol Alcohol 28.90% Myelinating 71.10% Pre-myelinating E MalesCg1 F FemalesCg1 56 1223 1224 1225 Figure 8. High drinking males have fewer myelinating oligodendrocytes than low drinking males in the cingulate cortex. LowHigh 0 5 10 15 20Alcohol intake (g/kg) Cycle 4 ✱✱ Females 0 5 10 15 20 0 100 200 300 400 Alcohol Intake (g/kg) Myelinating OLs(QKI7+/ASPA+ cells/mm2 ) Cg1 Females LowHigh 0 100 200 300 400 Myelinating OLs(QKI7+/ASPA+ cells/mm2 ) Cg1 Females 0 3 6 9 12 0 100 200 300 400 Alcohol Intake (g/kg) Myelinating OLs(QKI7+/ASPA+ cells/mm2 ) Cg1 Males r2 = 0.81, p = 0.0025 LowHigh 0 5 10 15 20Alcohol intake (g/kg) Cycle 4 ✱ Males LowHigh 0 100 200 300 400 Myelinating OLs(QKI7+/ASPA+ cells/mm2 ) Cg1 ✱✱ Males 1.7 mm Total (SUM) intake of Cycle 4 A B C 57 1226 1227 1228 Figure 9. Working model illustrating how binge drinking disrupts myelination of axons during adolescent development in males. 58 1229 1230 1231 Supplemental Figure 1. Alcohol intake across adolescent development. 1-23-4 1-23-4 0 2 4 6 8 10 DID cycle Average alcohol intake (g/kg) Males Baseline Binge day ✱✱ ✱✱ ✱✱✱ 1-23-4 1-23-4 0 2 4 6 8 10 DID cycle Average alcohol intake (g/kg) Females Baseline Binge day ✱✱ A B 59 1232 1233 1234 Supplemental Figure 2. Water intake of adolescent male and female mice. 0 5 10 15 20 25 0 2 4 6 8 10 Time (days) Water intake (g/kg) Males Baseline Binge 0 5 10 15 20 25 0 2 4 6 8 10 Time (days) Water intake (g/kg) Females Baseline Binge✱ 1 2 3 4 0 10 20 30 DID cycle Total water intake (g/kg) Males 1 2 3 4 0 10 20 30 1 2 3 4 0 10 20 30 DID cycle Total water intake (g/kg) Females 1 2 3 4 0 10 20 30 A B C D 60 1235 1236 1237 Supplementary Fig 3. Alcohol decreased the total population density of myelinating OLs with no change in the total population of mature OLs in the CCFM region of male mice. 2.01.70.90 1000 2000 3000 4000QKI7+ (cells/mm2 ) CCFM 2.01.70.90 1000 2000 3000 4000ASPA+ (cells/mm2) ✱ ✱ ✱ CCFM 2.01.70.90 1000 2000 3000 4000 All QKI7+ and ASPA+ (cells/mm2) CCFM 2.01.70.9 Control AlcoholCg1 2.01.70.9 Control Alcohol Cg1 2.01.70.9 ControlAlcohol Cg1 2.01.70.90 1000 2000 3000 4000QKI7+ (cells/mm2 ) CCFM 2.01.70.90 1000 2000 3000 4000ASPA+ (cells/mm2) CCFM 2.01.70.9 Control Alcohol Cg1 2.01.70.9 Control AlcoholCg1 2.01.70.90 1000 2000 3000 4000 All QKI7+ and ASPA+ (cells/mm2) CCFM 2.01.70.9 Control AlcoholCg1 A C Total Mature OLs BTotal Myelinating OLs Total Mature Lineage OLs Anterior → Posterior (mm from bregma) Anterior → Posterior (mm from bregma) Anterior → Posterior (mm from bregma) Anterior → Posterior (mm from bregma) Anterior → Posterior (mm from bregma) Anterior → Posterior (mm from bregma) Males Males Males Females Females Females