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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Alcohol Clin Exp Res. 2021 Dec 15;46(1):77–86. doi: 10.1111/acer.14752

Enduring myelin abnormalities in a third trimester-equivalent mouse model of fetal alcohol spectrum disorder

Jessie Newville 1, Tamara A Howard 2, Glenna J Chavez 1, C Fernando Valenzuela 1, Lee Anna Cunningham 1
PMCID: PMC8799509  NIHMSID: NIHMS1759629  PMID: 34825395

Abstract

Background:

Abnormal diffusion within white matter (WM) tracts has been linked to cognitive impairment in children with FASD. Whether changes to myelin organization and structure underlie the observed abnormal diffusion patterns remains unknown. Using a third trimester-equivalent mouse model of alcohol (EtOH) exposure, we previously demonstrated acute loss of oligodendrocyte lineage cells with persistent loss of myelin basic protein and lower fractional anisotropy (FA) in the corpus callosum (CC). Here, we tested whether these WM deficits are accompanied by changes in: (i) axial diffusion (AD) and radial diffusion (RD), (ii) myelin ultrastructure, or (iii) structural components of the node of Ranvier.

Methods:

Mouse pups were exposed to EtOH or air vapor for four hours daily from postnatal day (P)3 to P15 (BEC: 160.4±12.0 mg/dl; range=128.2–185.6 mg/dl). Diffusion tensor imaging (DTI) and histological analyses were performed on brain tissue isolated at P50. Diffusion parameters were measured in Paravision™ 5.1 software (Bruker) following ex vivo scanning in a 7.0 T MRI. Nodes of Ranvier were identified using high-resolution confocal imaging of immunofluorescence for Nav1.6 (nodes) and Caspr (paranodes) and measured using Imaris™ imaging software (Bitplane). Myelin ultrastructure was evaluated by calculating G-ratio (axonal diameter/myelinated fiber diameter) on images acquired using transmission electron microscopy (TEM).

Results:

Consistent with our previous study, high resolution DTI at P50 showed lower FA in the CC of EtOH-exposed mice (p=0.0014). Here, we show that while AD (diffusion parallel to CC axons) was similar between treatment groups (p=0.3087), RD (diffusion perpendicular to CC axons) in EtOH-exposed subjects was significantly higher compared to controls (p=0.0087). In the posterior CC, where we identified the highest degree of abnormal diffusion, node of Ranvier length did not differ between treatment groups (p=0.4137); however, the G-ratio of myelinated axons was significantly higher in EtOH-exposed animals compared to controls (p=0.023).

Conclusions:

High resolution DTI revealed higher RD at P50 in the CC of EtOH exposed animals suggesting less myelination of axons, particularly in the posterior regions. In agreement with these findings, ultrastructural analysis of myelinated axons in the posterior CC showed reduced myelin thickness in EtOH animals, evidenced by higher G-ratio.

Keywords: Diffusion Tensor Imaging, White Matter, Oligodendrocyte, Node of Ranvier, Corpus Callosum

Introduction

Fetal alcohol spectrum disorder (FASD) describes a range of phenotypes observed in individuals who have been exposed to alcohol during their gestational development. FASD encompasses an array of disorders including fetal alcohol syndrome, partial fetal alcohol syndrome, and alcohol-related neurodevelopmental disorder. Recent reports estimate that the prevalence of FASD may be as high as 1.1–5.0% for first grade school children in the United States (May et al., 2018). Individuals with FASD face life-long disability, often with the added burden of comorbidities, the most prevalent of which include congenital malformations, chromosomal abnormalities, and mental and behavior disorders (Popova et al., 2016). Individuals with FASD can experience a range of cognitive challenges including intellectual disability, attention deficit hyperactivity disorder, and learning disability, functional impairments to working memory, impulse control, and problem solving (Mela et al., 2020, Weyrauch et al., 2017). Understanding the harmful effects of alcohol on the developing brain is critical to develop therapies that mitigate these adverse cognitive outcomes and improve quality of life for individuals with FASD.

Evidence is building that the white matter of the central nervous system is particularly vulnerable to alcohol exposure during prenatal development. The largest white matter tract in the brain, the corpus callosum, is responsible for conduction of nerve impulses across the left and right hemispheres, and plays an important role in cognition (Hinkley et al., 2012). Intriguingly, the corpus callosum has been consistently implicated in the pathogenesis of prenatal alcohol exposure (Nunez et al., 2011). Early morphological assessments that utilized magnetic resonance imaging (MRI) describe partial or complete agenesis of the corpus callosum in individuals exposed to high levels of alcohol in utero (Riley et al., 1995, Swayze et al., 1997, Sowell et al., 2001, Bookstein et al., 2001). Indeed, using MRI to assess the shape and size of the corpus callosum in individuals with FAS, researchers found decreased corpus callosum area (Riley et al., 1995, Sowell et al., 2001), and increased variability in corpus callosum shape (Bookstein et al., 2001). Intriguingly, the posterior region of the corpus callosum exhibits heightened vulnerability to prenatal alcohol exposure, with high levels inducing structural anterior-inferior displacement of the splenium compared to healthy controls (Sowell et al., 2001). With the advent of diffusion tensor imaging (DTI), investigators have been able to further characterize more subtle forms of white matter injury through analysis of diffusion patterns within specific tissue regions that infer microstructural properties (Assaf and Pasternak, 2008). Indeed, clinical studies of FASD that employ DTI have found that white matter integrity is reduced in the corpus callosum, with more posterior regions being most affected (Riley et al., 2004, Fryer et al., 2009, Lebel et al., 2008, Wozniak et al., 2009). Abnormal corpus callosum microstructure resulting from prenatal alcohol exposure has been correlated with aberrant interhemispheric functional connectivity (Wozniak et al., 2011). This prenatal alcohol exposure induced white matter injury has been linked to a multitude of cognitive deficits such as diminished interhemispheric transfer of tactile information (Dodge et al., 2009), poor verbal learning and visuospatial processing (Sowell et al., 2001), reduced working memory (Wozniak et al., 2009, Malisza et al., 2012), and reduced processing speed (Ma et al., 2005).

Congruent with clinical findings, we previously demonstrated acute loss of oligodendrocyte lineage cells in the corpus callosum, followed by long term changes in myelin protein expression and diffusion properties using a third-trimester equivalent mouse model of FASD, in which mouse pups were exposed to EtOH vapor during peak myelination (Newville et al., 2017). Here, we sought to elucidate the structural underpinnings of this abnormal diffusion pattern by performing higher resolution ex vivo DTI coupled with ultrastructural analysis of myelin wrapping and node of Ranvier architecture. Measurement of diffusion changes throughout the anterior-posterior extent of the corpus callosum highlighted the posterior region as most susceptible to early postnatal EtOH-exposure. Diffusion abnormalities were associated with diminished myelin thickness without significant changes in the length of nodes of Ranvier.

Materials & Methods

All animal procedures were approved by the University of New Mexico Health Sciences Center Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health (NIH) Animal Welfare Regulations and Public Health Service Policy on Humane Care and Use of Laboratory Animals guidelines. All mice were housed in a reverse 12-h dark/light, humidity, and temperature controlled facility, where food and water were available ad libitum. For these studies, we utilized nestin-CreERT2:tdTomato reporter mice, maintained on a C57Bl/J6 background, to be consistent with our prior study (Newville et al., 2017). In this strain, reporter gene expression can be selectively induced in nestin+ progenitors by tamoxifen administration, as previously described and characterized in detail (Newville et al., 2017)(Lagace et al., 2007, Madisen et al., 2010, Chow et al., 2015, Patzlaff et al., 2017, Gustus et al., 2020, Gustus et al., 2019). However, since fate mapping of progenitors was not required for the current study, tamoxifen was not administered at any time. The brains of only male mice were studied in the present investigation, as previous work has demonstrated sex-specific differences in the corpus callosum of rodents (Cerghet et al., 2009), as well as humans (Dubb et al., 2003).

Alcohol Administration

To recapitulate a scenario of prenatal EtOH exposure in which exposure occurs during developmental myelination and peak oligodendrogenesis, mouse pups with their respective mothers were exposed to EtOH vapor from postnatal day (P)3 to P15 for four hours each day (1000 to 1400 hours), control litters were exposed to air under the same exposure paradigm (Morton et al., 2014, Zamudio-Bulcock et al., 2014, Newville et al., 2017). Exposure to air or EtOH vapor was accomplished by means of custom-built enclosures, in which the individual cages of dams and pups were placed. To minimize disturbance to the newly born litters and respective dams, animals remained within the vapor chambers over the course of the 13 days, P3-P15. To determine the concentration of EtOH within the enclosures, measurements of EtOH vapor concentration were taken daily throughout the duration of the exposure with a Breathalyzer instrument (Intoximeters, St. Louis, MO 12-0050-00). Over the course of the exposure, the concentration of EtOH vapor was gradually increased from P3 to P9, and then from P10 to P15 the concentration of EtOH vapor was maintained at a relatively constant level. As estimated based on our prior publication, this paradigm of EtOH vapor exposure results in mean pup blood alcohol concentration of 160 ± 12 mg/dl, ranging from 128 – 186 mg/dl (Newville et al., 2017), as measured by standard alcohol dehydrogenase-based assay (Galindo and Valenzuela, 2006). It should be noted that pup blood alcohol concentrations were not directly measured in this study, but were extrapolated from prior work characterizing this model (Newville et al., 2017). Following the last day of exposure, animals were placed back on normal housing racks and weaned on P25.

Ex vivo Diffusion Tensor Imaging and Analysis

To investigate the long-term impact of EtOH exposure during white matter development on cerebral white matter integrity and microstructure, we performed ex vivo MRI on P50 mouse brains (n = 4 per treatment group, each from separate litter). The P50 mouse brain roughly corresponds to a young adult human brain in regard to white matter development (Semple et al., 2013). Following transcardial perfusion with 1 M phosphate buffered saline (PBS) containing 0.1% procaine hydrochloride and 2 U/ml heparin, and then perfusion with 4% paraformaldehyde, the brain was removed from the skull, post-fixed overnight, and embedded in 2% agarose containing 3 mM NaN3 (Robinson et al., 2018), and stored at 4oC for up to one month. Scanning was accomplished with a 7.0 T small animal MRI (Bruker BioSpec 70/30 USR MRI, Billerica, MA). Twenty-eight sequential diffusion images in the coronal plane were acquired with echo-planar diffusion tensor imaging as previously described (Newville et al., 2017) with the following scan parameters: sequence = EPI-DTI, slice thickness = 0.5 mm, field of view (FOV) = 2 × 2 cm2, acquisition matrix (MTX) = 192 × 192, echo time (TE) = 38.0 ms, repetition time (TR) = 7000 ms; acquisition time = 9 hrs 48 mins. After imaging, the three eigenvalues of diffusion (ʎ1, ʎ2, and ʎ3) were calculated.

Region of interest analysis was performed on processed scans using Bruker’s Paravision 5.1 software (Robinson et al., 2018b, Yellowhair et al., 2019, Robinson et al., 2018a). For each scan slice containing conjoined corpus callosum (6 slices per brain; Bregma 1.10 to − 2.46 mm) (FIGURE 1B), fractional anisotropy (FA), axial diffusion (ʎ1), radial diffusion ((ʎ2 + ʎ3) / 2), and mean diffusion ((ʎ1 + ʎ2 + ʎ3) / 3) were calculated for selected ROIs encompassing the most medial crossing fibers of the corpus callosum. FA and diffusion encoded color maps were generated within Paravision. To better visualize the shift toward isotropic diffusion following EtOH exposure, the means of each eigenvalue (ʎ1, ʎ2, ʎ3) across the six sections were calculated for each treatment group and plotted as ellipsoids within MATLAB R2019a software (MathWorks, Natick, MA).

Figure 1.

Figure 1

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A. Representative DTI directionally encoded color maps in the coronal plane from P50 Air and EtOH exposed animals (red = transverse, green = vertical, blue = orthogonal to the plane, scale bar = 0.5 cm).

B. Diffusion parameters were measured across six regions of interest (ROI, purple) encompassing the crossing fibers of conjoined corpus callosum anterior to posterior for each animal.

C. Mean diffusion from anterior to posterior was significantly higher in EtOH-exposure mice. A main effect of treatment was detected with two-way ANOVA (n = 4/group, F[5,36] = 3.610, p=0.0095. Šidák’s multiple comparisons test revealed significance at level 5 (* p<0.05).

D. Averaged eigenvalues plotted as ellipsoids demonstrate the EtOH-induced shift toward isotropic diffusion.

Immunohistochemistry and Confocal Imaging

Following MRI scanning, brains were removed from agarose, saturated with 30% sucrose in PBS solution at 4°C, and cut in the coronal plane at 30 μm with a freezing sliding knife microtome (Fisher Scientific, Hampton, NH, BP2201). Sections containing dorsal hippocampus (Bregma −1.34 to −2.18) were selected for immunohistochemical co-labeling of Node of Ranvier (Nav1.6) and paranode (Caspr) markers. Contactin-associated protein (Caspr1) is a transmembrane protein crucial for the formation and stability of myelinated axons, specifically localized at the paranode region of the axon flanking the borders of each Node of Ranvier (Zou et al., 2017). Nodes of Ranvier within the central nervous system were readily identified by Nav1.6 localization. Nav1.6 is a subtype of sodium channel concentrated within the axonal membrane at Nodes of Ranvier (Caldwell et al., 2000). All immunofluorescence procedures were performed as previously published (Li et al., 2010). Briefly, floating tissue sections were permeabilized with 0.4% Triton-X PBS solution and blocked in 1% bovine serum albumin with 10% normal donkey serum in PBS. Following overnight incubation with primary antibodies at 4°C, tissue sections were incubated with FITC and Cy5 conjugated secondary antibodies (1:250; Jackson ImmunoResearch Laboratories, West Grove, PA) and counterstained with DAPI (D1306, Thermofisher scientific, Carlsbad, CA). The following primary antibodies were used in this study: mouse anti-caspr, clone K65/35 (1:100, Neuromab, UC Davis, Davis, CA); rabbit anti-Nav1.6 (1:500, ASC-009, Alomone Labs, Jerusalem, Israel). We observed no histological evidence for loss of tissue integrity due to prior storage in agarose.

Operating a Leica DMi8 TCS SP8 confocal microscope with LASX acquisition software (Wetzlar, Germany), sequential imaging was performed at 63x immersion objective to generate confocal z-stacks of nodes of Ranvier within the crossing fiber region of the corpus callosum. The following imaging parameters were uniform across scans: gain (100), zoom (5.02), pinhole size (95.6), scan speed (400 Hz), resolution (512 × 512), and laser intensity of the three separate channels (DAPI 405 nm:1.3%; paranode 499 nm: 14.5%; node 578 nm: 17.1%).

Within Imaris 9.3 software (Bitplane, Belfast, UK), measurement of node length (Nav1.6 immunofluorescence) was accomplished in 3D rendered confocal image stacks using the measurement points tool. For each node measurement, points were placed at the junctions of Nav1.6 and Caspr to span the width of a single node region, and the length between the two points was recorded. For each animal, 200–300 nodes were quantified.

Electron Microscopy and Quantification

To examine myelinated axons in the corpus callosum at the ultrastructural level, P50-P60 mice from air- and EtOH-exposed litters (n = 4 per treatment group each from a different litter) were anesthetized and transcardially perfused with fixative solution containing 3% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer. After whole brains were sectioned at 200 μm in the coronal plane, approximately 1 mm by 1 mm regions of midline corpus callosum at the level of dorsal hippocampus were isolated and washed in fixative solution. Following incubation for 1 hour in 1% OsO4 with 1.5% K4Fe(CN)6 in calcodylate buffer, tissue was washed and incubated in 1% uranyl acetate for 1 hour. Tissue blocks were dehydrated with a graded series of acetone, embedded in resin, sectioned in the sagittal plane (70–90 nm) with a Leica Ultramicrotome using a diamond knife. Tissue sections were positioned on copper grids for imaging using a Hitachi HT7700 transmission electron microscope (Tokyo, Japan) equipped with an 8 M pixel camera. Three 10 μm by 10 μm fields of view across the transected corpus callosum were captured for analysis for each animal.

To calculate the G-ratio (axonal diameter/myelinated fiber diameter) images were processed within ImageJ 1.8.0_122 software using the http://gratio.efil.de plug-in (Goebbels et al., 2010). 120 axons were counted across 3 different fields of view. For each animal, the total area assessed was 70 μm2. Use of the aforementioned plug-in allowed for unbiased selection and quantification of axon perimeter and myelin perimeter, measurements used to calculate G-ratio.

Statistics

Statistical comparisons were made between treatment groups with Students t test, two-way ANOVA with Šidák post-hoc for multiple comparisons, confirmed by non-parametric Mann-Whitney tests, using Prism 8.1.2 (GraphPad Software, La Jolla, CA). Data are expressed as means ± S.E.M, with the statistical unit of determination (n) defined as one pup, and p < 0.05 significance.

Results

EtOH exposure alters diffusion patterns within the corpus callosum at P50

To determine if EtOH exposure during the early postnatal period produced lasting changes to myelin microstructure of the corpus callosum, we compared diffusion tensor imaging of 4 mouse brains from EtOH-exposed mice and 4 mouse brains from air-exposed control mice, each from a separate litter. Representative directionally encoded color maps (FIGURE 1A) highlight the diversity of diffusion patterns within distinct regions of the brain. Comparison of the corpus callosum between treatment groups reveals distinct diffusion patterns. Specific ROI analysis was performed for each scan that contained a conjoined corpus callosum at A-P levels 1 through 5 (FIGURE 1B) to quantitatively assess differences in directional diffusion within the crossing fiber region. Mean diffusion (MD), which represents the overall diffusion within a ROI was calculated by averaging the three eigenvalues. Notably, mean diffusion was significantly higher in the corpus callosum of EtOH-exposed mice compared to air-exposed controls. Two-way ANOVA revealed main effects of alcohol treatment (n = 4/group, p = 0.0195), as well as a main effect of level along anterior-posterior (A-P) axis of the corpus callosum (p = 0.0095), without significant interaction (p = 0.1871) (FIGURE 1C). Individual comparison between MD values at each level by Šidák’s multiple comparisons test revealed significantly higher MD at more posterior levels (level 5, p = 0.0463).

To aid understanding of higher MD in EtOH-exposed mice, we plotted the three eigenvalues (averaged across all A-P levels of the medial corpus callosum) as an ellipsoid on a multidimensional plot for each treatment group. The eigenvalue ellipsoids allow visualization and comparison of the overall diffusion pattern, averaged across the A-P extent of the crossing fibers of the corpus callosum, between treatment groups. These ellipsoids highlight that the observed change in mean diffusion in the corpus callosum of EtOH-exposed mice is the result of more diffusion in ʎ2 and ʎ3 (radial diffusion, diffusion perpendicular to the fiber tract), with relatively no change in ʎ1 (axial diffusion, diffusion parallel to the fiber tract). Importantly, this pattern of disrupted diffusion at P50 following EtOH-exposure is suggestive of myelination deficits (Song et al., 2002).

Abnormal isotropic diffusion in EtOH-exposed mice at P50 suggests enduring white matter injury

To more closely investigate the nature of this change in diffusion pattern, we examined FA, a measure of organized diffusion within tissue. FA equal to 0 represents perfect isotropic diffusion, which occurs when all of the eigenvalues are of the same magnitude, in all directions. In contrast, FA equal to 1 represents perfect anisotropic diffusion, in which diffusion occurs in one direction only. Myelin restricts diffusion; thus, in more myelinated regions of the CNS, FA approaches 1. Color encoded FA color maps across each of the six A-P coronal sections containing conjoined corpus callosum show discernable differences in FA between treatment groups (FIGURE 2A). Specifically, diminished FA within EtOH-exposed animals compared to air-exposed controls is apparent. Indeed, two-way ANOVA revealed main effects of treatment (p < 0.0001, n = 4/group), and A-P level (p < 0.001), without significant interaction (p = 0.4004). Interestingly, EtOH induced diminishment of FA was more pronounced in posterior regions of the corpus callosum, as demonstrated by Šidák’s multiple comparisons showing statistically significantly lower FA at A-P level 3, level 5 and level 6 (p = 0.0487, p = 0.0027, p = 0.0016, respectively; FIGURE 2B).

Figure 2.

Figure 2

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A. Representative FA encoded color maps from P50 Air and EtOH exposed animals (perfect anisotropic diffusion = 1, perfect isotropic diffusion = 0, scale bar = 0.5 cm).

B. FA was significantly lower following EtOH exposure. A main effect of treatment was detected with two-way ANOVA (n = 4/group, F[5,36] = 48.12, p<0.0001, * p<0.05, **p<0.01).

C. & D. While axial diffusion was not statistically different between air and EtOH-exposed animals (two-way ANOVA, n = 4/group, F[5,36] = 3.386, p = 0.0740), RD was significantly higher (two-way ANOVA, n = 4/group, F[5,36] = 28.64, p<0.0001,* p<0.05, **p<0.01).

Next, we asked whether the reduced isotropic diffusion in the crossing fibers of the corpus callosum of EtOH-exposed mice was due to changes in axial (ʎ1) or radial (ʎ2 and ʎ3) diffusion. As previously suggested by the ellipsoids, axial diffusion did not significantly differ between treatment groups (two-way ANOVA, n = 4/group, p = 0.0740; FIGURE 2C). However, radial diffusion was significantly higher in EtOH-exposed animals (two-way ANOVA, p < 0.0001), an effect that was greater in more posterior regions (FIGURE 2D). For both axial and radial diffusion, two-way ANOVA determined significance across A-P level (p ≤ 0.0001), without significant interaction between treatment and level (p > 0.05). In support of our previous findings, these new data of lower FA, coupled with unchanged AD, and significantly higher RD, reflect a pattern of diffusion change that strongly suggests white matter injury following early-postnatal EtOH exposure.

EtOH exposure reduces myelin thickness of axons in the posterior corpus callosum at P50

To confirm on a cellular level whether early postnatal EtOH-exposure produces long-term damage to the white matter of the posterior corpus callosum, we next compared the wrapping of myelinated axons between EtOH-exposed animals to those of controls at the ultrastructure level. Sections which transected crossing fiber axons within posterior corpus callosum, where we observed the greatest change in diffusion, were processed for imaging with transmission electron microscopy, allowing for high-resolution visualization of myelin wrapping around each axon (FIGURE 3A). The thickness of myelin wrapping as a function of axon caliber was determined by calculating the G-ratio. In both air- and EtOH-exposed groups, the most abundant caliber of myelinated axon within this specific region of interest, were those with the diameter of 2–3.9 μm (FIGURE 3B), making up approximately 70% of the total myelinated axon population. The G-ratio within this majority population was significantly higher in EtOH-exposed animals compared to air controls (unpaired t test, n= 4/group, p = 0.023) (FIGURE 3C). Higher G-ratio indicates diminished thickness of myelin wrapping. These data showing diminished myelin wrapping support our findings of higher radial diffusion, and taken together, strongly indicate enduring white matter injury in young-adult mice following perinatal EtOH-exposure.

Figure 3.

Figure 3

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A. TEM of crossing fiber axons of the posterior CC at P50 (scale bars = 2 μm).

B. In both treatment groups 70% of myelinated axons fell in the 2–3 μm caliber range.

C. Within this majority population, the G-ratio of myelinated axons was significantly elevated in EtOH-exposed mice (unpaired t test, n = 4/group, *p=0.023).

Node length was not disrupted in the posterior corpus callosum of EtOH-exposed mice

In determining that myelin wrapping was affected long-term by perinatal EtOH-exposure, we next sought to determine if other aspects of myelin that contribute to proper propagation and conduction of action potentials were disrupted, such as node of Ranvier length (Arancibia-Carcamo et al., 2017). In histological sections containing posterior corpus callosum, we immunofluorescently labeled for Caspr (paranode) and Nav1.6 (node) proteins (FIGURE 4A), and determined node length by measuring the distance between pairs of paranodes, joined by a single node (FIGURE 4B). Within this specific region of interest the node length of EtOH-exposed animals at P50, 1.076 ± 0.027 μm, was not significantly different compared to the node length of air-exposed controls, 1.128 ± 0.046 μm (unpaired t test, n = 3–4/group, p = 0.4101). Interestingly, we did observe increased variance in node length within the EtOH-exposed mice (0.008 μm2) compared to controls (0.002 μm2), however, this did not reach statistical significance (F test, p = 0.4137).

Figure 4.

Figure 4

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A. Fluorescently labeled Nodes of Ranvier in the crossing fiber region of the corpus callosum from P50 Air and EtOH mice.

B. Node of Ranvier length was determined by measuring the distance (μm) between pairs of paranodes (red, Caspr), joined by a single node (green, Nav1.6).

C. Node Length was not significantly disrupted in EtOH-exposed animals compared to Air-exposed controls (unpaired t test, n = 3–4/group, p= 0.4101) (F test, p=0.4137).

Discussion

Using an early postnatal model of EtOH vapor exposure, in which mice are exposed to EtOH during peak myelination, our study revealed microstructural and ultrastructural changes in the corpus callosum, consistent with long lasting white matter injury. Here, we elaborate on our previous work demonstrating less myelin basic protein expression and lower FA in the corpus callosum of young adult mice (Newville et al., 2017), by performing DTI at higher resolution (7T) and by utilizing TEM and confocal microscopy to further characterize myelin abnormalities in this important white matter tract.

Our results show EtOH exposure during developmental myelination results in significant microstructural abnormalities in the corpus callosum that strongly reflect white matter injury. White matter injury is indicated by changes in corpus callosum diffusivity, higher MD with lower FA and higher RD. This pattern of diffusion, extra diffusion perpendicular to the axon, suggests that injury to myelin rather than axonal injury is a primary feature of EtOH-induced pathology (Song et al., 2002, Winklewski et al., 2018). Interestingly, these observed diffusion changes become more significant at posterior levels of the corpus callosum, a pattern consistent with clinical imaging reports of children and adolescence with FASD (Lebel et al., 2008, Li et al., 2009, Ma et al., 2005, Sowell et al., 2001, Wozniak et al., 2009). The corpus callosum is composed of axons responsible for interhemispheric integration and transfer of information. Callosal axons in the anterior regions interconnecting the frontal lobes provide for the transfer of motor information, whereas axons in the posterior fibers that connect the parietal, temporal, and occipital lobes support transfer and integration of somatosensory, auditory, and visual information (Fabri and Polonara, 2013, Tanaka-Arakawa et al., 2015). Thus, injury to myelin wrapping around posterior callosal axons could, in part, contribute to the impaired cognitive processing observed in individuals with FASD (Coffman et al., 2020, Fjeldsted and Xue, 2019, Hen-Herbst et al., 2020, Inkelis et al., 2020, Stephen et al., 2012, Tesche et al., 2015).

To further investigate the structural deficits underlying our MRI findings, we performed G-ratio ultrastructural analysis in the posterior region of the corpus callosum to more definitively determine if diminished myelin wrapping was underlying the abnormal diffusion pattern in this region. Indeed, by measuring the G-ratio of myelinated axons in the posterior corpus callosum we observed that animals exposed to EtOH had significantly less myelin wrapping, as evidenced by higher G-ratio compared to air-exposed controls. Reduced myelin wrapping in this region is consistent with our previous reports of diminished myelin basic protein in the corpus callosum of P50 EtOH-exposed mice (Newville et al., 2017). Diminished myelin thickness in the corpus callosum has important implications for CNS function, as efficient propagation and velocity of action potentials down axons is dependent on myelin thickness, length, and patterns of axonal coverage by myelin (Chang et al., 2017). Structural deficits in within the white matter of the corpus callosum and other brain regions has been linked to impairment of cognitive skills in clinical FASD (Lebel et al., 2010). Although beyond the scope of this imaging study, it will be important in future work to determine the extent to which abnormal myelination in our prenatal alcohol exposure model translates to impaired electrophysiological conduction properties and behavioral deficits.

To further investigate the possibility of other EtOH-induced changes to myelin with potential functional consequences, we measured Node of Ranvier length in the posterior region of the corpus callosum (Babbs and Shi, 2013, Susuki et al., 2013). Although other models of developmental injury with oligodendrocyte and myelin deficits report changes to node parameters in mature animals (Ritter et al., 2013), we did not identify significant changes in node length in this study. This suggests that although oligodendrocytes produce thinner myelin sheaths, they are able to produce myelinated segments spaced similarly to controls. While Node of Ranvier length is not affected at P50 in EtOH animals, myelin thickness alone could affect conduction velocity and CNS functioning.

It is important to note several limitations of this study. First, is the use of only male mice for our analyses to avoid variability associated with sex-specific differences in white matter development and EtOH toxicity. Sex-dependent effects of perinatal EtOH exposure have been reported in the literature (Paolozza et al., 2015; Tesche et al., 2015), and it will be important in future studies to determine the impact of sex as a biological variable in our mouse model. Another limitation is the relatively small sample size for this study. Although the MRI and ultrastructural differences achieved statistical significance sampled across multiple litters, there was significant variation in node of Ranvier length in the alcohol-exposed group. Overall, the study could have benefited from a larger sample size, which would likely reduce this variability. Additionally, although we infer functional deficits from our DTI and TEM studies we did not measure conduction velocity across the corpus callosum to determine the extent to which these structural changes in white matter predict impaired conduction across the corpus callosum in this mouse model. Finally, it would be of interest to assess additional white matter tracts in this mouse model of early postnatal alcohol exposure, to determine whether the white matter changes we have observed in corpus callosum also occur in other brain regions.

In sum, this study demonstrates that early postnatal EtOH exposure in mouse leads to long-lasting white matter structural deficits, as assessed by DTI analysis of the corpus callosum at postnatal day 50. Our imaging analysis points to impaired myelination, which was confirmed by ultrastructural evidence of reduced myelin thickness. Taken together with our previous findings of reduced myelin basic protein within corpus callosum, these studies further validate this EtOH exposure model to mimic white matter injury in clinical FASD, and thus provide a model for testing therapeutic intervention that may abrogate these long term white matter deficits.

Acknowledgements

This research was supported by funding from NIH-NIAAA P50-AA022534, NIH-NIAAA R01AA027462, NIH-NIGMS P20GM109089, and R37AA015614. We would like to thank Dr. Yirong Yang at the UNM School of Medicine’s BRaIN Imaging Center for his MRI expertise and technical assistance, as well as the UNM Cancer Center Fluorescence Microscopy Shared Resource, funded as detailed on http://hsc.unm.edu/crtc/microscopy/acknowledgement.shtml

References

  1. ARANCIBIA-CÁRCAMO IL, FORD MC, COSSELL L, ISHIDA K, TOHYAMA K, ATTWELL D 2017. Node of Ranvier length as a potential regulator of myelinated axon conduction speed. eLife, e23329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. ARAUJO CM, HUDZIAK J, CROCETTI D, WYMBS NF, ORTIZ JLM, ORR C, ALBAUGH MD, ALTHOFF RR, O’LOUGHLIN K, HOLBROOK H, GARAVAN H, YANG BZ, MOSTOFSKY S, JACKOWSKI A, LEE RS, GELERNTER J & KAUFMAN J 2020. Tubulin Polymerization Promoting Protein (TPPP) gene methylation and corpus callosum measures in maltreated children. Psychiatry Research: Neuroimaging, 298, 111058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. ARANCIBIA-CARCAMO IL, FORD MC, COSSELL L, ISHIDA K, TOHYAMA K & ATTWELL D 2017. Node of Ranvier length as a potential regulator of myelinated axon conduction speed. Elife, 6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. ASSAF Y & PASTERNAK O 2008. Diffusion Tensor Imaging (DTI)-based White Matter Mapping in Brain Research: A Review. Journal of Molecular Neuroscience, 34, 51–61. [DOI] [PubMed] [Google Scholar]
  5. BABBS CF & SHI R 2013. Subtle paranodal injury slows impulse conduction in a mathematical model of myelinated axons. PLoS One, 8, e67767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. BOOKSTEIN FL, SAMPSON PD, STREISSGUTH AP & CONNOR PD 2001. Geometric morphometrics of corpus callosum and subcortical structures in the fetal-alcohol-affected brain. Teratology, 64, 4–32. [DOI] [PubMed] [Google Scholar]
  7. CALDWELL JH, SCHALLER KL, LASHER RS, PELES E & LEVINSON SR 2000. Sodium channel Na&lt;sub&gt;v&lt;/sub&gt;1.6 is localized at nodes of Ranvier, dendrites, and synapses. Proceedings of the National Academy of Sciences, 97, 5616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. CERGHET M, SKOFF RP, SWAMYDAS M & BESSERT D 2009. Sexual dimorphism in the white matter of rodents. Journal of the neurological sciences, 286, 76–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. CHANG EH, ARGYELAN M, AGGARWAL M, CHANDON T-SS, KARLSGODT KH, MORI S & MALHOTRA AK 2017. The role of myelination in measures of white matter integrity: Combination of diffusion tensor imaging and two-photon microscopy of CLARITY intact brains. NeuroImage, 147, 253–261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. CHOW CL, GUO W, TRIVEDI P, ZHAO X & GUBBELS SP 2015. Characterization of a unique cell population marked by transgene expression in the adult cochlea of nestin-CreER(T2)/tdTomato-reporter mice. The Journal of comparative neurology, 523, 1474–1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. COFFMAN BA, CANDELARIA-COOK FT & STEPHEN JM 2020. Unisensory and Multisensory Responses in Fetal Alcohol Spectrum Disorders (FASD): Effects of Spatial Congruence. Neuroscience, 430, 34–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DODGE NC, JACOBSON JL, MOLTENO CD, MEINTJES EM, BANGALORE S, DIWADKAR V, HOYME EH, ROBINSON LK, KHAOLE N, AVISON MJ & JACOBSON SW 2009. Prenatal alcohol exposure and interhemispheric transfer of tactile information: Detroit and Cape Town findings. Alcohol Clin Exp Res, 33, 1628–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DUBB A, GUR R, AVANTS B & GEE J 2003. Characterization of sexual dimorphism in the human corpus callosum. Neuroimage, 20, 512–9. [DOI] [PubMed] [Google Scholar]
  14. FABRI M & POLONARA G 2013. Functional topography of human corpus callosum: an FMRI mapping study. Neural Plast, 2013, 251308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. FJELDSTED B & XUE L 2019. Sensory Processing in Young Children with Fetal Alcohol Spectrum Disorder. Phys Occup Ther Pediatr, 39, 553–565. [DOI] [PubMed] [Google Scholar]
  16. FRYER SL, SCHWEINSBURG BC, BJORKQUIST OA, FRANK LR, MATTSON SN, SPADONI AD & RILEY EP 2009. Characterization of white matter microstructure in fetal alcohol spectrum disorders. Alcohol Clin Exp Res, 33, 514–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. GALINDO R & VALENZUELA CF 2006. Immature hippocampal neuronal networks do not develop tolerance to the excitatory actions of ethanol. Alcohol, 40, 111–118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. GOEBBELS S, OLTROGGE JH, KEMPER R, HEILMANN I, BORMUTH I, WOLFER S, WICHERT SP, MOBIUS W, LIU X, LAPPE-SIEFKE C, ROSSNER MJ, GROSZER M, SUTER U, FRAHM J, BORETIUS S & NAVE KA 2010. Elevated phosphatidylinositol 3,4,5-trisphosphate in glia triggers cell-autonomous membrane wrapping and myelination. J Neurosci, 30, 8953–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. GUSTUS K, LI L, NEWVILLE J & CUNNINGHAM LA 2020. Functional and Structural Correlates of Impaired Enrichment-Mediated Adult Hippocampal Neurogenesis in a Mouse Model of Prenatal Alcohol Exposure. Brain Plasticity, 6, 67–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. GUSTUS K, LOZANO E, NEWVILLE J, LI L, VALENZUELA CF & CUNNINGHAM LA 2019. Resistance of Postnatal Hippocampal Neurogenesis to Alcohol Toxicity in a Third Trimester-Equivalent Mouse Model of Gestational Alcohol Exposure. Alcoholism: Clinical and Experimental Research, 43, 2504–2513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. HEN-HERBST L, JIRIKOWIC T, HSU LY & MCCOY SW 2020. Motor performance and sensory processing behaviors among children with fetal alcohol spectrum disorders compared to children with developmental coordination disorders. Res Dev Disabil, 103, 103680. [DOI] [PubMed] [Google Scholar]
  22. HINKLEY LBN, MARCO EJ, FINDLAY AM, HONMA S, JEREMY RJ, STROMINGER Z, BUKSHPUN P, WAKAHIRO M, BROWN WS, PAUL LK, BARKOVICH AJ, MUKHERJEE P, NAGARAJAN SS & SHERR EH 2012. The role of corpus callosum development in functional connectivity and cognitive processing. PloS one, 7, e39804–e39804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. INKELIS SM, MOORE EM, BISCHOFF-GRETHE A & RILEY EP 2020. Neurodevelopment in adolescents and adults with fetal alcohol spectrum disorders (FASD): A magnetic resonance region of interest analysis. Brain Res, 1732, 146654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. LAGACE DC, WHITMAN MC, NOONAN MA, ABLES JL, DECAROLIS NA, ARGUELLO AA, DONOVAN MH, FISCHER SJ, FARNBAUCH LA, BEECH RD, DILEONE RJ, GREER CA, MANDYAM CD & EISCH AJ 2007. Dynamic Contribution of Nestin-Expressing Stem Cells to Adult Neurogenesis. The Journal of Neuroscience, 27, 12623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. LEBEL C, RASMUSSEN C, WYPER K, ANDREW G & BEAULIEU C 2010. Brain microstructure is related to math ability in children with fetal alcohol spectrum disorder. Alcohol Clin Exp Res, 34, 354–63. [DOI] [PubMed] [Google Scholar]
  26. LEBEL C, RASMUSSEN C, WYPER K, WALKER L, ANDREW G, YAGER J & BEAULIEU C 2008. Brain diffusion abnormalities in children with fetal alcohol spectrum disorder. Alcohol Clin Exp Res, 32, 1732–40. [DOI] [PubMed] [Google Scholar]
  27. LI L, COLES CD, LYNCH ME & HU X 2009. Voxelwise and skeleton-based region of interest analysis of fetal alcohol syndrome and fetal alcohol spectrum disorders in young adults. Hum Brain Mapp, 30, 3265–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. LI L, HARMS KM, VENTURA PB, LAGACE DC, EISCH AJ & CUNNINGHAM LA 2010. Focal cerebral ischemia induces a multilineage cytogenic response from adult subventricular zone that is predominantly gliogenic. Glia, 58, 1610–1619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. MA X, COLES CD, LYNCH ME, LACONTE SM, ZURKIYA O, WANG D & HU X 2005. Evaluation of corpus callosum anisotropy in young adults with fetal alcohol syndrome according to diffusion tensor imaging. Alcohol Clin Exp Res, 29, 1214–22. [DOI] [PubMed] [Google Scholar]
  30. MADISEN L, ZWINGMAN TA, SUNKIN SM, OH SW, ZARIWALA HA, GU H, NG LL, PALMITER RD, HAWRYLYCZ MJ, JONES AR, LEIN ES & ZENG H 2010. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature Neuroscience, 13, 133–140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. MALISZA KL, BUSS JL, BOLSTER RB, DE GERVAI PD, WOODS-FROHLICH L, SUMMERS R, CLANCY CA, CHUDLEY AE & LONGSTAFFE S 2012. Comparison of spatial working memory in children with prenatal alcohol exposure and those diagnosed with ADHD; A functional magnetic resonance imaging study. J Neurodev Disord, 4, 12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. MAY PA, CHAMBERS CD, KALBERG WO, ZELLNER J, FELDMAN H, BUCKLEY D, KOPALD D, HASKEN JM, XU R, HONERKAMP-SMITH G, TARAS H, MANNING MA, ROBINSON LK, ADAM MP, ABDUL-RAHMAN O, VAUX K, JEWETT T, ELLIOTT AJ, KABLE JA, AKSHOOMOFF N, FALK D, ARROYO JA, HERELD D, RILEY EP, CHARNESS ME, COLES CD, WARREN KR, JONES KL & HOYME HE 2018. Prevalence of Fetal Alcohol Spectrum Disorders in 4 US CommunitiesPrevalence of Fetal Alcohol Spectrum Disorders in 4 US CommunitiesPrevalence of Fetal Alcohol Spectrum Disorders in 4 US Communities. JAMA, 319, 474–482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. MELA M, FLANNIGAN K, ANDERSON T, NELSON M, KRISHNAN S, CHIZEA C, TAKAHASHI S & SANJANWALA R 2020. Neurocognitive Function and Fetal Alcohol Spectrum Disorder in Offenders with Mental Disorders. Journal of the American Academy of Psychiatry and the Law Online, JAAPL.003886–20. [DOI] [PubMed] [Google Scholar]
  34. MORTON RA, DIAZ MR, TOPPER LA & VALENZUELA CF 2014. Construction of vapor chambers used to expose mice to alcohol during the equivalent of all three trimesters of human development. J Vis Exp. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. NEWVILLE J, VALENZUELA CF, LI L, JANTZIE LL & CUNNINGHAM LA 2017. Acute oligodendrocyte loss with persistent white matter injury in a third trimester equivalent mouse model of fetal alcohol spectrum disorder. Glia, 65, 1317–1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. NUNEZ CC, ROUSSOTTE F & SOWELL ER 2011. Focus on: structural and functional brain abnormalities in fetal alcohol spectrum disorders. Alcohol Res Health, 34, 121–31. [PMC free article] [PubMed] [Google Scholar]
  37. PAOLOZZA A, MUNN R, MUNOZ DP, & REYNOLDS JN 2015. Eye movements reveal sexually dimorphic deficits in children with fetal alcohol spectrum disorder. Frontiers in Neurosciences 9: 76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. PATZLAFF NE, NEMEC KM, MALONE SG, LI Y & ZHAO X 2017. Fragile X related protein 1 (FXR1P) regulates proliferation of adult neural stem cells. Human molecular genetics, 26, 1340–1352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. POPOVA S, LANGE S, SHIELD K, MIHIC A, CHUDLEY AE, MUKHERJEE RAS, BEKMURADOV D & REHM J 2016. Comorbidity of fetal alcohol spectrum disorder: a systematic review and meta-analysis. The Lancet, 387, 978–987. [DOI] [PubMed] [Google Scholar]
  40. RILEY EP, MATTSON SN, SOWELL ER, JERNIGAN TL, SOBEL DF & JONES KL 1995. Abnormalities of the corpus callosum in children prenatally exposed to alcohol. Alcohol Clin Exp Res, 19, 1198–202. [DOI] [PubMed] [Google Scholar]
  41. RILEY EP, MCGEE CL & SOWELL ER 2004. Teratogenic effects of alcohol: a decade of brain imaging. Am J Med Genet C Semin Med Genet, 127c, 35–41. [DOI] [PubMed] [Google Scholar]
  42. RITTER J, SCHMITZ T, CHEW LJ, BUHRER C, MOBIUS W, ZONOUZI M & GALLO V 2013. Neonatal hyperoxia exposure disrupts axon-oligodendrocyte integrity in the subcortical white matter. J Neurosci, 33, 8990–9002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. ROBINSON S, CONTEH FS, OPPONG AY, YELLOWHAIR TR, NEWVILLE JC, DEMERDASH NE, SHROCK CL, MAXWELL JR, JETT S, NORTHINGTON FJ & JANTZIE LL 2018a. Extended Combined Neonatal Treatment With Erythropoietin Plus Melatonin Prevents Posthemorrhagic Hydrocephalus of Prematurity in Rats. Frontiers in Cellular Neuroscience, 12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. ROBINSON S, CORBETT CJ, WINER JL, CHAN LAS, MAXWELL JR, ANSTINE CV, YELLOWHAIR TR, ANDREWS NA, YANG Y, SILLERUD LO & JANTZIE LL 2018b. Neonatal erythropoietin mitigates impaired gait, social interaction and diffusion tensor imaging abnormalities in a rat model of prenatal brain injury. Experimental Neurology, 302, 1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. SEMPLE BD, BLOMGREN K, GIMLIN K, FERRIERO DM & NOBLE-HAEUSSLEIN LJ 2013. Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species. Prog Neurobiol, 106–107, 1–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. SONG SK, SUN SW, RAMSBOTTOM MJ, CHANG C, RUSSELL J & CROSS AH 2002. Dysmyelination revealed through MRI as increased radial (but unchanged axial) diffusion of water. Neuroimage, 17, 1429–36. [DOI] [PubMed] [Google Scholar]
  47. SOWELL ER, MATTSON SN, THOMPSON PM, JERNIGAN TL, RILEY EP & TOGA AW 2001. Mapping callosal morphology and cognitive correlates: effects of heavy prenatal alcohol exposure. Neurology, 57, 235–44. [DOI] [PubMed] [Google Scholar]
  48. STEPHEN JM, KODITUWAKKU PW, KODITUWAKKU EL, ROMERO L, PETERS AM, SHARADAMMA NM, CAPRIHAN A & COFFMAN BA 2012. Delays in auditory processing identified in preschool children with FASD. Alcohol Clin Exp Res, 36, 1720–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. SUSUKI K, CHANG K-J, ZOLLINGER DANIELR, LIU Y, OGAWA Y, ESHED-EISENBACH Y, DOURS-ZIMMERMANN MARÍAT, OSES-PRIETO JUANA, BURLINGAME ALMAL, SEIDENBECHER CONSTANZEI, ZIMMERMANN DIETERR, OOHASHI T, PELES E & RASBAND MATTHEWN 2013. Three Mechanisms Assemble Central Nervous System Nodes of Ranvier. Neuron, 78, 469–482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. SWAYZE VW 2ND, JOHNSON VP, HANSON JW, PIVEN J, SATO Y, GIEDD JN, MOSNIK D & ANDREASEN NC 1997. Magnetic resonance imaging of brain anomalies in fetal alcohol syndrome. Pediatrics, 99, 232–40. [DOI] [PubMed] [Google Scholar]
  51. TANAKA-ARAKAWA MM, MATSUI M, TANAKA C, UEMATSU A, UDA S, MIURA K, SAKAI T & NOGUCHI K 2015. Developmental changes in the corpus callosum from infancy to early adulthood: a structural magnetic resonance imaging study. PLoS One, 10, e0118760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. TESCHE CD, KODITUWAKKU PW, GARCIA CM & HOUCK JM 2015. Sex-related differences in auditory processing in adolescents with fetal alcohol spectrum disorder: A magnetoencephalographic study. Neuroimage Clin, 7, 571–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. WEYRAUCH D, SCHWARTZ M, HART B, KLUG MG & BURD L 2017. Comorbid Mental Disorders in Fetal Alcohol Spectrum Disorders: A Systematic Review. Journal of Developmental & Behavioral Pediatrics, 38. [DOI] [PubMed] [Google Scholar]
  54. WINKLEWSKI PJ, SABISZ A, NAUMCZYK P, JODZIO K, SZUROWSKA E & SZARMACH A 2018. Understanding the Physiopathology Behind Axial and Radial Diffusivity Changes-What Do We Know? Front Neurol, 9, 92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. WOZNIAK JR, MUELLER BA, MUETZEL RL, BELL CJ, HOECKER HL, NELSON ML, CHANG PN & LIM KO 2011. Inter-hemispheric functional connectivity disruption in children with prenatal alcohol exposure. Alcohol Clin Exp Res, 35, 849–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. WOZNIAK JR, MUETZEL RL, MUELLER BA, MCGEE CL, FREERKS MA, WARD EE, NELSON ML, CHANG PN & LIM KO 2009. Microstructural corpus callosum anomalies in children with prenatal alcohol exposure: an extension of previous diffusion tensor imaging findings. Alcohol Clin Exp Res, 33, 1825–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. YELLOWHAIR TR, NEWVILLE JC, NOOR S, MAXWELL JR, MILLIGAN ED, ROBINSON S & JANTZIE LL 2019. CXCR2 Blockade Mitigates Neural Cell Injury Following Preclinical Chorioamnionitis. Frontiers in Physiology, 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. ZAMUDIO-BULCOCK PA, MORTON RA & VALENZUELA CF 2014. Third trimester-equivalent ethanol exposure does not alter complex spikes and climbing fiber long-term depression in cerebellar Purkinje neurons from juvenile rats. Alcohol Clin Exp Res, 38, 1293–300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. ZOU Y, ZHANG W-F, LIU H-Y, LI X, ZHANG X, MA X-F, SUN Y, JIANG S-Y, MA Q-H & XU D-E 2017. Structure and function of the contactin-associated protein family in myelinated axons and their relationship with nerve diseases. Neural Regeneration Research, 12, 1551–1558. [DOI] [PMC free article] [PubMed] [Google Scholar]

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