Fetal alcohol spectrum disorders (FASDs), caused by exposure to alcohol during pregnancy, is an umbrella term for constellation of permanent defects in several body systems including notably the nervous system. FASDs range in severity depending on the amount, timing, and frequency of alcohol exposure. Fetal alcohol syndrome, the most commonly known and severest form of FASD, is easily detected because it is accompanied by characteristic physical features such as craniofacial abnormalities and retarded growth 1. FASD cases lacking these hallmarks can go undiagnosed for several years. A common nervous system abnormality reported in FASD is sensorimotor defects 2,3 2,3. Sensorimotor information travels to and from the cortex through the internal capsule, which consists of thalamocortical, corticothalamic, and corticospinal axons. Each of these tracts has a distinct role in sensorimotor communication: thalamocortical axons input sensory information to the neurons in layer 4 of the cortex, corticothalamic axons extend from layer 6 cortical neurons and coordinate the response to the sensory information provided by the thalamus, and corticospinal axons extend from neurons in layers 5 and 6 of the cortex to regulate motor responses 4. Alcohol-induced alterations in neural development of these tracts may cause the sensorimotor defects evident in FASD.
Rodent models of FASD show differential effects on thalamocortical, corticothalamic, and corticospinal neurons and axons. Prenatal ethanol exposure results in reduced 5 or misrouted 6,7 6,7 thalamocortical axons with no effect on ventrobasal thalamic neuron generation or survival 7,8 7,8, suggesting effects at the level of axon guidance, fasciculation, or myelination. In terms of cortical development, prenatal ethanol exposure alters multiple aspects including neuron generation 9, differentiation 10, migration 9, axon arborization 7, and myelination 11, with the nature and severity of effects depending on cortical layer/region and developmental timing of exposure.
Studies in humans and rodent models show that prenatal binge ethanol exposure can be as deleterious as chronic exposure, but again outcomes vary depending on the nature of the exposure. Several studies suggest that ethanol impact is greatest when exposure coincides with the time of neuron generation 7,12,13 7,12,13 7,12,13. We are particularly interested in the effects of binge drinking during the first-trimester equivalent in rodents, because this is when the majority of forebrain neurons are being generated, migrating, and differentiating. Reports differ with regard to the outcome of brief prenatal ethanol exposure during this time period of cortical development 7,13–15 7,13–15 7,13–15 7,13–15 likely due to differences in exposure paradigm or animal model used. In this study, we hypothesized that prenatal binge ethanol exposure on embryonic days (E) 11–13 in Swiss Webster outbred mice would alter corticothalamic neuron generation, number, or laminar location without an accompanying reduction in the brain size. To identify corticothalamic neurons, we used T-box brain 1 (Tbr1) immunostaining. Tbr1 is a transcription factor expressed in Cajal–Retzius cells, subplate, and layer 6 corticothalamic neurons 4. To investigate corticothalamic neurogenesis, we carried out bromodeoxyuridine (BrdU) birthdating on E11.5 at the onset of corticothalamic neuron generation 16. On the basis of other reports, we did not expect our paradigm to affect ventrobasal thalamic neurogenesis, which are also generated during this time 7,17 7,17. The timing of our paradigm corresponds to weeks 7–9 of human pregnancy 18.
Pregnant Swiss Webster mice purchased from Charles River Laboratories were shipped to the mouse facility at Ursinus College on E2.5. Mice were individually housed in standard shoebox mouse cages. On E7.5 and E11.5, mice were weighed again to determine whether they had gained weight since mating. Mice that had gained 2 g or more by E7.5 and continued to gain by E11.5 were presumed pregnant and experimental procedure was begun. Food and water were freely available at all times. The mouse facility was maintained on a reverse 12-h light–dark cycle with lights out at 1:30 p.m. The temperature and humidity was maintained at 21±1°C and 60±5%, respectively. All procedures were approved by Ursinus College’s Institutional Animal Care and Use Committee (IACUC, permit #A4347-01) and are in accordance with NIH guidelines on the care and use of animals. All intraperitoneal injections were performed with proper animal handling techniques and every effort was made to minimize suffering.
Experimental paradigm, blood ethanol determination, and tissue preparation
Two groups containing six and seven pregnant mice were randomly assigned as either control or ethanol, respectively. On E11.5, E12.5, and E13.5 mice in the ethanol group were injected intraperitoneally with 2.9 g/kg ethanol (20%, v/v in sterile PBS, pH 7.3–7.5; #BP661-10; Fisher Scientific, Pittsburgh, Pennsylvania, USA). Two hours later, they received a second injection of 1.45 g/kg ethanol (10%, v/v diluted from 200 proof in sterile PBS) 19. Mice in the control group also received two injections on the same days, 2 h apart, of equivalent volumes of PBS. For BrdU birthdating, control and experimental mice were also injected with BrdU (#B5002; Sigma, St. Louis, Missouri, USA) at a final concentration of 50 mg/kg in the second injection on E11.5.
At birth (postnatal day zero, P0), pups were removed for analysis. Each P0 pup was weighed and examined for gross anatomical abnormalities. Pups were then euthanized by decapitation. Brains were removed, weighed, and fixed overnight in 4% paraformaldehyde (20% v/v in PBS; #157-SP; Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). Brains to be paraffin sectioned were shipped to AML Laboratories (Baltimore, Maryland, USA) for further processing. Paraffin sections were cut at 5 μm thickness.
A separate study was conducted to determine blood ethanol concentration (BEC). Two groups of Swiss Webster female mice purchased from Ace Animals, each containing six mice, were randomly assigned as either control or ethanol. On E12.5 and E13.5, 30 min after the second injection, a blood sample was collected from each mouse through submandibular bleed. The BEC of whole blood sample was determined using a kit according to the manufacturer’s instructions (#N7160; Sigma). None of the offspring from these dams were used for further analysis.
Immunohistochemistry and photomicrograph production
For immunohistochemistry, paraffin sections were dewaxed and subjected to antigen retrieval (#H-3300; Vector Laboratories Inc., Burlingame, California, USA). Sections immunostained with BrdU were treated with 4 M HCl (#320331; Sigma) then neutralized with 0.1 M boric acid (#A73-1; Fisher Scientific). Sections were blocked in PBS containing 2% normal goat serum (#005-000-121; Jackson Immunoresearch Laboratories Inc., West Grove, Pennsylvania, USA) and 0.1% Triton X-100 (#22145; Electron Microscopy Sciences) and incubated overnight with primary rat polyclonal antibody against BrdU (#OBT0030G, Axyll; Accurate Chemical & Scientific Corporation, Westbury, New York, USA) diluted 1 : 100 and rabbit polyclonal antibody against Tbr1 (#ab31940; AbCam, Cambridge, Massachusetts, USA) diluted 1 : 500. The next day, sections were incubated with Alexa Fluor 546 goat anti-rat (BrdU) and Alexa Fluor 488 goat anti-rabbit (Tbr1) secondaries diluted 1 : 200 (Molecular Probes, Grand Island, New York, USA). Sections were coverslipped with Fluoro-gel with Tris buffer (#17985-11; Electron Microscopy Sciences).
Images were collected using a Nikon Eclipse Ni confocal microscope equipped with a Nikon C2-SH digital camera, and Nikon C-HGFI Intensilight fluorescent light source (Nikon, Melville, NY, USA). NIS-Elements AR software (Nikon) was used to capture images.
Lamination and cell count analysis
Laminar position of BrdU+ and Tbr1+ neurons was analyzed as previously described 20. The number of BrdU+, Tbr1+, and Brdu+Tbr1+ neurons was counted using ImageJ (ImageJ, Bethesda, Maryland, USA). Briefly, the channels of each image were split into red and green, background of each color was subtracted with a 50 pixel rolling ball radius, and each color image was binarized. To determine the number of BrdU+Tbr1+ neurons, a composite red and green image was created using the image calculator. Immunolabeled nuclei sized 35–200 square pixels with 0.5–1.00 circularity were counted using Analyze Particles. The cortex was partitioned with a grid of 10 equally sized horizontal rows (hereafter called bins) from the pial surface to the ventricular surface. The number of immunolabeled nuclei in each bin was determined, the percent of total cells in each bin was calculated, and the average percent of total cells in each bin was calculated for each group (control and experimental). To obtain total cell counts of BrdU+, Tbr1+, and BrdU+Tbr1+ nuclei, the number of total cells within all 10 bins for each molecular marker in the image was calculated. To obtain cell counts of BrdU+ nuclei in the thalamus, the number of nuclei within a given area of ventrobasal thalamus was counted and the average cell count was calculated for each group.
Data are expressed as mean±SEM. For analysis of gestational and neonatal outcomes, a t-test between the experimental and the control group averages for all pups in the litter was performed using Excel (Microsoft Office, Redmond, Washington, USA). For cell counts, a t-test between the experimental and the control group litter averages for 12 pups and 11 pups, respectively, was performed using Excel.
Binge prenatal ethanol exposure from E11.5 to E13.5 does not affect pups at birth
First, we wanted to determine the effects of binge prenatal ethanol exposure from E11.5 to E13.5 on neonatal outcome. The maternal BEC of ethanol injected females was 294.8±15.4 mg/dl on E12.5 and 258.3±22.2 mg/dl on E13.5. No gross anatomic abnormalities were noted at birth in ethanol-exposed pups. We did not see increased gestational period (control: 19.33±0.21 days; ethanol: 19.14±0.14 days; P=0.46) with our exposure paradigm. At birth, there were also no differences in litter size (control: 10.5±0.81 pups; ethanol: 9.57±0.78 pups; P=0.43), pup weight (control: 1.65±0.05 g; ethanol: 1.62±0.04 g; P=0.65), brain weight (control: 0.09±0.002 g; ethanol: 0.09±0.003 g; P=0.39), or brain/body weight ratio (control: 0.054±0.002; ethanol 0.057±0.002; P=0.36).
Binge prenatal ethanol exposure from E11.5 to E13.5 does not alter E11.5 neurogenesis in the ventrobasal thalamus
The cell bodies of the neurons whose axons form the internal capsule are located in the ventrobasal (aka ventral posterior) nucleus of the thalamus (thalamocortical) and the deep layers of the cerebral cortex (corticothalamic and corticospinal). We used BrdU birthdating to investigate the effect of our ethanol prenatal exposure paradigm on the number thalamic neurons generated on E11.5, the earliest onset of generation for these neurons. The number of BrdU immunolabeled neurons did not differ between the two groups in the ventrobasal thalamus (Fig. 1).
Binge prenatal ethanol exposure from E11.5 to E13.5 does not change corticothalamic neuron genesis, number, or laminar location
To specifically investigate the effects of prenatal ethanol exposure on corticothalamic neuron fate, we coimmunolabeled P0 brain sections with antibodies against Tbr1 and BrdU. Similar to our results in the ventrobasal thalamus, we found no differences in the number or pattern of BrdU labeling between groups (Fig. 2). We also found no differences in the laminar location or number of Tbr1+ or BrdU+Tbr1+ cortical neurons between ethanol-exposed and control groups (Fig. 2). In both ethanol-exposed and control P0 pup cortices, the majority of Tbr1+-immunolabeled neurons were found in the deep layers (bins 5–7) as expected (Fig. 2d).
Expanding animal models used in fetal alcohol spectrum disorder research
In this study, we administered binge levels of ethanol during the first trimester equivalent in Swiss Webster mice. Swiss Webster mice are an outbred strain widely used for studies on ethanol exposure in adult mice, but the effects of developmental ethanol exposure using this strain are not well characterized. Most reports of prenatal ethanol exposure are conducted during the entirety of gestation and show deleterious effects on neonatal outcome exhibited as increased gestational period, or reductions in litter size, pup weight, or brain weight. Recent reports using shorter exposure times (single day 15 or 4 days 21) also show developmental delays 21 and reduced brain or body weights 15. In contrast, the paradigm we used did not result in any such changes. The primary differences between our study and previous reports are ethanol administration route, ethanol dose, and rodent strain.
Binge prenatal ethanol exposure from E11.5 to E13.5 doesn’t alter neuron generation in the ventrobasal thalamus or cortex
The timing of exposure occurred at the onset of generation for neurons extending axons to form the internal capsule. We saw no evidence that binge ethanol exposure affected neurogenesis in the ventrobasal thalamus (Fig. 1) or cortex (Fig. 2) as assessed by BrdU birthdating on E11.5. Our findings within the ventrobasal thalamus are similar to studies using different exposure paradigms in rats [administration from gestational day (G) 14 to 19 through oral gavage 7 and from G6 to birth through pair feeding 8]. In contrast, many groups suggest that ethanol exposure coincident with neurogenesis has a profound impact in the cortex 7,13 7,13, but to our knowledge there are only three other in-vivo reports that have examined this concept directly 9,14,22 9,14,22 9,14,22. Skorput and Yeh 22 showed increased BrdU immunolabeling in E12.5 mouse cortices with exposure starting at E9.5 and Miller 9 showed fewer tritiated thymidine labeled neurons in 3-month-old rat cortices ethanol exposed from G6 to birth; both of these studies used pair-feeding paradigms. In contrast, Kennedy and Elliott 14 found no difference in the number of mitotic figures in the E15 mouse cortices with exposure by oral gavage starting at E13. We believe the conflicting findings of these reports are due to differences in developmental timing of ethanol exposure. Similar to the study by Kennedy and Elliott 14, ethanol exposure in our study occurred at the onset of generation for deep layer neuron populations and we found no effect on the number or location of BrdU+ cells in the cortex. Thus, we speculate that exposure must occur prior to neurogenesis to alter cortical neuron progenitors. We did not label neurons born on E12.5 and E13.5 with BrdU, an acknowledged limitation of our study; hence, we do not know the full extent of ethanol exposure on all neurons generated during our exposure paradigm.
Binge prenatal ethanol exposure from E11.5 to E13.5 doesn’t change Tbr1+ neuron genesis, number, or laminar location
We believe that alcohol-induced alterations in internal capsule formation may cause the sensorimotor defects evident in human FASD. The internal capsule is formed by thalamocortical, corticospinal, and corticothalamic axons. Effects of ethanol exposure on thalamocortical and corticospinal neurons are described previously 17,23 17,23, therefore, our study focused on corticothalamic neurons. Corticothalamic and corticospinal neurons share generation time and laminar location. Prenatal ethanol exposure alters corticospinal neurons: the time when they are generated, their number, and lamination 23. Therefore, it is of interest that none of these parameters for corticothalamic neurons were affected by our paradigm. In contrast, Granato et al. 7 saw a significant reduction of layer 6 areal numeric density in sensorimotor cortex of rats prenatally exposed to ethanol from G14 to G19. In this study, layer 6 neurons were validated as corticothalamic neurons through retrograde labeling by wheat germ agglutinin-horseradish peroxidase injections in the thalamus. The offspring examined in the study came from alcohol-dependent dams suggesting that the vulnerability of corticothalamic neurons observed is the result of increased severity of ethanol exposure in combination with timing at the onset of generation for these neurons.
Two recent studies further demonstrate that there are differences in sensitivity of cortical neurons to ethanol exposure, with neurons generated early in cortical neurogenesis, namely progenitors and pioneers, being the most susceptible. Using a binge paradigm very similar to ours (daily intraperitoneal injections from E14 to E16 in pregnant CD1 outbred mice), Hashimoto-Torii et al. 13 found less Tbr2+ cortical neurons in offspring. This study also looked at gene expression of cortical layer markers and found no difference in Foxp2 expression, a marker of deep layer neurons including corticothalamic neurons, but decreases in Satb2, BHLHB5, and Foxp1. As these genes are involved in the genesis (Tbr2) and postmitotic identity (Satb2, BHLHB5, Foxp1) of upper layer neurons, these findings suggest that these processes are highly sensitive to ethanol exposure. Skorput and Yeh 22 recently investigated the effects of ethanol exposure starting on E9.5 on Cajal–Retzius cells using pregnant Ebf2-EGFP mice. Together with subplate neurons, the onset of neurogenesis for Cajal–Retzius cells just precedes corticothalamic neurons. All three cell populations express Tbr1 and should be labeled in our study. The conflict in our results with this study further supports our conclusion that developmentally early ethanol exposure is required to affect Tbr1+ neurons.
Our findings suggest corticothalamic neurogenesis is resistant to prenatal ethanol exposure. We cannot rule out the possibility that our paradigm affected development in other regions of the brain that we did not measure. Growing evidence from human cases 24 and animal studies 5–7,11,25 5–7,11,25 5–7,11,25 5–7,11,25 5–7,11,25 shows cortical and thalamocortical axons are particularly sensitive to ethanol. During development, misguided axons making aberrant connections within their target regions substantially impact behavior 6. Using a voluntary binge drinking exposure paradigm, we have seen sensorimotor neurobehavioral abnormalities characteristic of FASD in ethanol-exposed Swiss Webster mice prior to weaning (Favero, submitted). This further underscores the importance of expanding the rodent strains used to model FASD in order to identify the parameters that confer vulnerability or resistance, even within a given strain or brain region. Ongoing studies in our lab are investigating the impact of ethanol exposure during internal capsule formation. This work will enhance our understanding of ethanol-induced disruptions of sensorimotor function evident in FASD and the types of maternal ethanol abuse that are most detrimental to neural development.
The authors thank Brenna Louise Rasmussen, Jillian Bernice Alacci, and Kayla Elizabeth Waits for pilot data leading to the formation of the studies cited in this manuscript. They also thank Tania V. Hanna for her care, skill, and professionalism during mouse injections.
This study was supported by the Ursinus College Biology Department.
Conflicts of interest
There are no conflicts of interest.
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