Effects of Ethanol Exposure During Early Pregnancy ...

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hyper locomotive activity, attention deficit and impulsivity. EtOH group also ... Pitna Kim and Jin Hee Park are equally contributed to this work. P. Kim Á J. H. Park ..... were washed three times again and then mounted on coated slides, and slides ..... FT, Maes LI, Rosenberg M, Valenzuela CF, Savage DD (2010). Neurochem ...

Neurochem Res DOI 10.1007/s11064-012-0960-5

ORIGINAL PAPER

Effects of Ethanol Exposure During Early Pregnancy in Hyperactive, Inattentive and Impulsive Behaviors and MeCP2 Expression in Rodent Offspring Pitna Kim • Jin Hee Park • Chang Soon Choi • Inah Choi • So Hyun Joo Min Kyoung Kim • Soo Young Kim • Ki Chan Kim • Seung Hwa Park • Kyoung Ja Kwon • Jongmin Lee • Seol-Heui Han • Jong Hoon Ryu • Jae Hoon Cheong • Jung Yeol Han • Ki Narm Ko • Chan Young Shin



Received: 11 September 2012 / Revised: 22 November 2012 / Accepted: 19 December 2012 Ó Springer Science+Business Media New York 2013

Abstract Prenatal exposure to alcohol has consistently been associated with adverse effects on neurodevelopment, which is collectively called fetal alcohol spectrum disorder (FASD). Increasing evidence suggest that prenatal exposure to alcohol increases the risk of developing attention deficit/hyperactivity disorder-like behavior in human. In this study, we investigated the behavioral effects of prenatal exposure to EtOH in offspring mice and rats focusing on hyperactivity and impulsivity. We also examined

Pitna Kim and Jin Hee Park are equally contributed to this work. P. Kim  J. H. Park  C. S. Choi  I. Choi  S. H. Joo  M. K. Kim  S. Y. Kim  K. C. Kim  S. H. Park  K. J. Kwon  J. Lee  S.-H. Han  K. N. Ko  C. Y. Shin Department of Neuroscience, School of Medicine and Neuroscience Research Center, Institute SMART-IABS, Konkuk University, Seoul 143-701, Korea J. H. Ryu Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoeki-dong, Dongdaemoon-Gu, Seoul 130-701, Korea J. H. Cheong College of Pharmacy, Uimyong Research Institute for Neuroscience, Samyook University, Nowon-goo, Seoul 139-742, Korea J. Y. Han Korean Mother Risk Program, Department of Obstetrics and Gynecology, Cheil Hospital and Women’s Healthcare Center, Kwandong University College of Medicine, Seoul, Korea C. Y. Shin (&) Department of Pharmacology, School of Medicine, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 143-701, Korea e-mail: [email protected]

changes in dopamine transporter and MeCP2 expression, which may underlie as a key neurobiological and epigenetic determinant in FASD and hyperactive, inattentive and impulsive behaviors. Mouse or rat offspring born from dam exposed to alcohol during pregnancy (EtOH group) showed hyper locomotive activity, attention deficit and impulsivity. EtOH group also showed increased dopamine transporter and norepinephrine transporter level compared to control group in the prefrontal cortex and striatum. Prenatal exposure to EtOH also significantly decreased the expression of MeCP2 in both prefrontal cortex and striatum. These results suggest that prenatal exposure to EtOH induces hyperactive, inattentive and impulsive behaviors in rodent offspring that might be related to global epigenetic changes as well as aberration in catecholamine neurotransmitter transporter system. Keywords Fetal alcohol spectrum disorder (FASD)  Attention deficit/hyperactivity disorder (ADHD)  Neurotransmitter transporter  DNA methyltransferase 1 (DNMT1)  Methyl-CpG-binding protein 2(MeCP2)

Introduction Drinking alcohol during pregnancy has been implicated in a wide range of mental and physical developmental defects in human and animal offspring, which is collectively known as fetal alcohol spectrum disorder (FASD). In a study using an Ireland cohort of 61,241 women, 49,628 (81 %) was reported to experience some types of alcohol consumption during the peri-conceptional period [1]. Although most of the cases (43,455 women, 71 %) was in low intake group, 9.9 % was in moderate intake group and

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0.2 % was in high intake ([20 units per week) group [1], suggesting alcohol consumption during peri-pregnant periods is still common despite the continued warning of possible adverse health effects on offspring. Although initial researches were more focused on the severe aspects of FASD with characteristic facial malformation and severe mental retardation with structural and functional abnormalities such as reduction in volume of cerebellum and basal ganglia, callosal anomalies, hearing defects, which has been called fetal alcohol syndrome (FAS) [2–4], it is now recognized that a significant proportion of affected individuals only show minor form of neurological symptoms such as hyperactivity, inhibitory deficits and learning impairment [5] many of which is also appear in neurodevelopmental disorders such as attention deficit hyperactivity disorder (ADHD) [6], which costs more than $42.5 billion based on the prevalence rate of 5 % in a study reported in 2007 [7]. In a study using adult rats prenatally exposed to EtOH, behavioral analysis using choice reaction time (CRT) tasks revealed that affected rats show more variability in reaction time as well as high percentage of false alarm suggesting the role of prenatal alcohol exposure on inattentive and impulsive behavioral phenotype [8]. Similarly, altered social interactive behavior sometimes with aggression has been reported in rat offspring prenatally exposed to EtOH in sexually distinct manners [9, 10]. Initial studies hint prenatal exposure to EtOH may induce abnormalities in dopaminergic nervous system including the decrease in the number of dopaminergic neuron, dopamine contents and reuptake as well as the concentration of dopamine metabolites such as homovanillic acid and 3,4-dihydroxyphenylacetic acid [11–13] In addition, changes in the size of dopaminergic neuronal cell body and dendrite [14] as well as changes in spontaneous electrical activity of dopaminergic neuron have been reported [15]. Dopaminergic abnormality is believed at the core of pathologic aberrations seen in hyperactive and inattentive patients and stimulant or non-stimulant drugs affecting catecholaminergic nervous system effectively reverse the altered behavioral phenotypes. These results suggest that prenatal EtOH exposure may induce hyperactive, inattentive and impulsive behavior in offspring by inducing developmental changes in catecholaminergic nervous systems. However, the exact etiological factors as well as the mechanism of pathophysiogy affected by them are not completely understood yet. Just as in the case of many other environmental toxicants, exposure to EtOH may induce epigenetic changes such as altered DNA methylation and expression of DNA methyltransferase in alcoholics or alcohol-related liver disease patients as well as in developing embryos in vivo

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and in vitro [16–23]. The importance of epigenetic mechanism in understanding neuropsychiatric disorders such as schizophrenia, autism and ADHD has been reviewed by several groups [24–26]. These results suggest that epigenetic changes induced by alterations in key regulators for DNA methylation in prenatally EtOH exposed offspring may contribute to the manifestation of behavioral abnormalities in FASD. In this study, we investigated the behavioral effects of prenatal exposure to EtOH in offspring mice and rats focusing on hyperactivity and impulsivity. We also investigated the changes in the expression level of dopamine transporter and epigenetic factors such as DNMT1 and MeCP2 in the brain of mice offspring that has been prenatally exposed to EtOH.

Materials and Methods Maternal Alcohol Treatments The female ICR mice (28–30 g weight, typically 8 weeks of age) and Sprague–Dawley rats (SD, 250–300 g weight, typically 8 weeks old) used in the study were obtained from DBL Animal CO. (Seoul, Korea) at gestation day (GD) 2 and stabilized under environment-controlled rearing system, maintained in standard light–dark cycle (light turns on 08:00) at ambient temperature (22.2 °C) and humidity (55 ± 5 %) with free access to chow pellets and water. Stabilized animals were divided into six groups, and five groups were treated with EtOH (Hayman, UK; 0.5, 1, 2, 4 and 6 g/kg/day; 25 v/v%) diluted with normal saline from GD 6 to GD 15 via intragastric intubation. Control groups were treated with saline. Pregnant SD rats were similarly treated with 4 and 6 g/ kg/day EtOH. Treatment was divided into two equal doses 6 h apart, at 10:00 and 16:00. In each group, eight pregnant animals were treated with EtOH and the number of litters was adjusted to 10 for each dam. The day after parturition was regarded postnatal day (PD) 1. The offspring was weaned on PD 21. All efforts were made to minimize the number of animals used in this study as well as their suffering. All tests took place between 9:00 and 18:00 in dedicated test room. The growth profile of all the offspring were checked until weaning. The experimental groups, consisting of 8–10 animals in each group were chosen by means of a randomized schedule. Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and were approved by the Animal Care and Use Committee of Konkuk University, Korea.

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Behavioral Testing Procedure Open-Field Locomotor Activity The locomotive activity of prenatally EtOH-treated animals was assessed by open-field test. The equipment was located in animal room allowing the experimenter to observe the animals through a computer outside the room. The observation apparatus consisted of five plastic boxes (42 9 42 cm) with the field bordered by 42 cm high sidewalls. Animal were placed into test boxes 5 min before recording for habituation. The total distance moved and the duration of movement was monitored for 20 min [27, 28] and the behavior changes of animals were monitored automatically using computerized motion tracking apparatus and software equipped with CCD camera (EthoVision 3.1, Noldus information Technology, the Netherlands). Y-Maze Test Spontaneous alteration behavior requires attention (Katz, Schmaltz [29]) and working memory in a Y-maze was assessed by the methods of McGaughy and Sarter [30]. Y-maze test experiment was conducted on PD 28. The standard Y-maze apparatus was made of Plexiglas and the size of each arm was 3 cm wide, 42 cm long and 12 cm high, and both arms were positioned at equal angles (120°). Each animal was placed at the end of an arm and allowed to move freely for 8 min test session without reinforce such as food or electric shock. An arm entry was defined as the entry of all four paws and the tail into one arm. The sequence of arm entries was recorded with a computerized Noldus EthoVision system (EthoVision 3.1, Noldus information Technology, the Netherlands). The alternation behavior (actual alternation) was defined as the consecutive entry into three arms, i.e. the combination of three different arms, with stepwise combination in the sequence. We quantified % of spontaneous alternation for attentive ability and total arm entries. Spontaneous alternation was calculated by the following equation: Spontaneous alternation ¼ ðactual alternations= total arm entries  2 Þ  100 The Electro-Foot Shock Aversive Water Drinking Test (EFSDT) The test procedures were performed for mice as we previously reported with slight medication [31]. The EFSDT consisted of two phases; the training phase which lasted for 2 days and a testing phase which lasted for a day. Prior to training, animals were individually housed and deprived of water for at least 18 h. This is a moderate, but sufficient

deprivation for motivating the animal to drink water. During this phase, whenever a mouse licked water from the bottle for at least 5 s, an experimenter removed it from the water area and placed it back to the start area. Training lasted for 10 min, for two consecutive days. For test phase, animals were deprived of water overnight before testing. In addition, they were orally administered with 8 % NaCl solution (1 g/kg) 30 min before tests for further motivation to drink water. The procedure of the test phase is similar to those of the training phase. This time, however, an experimenter presented an electroshock punishment (2 mA, 0.5 s) whenever an animal has licked from the water bottle for at least 5 s. The number of drinking resulted in electroshocks (i.e. 5 s licking of the water bottle) was noted as the number of impulsive drinking and such data was used to demonstrate impulsivity in animals (i.e. persevering with drinking despite punishment). In addition, frequency of the animals in the water (shock) area the EFSDT box was also recorded via automated systems (Noldus Ethovision). Tissue Preparation For Western-blot analysis, 5 weeks old animals were decapitated and forebrain samples, including the striatum, hippocampus and frontal cortex were taken swiftly. These samples were then frozen rapidly using liquid nitrogen and stored at -70 °C until the experiments. For immunohistochemical analysis, 5 weeks old animals were fixed with 4 % paraformaldehyde solution (PFA; dissolved in PBS, Sigma St Louis, MO, USA) kept in ice. Fixed brain was put in cold PFA solution and 1 day later, brain was transferred to 30 % sucrose solution (dissolved in phosphate buffered saline) for a few days in order to dehydrate. Dehydrated tissues were cut into 40 lm thick coronal sections in a cryostat through the striatum, basal forebrain and stored in stock solution which contained glycerol, ethylene glycol and phosphate buffer at -20 °C. Western-Blot Analysis Brains samples were briefly homogenized in 3 volumes of ice-cold homogenization buffer (0.32 M sucrose, 4 mM Tris pH 7.4, protease inhibitors, phosphatase inhibitors). Protein concentrations were measured by the BCA assay. The relative amount of b-actin and Histone H3 were used as a loading control. Synaptosomal and Nuclear fractions were prepared as previously described (Blackstone et al. [32]. Equal amount of protein from homogenized samples was electrophoresed on 10 % SDS–polyacrylamide gel for 120 min and the proteins were electrically transferred to the nitrocellulose membranes (Whatman, Germany) for 90 min. The blot was blocked with 5 % nonfat dried milk

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at room temperature and incubated overnight at 4 °C with each antibody, which were diluted at 1:1000 in 5 % non-fat dried milk. After the membranes were incubated again with horseradish peroxidase (HRP)-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) at room temperature for 2 h, positive bands were detected with enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) and exposed to LAS-3000 image detection system (Fuji, Tokyo, Japan). Immunohistochemical Analysis Immunohistochemical analysis was conducted using freefloating method. Stored sections were dipped in 1.5 % normal horse serum in PBS with 0.1 % Triton X-100 solution (SigmaAldrich) and incubated overnight at 4 °C with anti-DAT (diluted 1:100), anti-norepinephrine transporter (NET, 1:100) (Santa Cruz Biotechnology Inc.), anti-DNMT1 (1:100) and anti-MeCP2 (1:500) (Cell Signaling Technology) antibodies. After incubation, sections were washed three times for 5 min with PBS, followed by incubation for 1 h with Alexa 594-labeled anti-rabbit (NET and MeCP2) and anti-rat (DAT) secondary antibody (Invitrogen). In case of DNMT1, HRPcoupled secondary antibody was used and the sections were color developed with 3,30 -Diaminobenzidine (DAB) as described previously (Dhawan et al. [33]. Stained sections were washed three times again and then mounted on coated slides, and slides were investigated using Olympus BX61 fluorescence microscope system (Olympus, Tokyo, Japan).

Data Analysis Data were expressed as the mean ± standard error of mean (SEM) and analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Dunnett’s test as a post hoc test. When appropriate, two-way ANOVA was used to identify treatment effects (dosage of prenatal ethanol exposure)

Fig. 1 a The body weight of ICR mice pups prenatally exposed to EtOH during lactation periods (n = 52). b The body weight of 3 weeks old SD rat offspring prenatally exposed to EtOH (n = 29). Asterisk represents significant difference as compared with control (Con) (*p \ 0.05, **p \ 0.01)

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or age effects, or interaction between the two factors. If significant effects were found in any one of the factors, post hoc comparisons were conducted using Bonferroni’s post-tests. Differences were considered statistically significant when the p value was \0.05 (p \ 0.05). All statistical analyses were conducted using Graph Pad Prism (Version 5, CA, USA).

Results Postnatal Growth In this study, we treated pregnant mice with EtOH between E6 and E15. All of them successfully delivered their offspring and no change in the number of live birth was observed (data not shown). To check whether EtOH exposure during pregnancy induces gross developmental problems to offspring, postnatal growth retardation was evaluated until weaning day (PD 21). No difference in body weight until weaning was observed between EtOH and control group (Fig. 1a). Interestingly, albeit small but a significant increase in body weight was observed in 3 weeks old, prenatally EtOH-exposed rat offspring (Fig. 1b).

Effects of Prenatal Exposure to EtOH on Hyperactivity of Offspring Exposure to EtOH during early pregnancy resulted in hyperactivity phenotype in mice offspring when they were investigated by open-field test at 3, 4 and 5 weeks of age (Fig. 2a, b). Specifically, two-way ANOVA revealed that moved distance in open-field was increased by EtOH dosage [F(5,216) = 12.57, p \ 0.0001], and decreased by age [F(2,216) = 34.45, p \ 0.0001]. Post-hoc comparisons showed that 6 g/kg/day EtOH-treated group in 3 weeks (p \ 0.05), 4 weeks (p \ 0.001), and 5 weeks of age (p \ 0.001) moved more in open-field than control group. There is no significant interaction between EtOH dosage 9 age [F(10,216) = 1.451, p = 0.1596]. Similarly, the time spent for moving in open-field

Neurochem Res Fig. 2 The effects of prenatal exposure to EtOH on locomotor activity of rodent offspring (mice: a, b. rats: c, d). The distance moved and movement duration was determined as described in materials and methods. Distance moved (a, c) and movement duration (b, d) was significantly increased in ICR mice (a, b) as well as in SD rat (c, d) by prenatal exposure to EtOH through pregnant dam. The data represent mean ± SEM. Asterisk represents significant difference as compared with control (*p \ 0.05, **p \ 0.01, n = 13 for mouse, n = 29 for rat)

Fig. 3 The effects of prenatal exposure of different concentrations of EtOH on Y-maze performance of 4 weeks old F1 pups of rodent. Total entry (a) was not different but the spontaneous alternation behavior (SAB) (b) was significantly and dosedependently decreased in EtOH group in ICR mouse offspring compared to the control group. Similar results were observed in 4 weeks old SD rats (c, d). The data represent mean ± SEM. Asterisk represents significant difference as compared with control (*p \ 0.05, **p \ 0.01, n = 12 for mouse, n = 29 for rat)

was also increased by EtOH dosage [F(5,238) = 7.652, p \ 0.0001] and decreased by age [F(2,238) = 24.74, p \ 0.0001]. Post-hoc comparisons showed that 6 g/kg/day EtOH-treated group in 4 weeks age moved longer in open-

field than control group (p \ 0.001). There is no significant interaction between EtOH dosage 9 age [F(10,238) = 1.380, p = 0.1902]. Similar hyperactivity was observed in prenatally EtOH-treated rat offspring (Fig. 2c, d).

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Neurochem Res Fig. 4 The effects of prenatal exposure to EtOH on impulsivity phenotype of 5 weeks old rodent offspring. Mouse (a, b) or Rat (c, d) offspring was trained and subjected to electro-foot shock aversive water drinking test (EFSDT) as described. Two parameters were determined as indices of impulsivity that is the frequency entering water area (a, c) and the number of impulsive drinking (b, d), which lasts for 5 s resulting in electroshock punishment during EFSDT. The data represent mean ± SEM. Asterisk represents significant difference as compared with control (*p \ 0.05, **p \ 0.01, ***p \ 0.001, n = 12 for mouse and n = 20 for rat)

Effects of Prenatal Exposure to EtOH on Attention-Related Behaviors in Offspring Spontaneous alternation behavior (SAB) in Y-maze test reflects not only spatial memory but also attention [34]. We measured SAB in mice offspring using Y maze test at PD 28 to determine whether EtOH group has the propensity of inattention. In this study, EtOH group exhibited significantly decreased SAB (Fig. 3b), and slightly increased explorative tendency as indicated by total arm entries (Fig. 3a) compared with control group. Similar decrease in SAB along with increased total entry was observed in Y-maze test of prenatally EtOH-exposed rat offspring (Fig. 3c, d). Effects of Prenatal Exposure to EtOH on Impulsivity in Offspring To measure the effects of EtOH on impulsivity behavior in mice offspring, we performed the EFSDT, as reported previously (see ‘‘Materials and Methods’’). Impulsivity is related to risk-taking, lack of planning, and making up one’s mind quickly [35]. In EFSDT, impulsivity is measured by the persistence of the animal’s action to obtain a biological reward (water) despite the presentation of an aversive consequence (electroshock). In this test, two measures were used to demonstrate impulsivity in the animal: (1) the number of impulsive attempt

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(the frequency entering water area of the EFSDT box), (2) the number of impulsive action (5 s water drinking resulted in electroshock punishment). Tendency to get a reward (fresh water for thirsty animals) in spite of a foreseeable punishment (electric foot shock) was measured as an indicator of impulsive behavior. As shown in Fig. 4, EtOH group displayed more impulsive tendency in a dose dependant manner. EtOH group entered the water area more frequently and showed higher impulsive drinking frequency than control group. The number of impulsive attempt (water area frequency) was significantly increased at the highest dose of EtOH (6 g/kg/ day, Fig. 4a). The number of impulsive action (water drinking frequency) in EtOH group was increased in the mid and high dose range in this study with statistically significant difference in 4 and 6 g/kg/day EtOH group in mice offspring (Fig. 4b). Similar impulsive behavior was also observed in rat offspring prenatally exposed to 4 or 6 g/kg/day EtOH (Fig. 4c, d). Changes of Neurotransmitter Transporter Expression in Mice Offspring Prenatally Exposed to EtOH We next examined the effects of prenatal exposure of EtOH on the protein expression of dopamine transporter (DAT) and norepinephrine transporter (NET) in prefrontal cortex and striatum by Western blot and immunohistochemistry. In Western blot analysis, increased expression of DAT and NET protein was observed in EtOH group. Densitometric quantification of Western blot revealed a significant increase in DAT

Neurochem Res Fig. 5 Effects of prenatal exposure to EtOH on the protein expression of dopamine transporter (DAT) and norepinephrine transporter (NET). Pregnant mice were orally treated with 0.5, 1, 2, 4 and 6 g/kg EtOH from E6 to E15. At 5 week after birth, offspring mice were sacrificed and perfused with saline. Brain tissues (prefrontal cortex and striatum) were collected and homogenized for the determination of DAT and NET protein expression by Western blot. The graph is the densitometric quantification data (a). The data represent mean ± SEM. Asterisk represents significant difference as compared with control (*p \ 0.05, n = 4–6). b For Immunohistochemistry, animals were perfused with 4 % paraformaldehyde and coronal brain sections were stained with DAT and NET antibody. Stained tissues were visualized by fluorescence microscopy. Bar size = 50 lm

and NET expression in frontal cortex and striatum at highest dose of EtOH (6 g/kg/day) (Fig. 5). In immunohistochemistry, prenatal EtOH exposure increased DAT and NET expression in frontal cortex as well as in striatum (Fig. 5). Changes of DNMT1 and MeCP2 Expression in Mice Offspring Prenatally Exposed to EtOH Exposure to EtOH during development may induce epigenetic changes such as one-carbon methylation metabolism, acetylation and methylation of DNA or histone. In this study, we investigated the effects of prenatal exposure

to EtOH on DNA methyltransferase 1 (DNMT1) and Methyl-CpG-binding protein 2 (MeCP2) expressions in mice brain, which are two important players of methylation-induced regulation of gene expression. The highest dose of EtOH used in this study (6 g/kg/day), significantly decreased the expression of MeCP2 in both cerebral cortex and striatum as determined by Western blot (Fig. 6). No changes in DNMT1 expression was observed in this study. Consistent with Western blot experiment, decreased expression of MeCP2 in frontal cortex and striatum was also observed in immunohistochemistry experiments (Fig. 6).

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Neurochem Res Fig. 6 Effects of prenatal exposure to EtOH on the protein expression of DNMT1 and MeCP2 in prefrontal cortex and striatum of ICR mice offspring. a The Western blot was performed with prefrontal and striatum homogenates obtained from 5 weeks old mice offspring as described above. The graph is the Western blot densitometric quantification data. The data represent mean ± SEM. Asterisk represents significant difference as compared with control (*p \ 0.05, **p \ 0.01, n = 4–6). b For immunohistochemistry, ICR mice were perfused with 4 % paraformaldehyde. Coronal brain sections were stained with DNMT1 and MeCP2 antibody. Tissue sections were visualized by diaminobenzidine for DNMT1 and by fluorescence for MeCP2. Bar size = 50 lm

Discussion In this study, 0.5–6 g/kg/day EtOH was treated to pregnant animals during E6-E15 and high concentration of EtOH induced hyperactivity, inattention, deficits in spatial learning memory and impulsive behavior in offspring, without major reproductive toxicity or gross developmental defects. The results suggested that prenatal EtOH exposure during early pregnancy affects the expression of catecholamine neurotransmitter transporters, which might modulate the function of catecholaminergic nervous system as well as behavioral phenotypes. The results from the present study supports the growing concerns about the association of fetal exposure to EtOH and manifestation of ADHD-like

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behavioral and neurodevelopmental abnormalities [36, 37]. In the analysis of charts of 2,231 youth referred for FASD, ADHD is the highest prevalence (41 %) disorders followed by learning disability, which is correlated with the general rank of risk tendency [36]. In a well designed twin study, it is also suggested that alcohol use disorder is not only a risk factor for ADHD but also may share heritable genetic components with ADHD [37]. Adopting the dose-conversion factor based on the differences in body surface area among different species (6:1 for rat vs human and 12:1 for mouse vs human), it is calculated that the highest dose used in mice and rat experiments corresponds to 30 and 60 g of EtOH per day for 60 kg human. This dose range is lower than so called binge

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drinking, suggesting the dose used in this study is in the physiologically feasible range, although species difference should be considered in the interpretation of the data. The dose conversion factor among different species also partly explains seemingly stronger effects of prenatal EtOH exposure on behavioral changes in rat compared to mice, although other factors such as generally higher locomotive activity in mice should be considered as well. In this study, we treated the pregnant mice or rats with EtOH during E6-E15, which is the period for neural tube closure and neural differentiation. In mice, initiation of neural tube closure occurs at E8 [38], and the neuropores eventually close and form an intact tube at E9.5 [39]. Primary neurulation ends at E10, when spinal neurulation is completed by closure of the posterior neuropore [39]. At E10 in mice, most of the pre-migratory neural crest are composed of stem cells [40], and the onset of neocortical neurogenesis occurs at E11 [41, 42]. The specific critical time window and minimum duration of alcohol exposure, which may induce altered behavioral phenotype in offspring animals should be experimentally determined in the future. In case of valproic acid-induced autism-like behavior in rats, we reported that a single injection of valproic acid at E12, but neither earlier nor later than that particular periods, can induce behavioral alterations in rats [28]. Epidemiological studies have suggested that alcohol use problem of parents might be related to the hyperactivity or inattention symptoms [43–46]. A recent study conducted using an Australian twin cohort suggested that descendants from sibling of twin who has alcohol overuse disorder showed higher prevalence of ADHD [37]. These reports suggest that ADHD and alcohol abuse or maybe FASD may share common heritable etiological determinants, especially during neuronal development and parental alcohol misuse during fetal and perinatal periods may induce ADHD-like phenotypes such as hyperactivity, inattention and impulsivity in offspring. Although, dozens of genes has been implicated in ADHD and no single gene can represent majority of ADHD cases in human, the dopamine transporter (DAT) is one of the principal and most widely studied candidates [47]. Besides, DAT along with NET is the major target of currently available anti-ADHD medication. DAT is expressed in dopamine neurons in substantia nigra and ventral tegmental area as well as in projections to the striatum, nucleus accumbens, prefrontal cortex, and hypothalamus [47]. Various forms of mutations in human DAT gene has been implicated in ADHD, and some studies have reported increased levels of the DAT in striatum of ADHD subjects, although discrepancy exists among different studies that used different experimental conditions [48–50]. At present, it is hard to conclude whether the observed

changes in DAT expression in human brain is a cause or a consequence of hyperactive, inattentive and impulsive phenotype. In animal experiments, DAT knock-out mice showed hyperactivity and inattention and DAT overexpressing mice showed modest hypoactivity [51], again make it difficult to draw a unifying picture by which alteration in DAT expression affects manifestation of ADHD-like behavioral phenotypes in human. Microdialysis studies conducted in hyperactive rodent brain suggested that perturbation of brain region-specific pattern of catecholamine efflux may underlie ADHD like behavior such as hyperactivity [52, 53]. These results suggest that aberrant expression of DAT or NET may govern the functional activity of catecholaminergic nervous system in brain regions such as prefrontal cortex, striatum and nucleus accumbens, which results in abnormal behaviors. In addition, it should be remembered that the expression level and function of DAT is dynamically regulated by multiple intracellular and extracellular signaling pathways and several types of protein–protein interactions. Moreover, functional DAT expression is regulated through the removal (internalization) and recycling of the protein from the cell surface [54]. Therefore, not only the level of DAT but also the functional status of DAT in neuronal membrane and synapse may ultimately govern the behavioral outcome, which should be investigated further in the future. It is also possible that developmental alteration in DAT level in either way may affect proper function of dopaminergic nervous system [47] and balance with other nervous systems such as NE or 5-HT nervous system might be important to the development of ADHD-like behaviors. At present, the mechanism by which prenatal exposure to EtOH affects DAT expression in adolescent mice brain is not clear. Although, the developmental modulation of dopaminergic cell number during embryonic dopaminergic differentiation is still a possible scenario, no difference in TH level in substantia nigra was observed in this study (data not shown) suggesting that there are no changes in overall number of dopaminergic neuron in adolescent mice brain prenatally exposed to EtOH. Interestingly, long-term treatment of female SD rats with EtOH before and during pregnancy modestly reduced DAT mRNA level in substantia nigra without affecting TH mRNA, which is specific to dopaminergic neurons [55] suggesting selective regulatory mechanism specific for DAT might exist in developing brain. Similar long-term EtOH treatment during peripregnancy and lactation periods induced a significant decrease in DAT binding without a significant alteration of D1 and D2 receptor binding in the striatum of 2 months old rats [56]. In contrast, it has been suggested that activity and cell surface expression of functional DAT is increased by acute EtOH treatment both in neuronal and non-neuronal cells [57–59].

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Compared to DAT, less is known for the role of NET in hyperactive and inattentive behavior, albeit it is the principle target of atomoxetine, a non-stimulant type ADHD medication. Although not much information is available for the effects of prenatal exposure to EtOH on NET expression, prolonged stress has been linked to the methylation of NET promoter, possibly affecting the expression of NET in essential hypertension [60]. Regulation of NET activity and expression by altered NET promoter methylation was also reported in cardiac tissue obtained from panic disorder patients [60, 61]. These results suggest that NET is another neural substrates regulated by epigenetic changes, probably by prenatal stress induced by many toxicants including EtOH. The role of epigenetic changes in EtOH-induced toxicity has been increasingly evident during last decade [16–18]. EtOH may affect one-carbon methylation metabolism, acetylation and methylation status of DNA or histone, which may affect the expression of specific or subsets of genes during development. EtOH also may affect individual participating enzymes and proteins for epigenetic regulation of gene expression. In an experiment using whole embryo culture, it has been suggested that binge-type alcohol exposure delayed the cellular DNA methylation pattern and changed MBD1 expression and also retarded embryonic growth [19]. Hypermethylation of specific genes encoding alpha-synuclein and HERP was reported [20, 21], and changes in DNA methyltransferase expression has been suggested to be related with altered DNA methylation in alcohol abuse patients [22]. MeCP2 is a methyl binding protein and a single gene cause of Rett syndrome [23], which is categorized as one of the Autism spectrum disorder. MeCP2 may play an important role during mammalian neuronal development by regulating dendritic and axonal growth [62] and acts as both repressor and activator of gene expression [63]. Although it has been suggested that the expression of MeCP2 is regulated by translational control involving microRNAs, little is known for the factors and pathways regulating the expression level of MeCP2 [63]. Although clear etiological links between MeCP2 and ADHD-like behaviors are not reported yet, a significant reduction in MeCP2 expression compared to age-matched controls was found in two ADHD patients, which might suggest the reduced level of MeCP2 might also contribute to other neurodevelopmental or psychiatric disorders including ADHD [64]. In a recent microarray experiments using SHR and PCB-exposed mice as model systems, decreased MeCP2 and DNMT3a expression was observed in both animal models of hyperactivity and inattention [65]. In our study, prenatal EtOH exposure induced down-regulation of MeCP2 in mice offspring. Taken together, these results may suggest the possible link of MeCP2 in FASD and

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ADHD-like behavioral phenotype. The regulation of target gene expression by MeCP2 is modulated by gene specific manner. Therefore, investigating the effects of altered expression of MeCP2 on the regulation of DAT and NET expression may provide more direct link to the EtOHinduced epigenetic modulation to the well known determinants of neurobehavioral regulators.

Conclusion We reported here that prenatal exposure to EtOH in physiologically relevant concentration induced hyperactive, inattentive and impulsive behavioral phenotypes in mouse and rat offspring with increased expression of DAT and decreased expression of MeCP2 in prefrontal cortex and striatum. The pathological role of epigenetic changes induced by EtOH, especially focusing on the modulation of the expression of catecholamine transporter system, should be given more attention as a possible link between FASD and ADHD-like behavioral phenotypes. Acknowledgments This research was supported by research grants (09162KFDA566, 11182KFDA556) from the Korea Food & Drug Administration in 2009 and 2011.

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