Proceedings of the 2008 annual meeting of the Fetal Alcohol Spectrum Disorders Study Group

The annual meeting of the Fetal Alcohol Spectrum Disorders Study Group (FASDSG) was held on June 28, 2008 in Washington DC, as a satellite to the Research Society on Alcoholism meeting. The FASDSG membership includes clinical, basic, and social scientists who meet to discuss recent advances and issues in FASD research. The main theme of the meeting was “Factors that Influence Brain and Behavioral Development: Implications for Prevention and Intervention.” Two keynote speakers, Dr. Stephen Suomi and Dr. Carl Keen addressed how early environment and nutrition may influence outcome after prenatal alcohol exposure. The final keynote speaker, Kathy Mitchell, addressed issues regarding the relationship between scientists and the families with children with FASD. Members of the FASDSG provided updates on new findings through brief (FASt) data reports and national agency representatives provided updates of activities and funding priorities. Presentations were also made by recipients of the Student Research Merit award and Rosett award.     

Acute ethanol administration affects zebrafish preference for a biologically inspired robot

Preclinical animal models constitute a cornerstone against which the reward processes involved in drug addiction are often studied and dissected. While rodents have traditionally represented the species of choice, a growing body of literature indicates that zebrafish are emerging as a valuable model organism. Specifically, several studies demonstrate that the effects of ethanol at the level of emotional- and cognitive-related domains can be reliably investigated using zebrafish. The rapidly evolving nature of these efforts allows substantial room for the development of novel experimental paradigms suited to this freshwater species. The field of ethorobotics may prove particularly beneficial, due to its ability to convey fully controllable and easily reproducible experimental tools. In this study, we addressed the possibility of using a biologically inspired robot to investigate the emotionally related properties of ethanol in a preference task in zebrafish. To this aim, we evaluated wild-type zebrafish preference toward a robotic stimulus and addressed whether ethanol administration (0.25% and 1.00% ethanol/water concentration) may alter such preferences. In accordance with our previous studies, we observed that zebrafish exhibit a natural attraction toward the robot. Additionally, in agreement with our predictions, we showed that ethanol administration abolishes such preferences. This work is the first to demonstrate that robotic stimuli can be used in zebrafish to investigate the reward-related properties of alcohol.     

Blood acetaldehyde response to ethanol ingestion during the reproductive cycle of the female rat

Acetaldehyde could mediate a number of the toxic effects of alcohol both in females and their offspring. Thus, we assessed the blood acetaldehyde response to ethanol (3 g/kg) at various stages of the female reproductive cycle. Blood levels were low throughout the various phases of the estrous cycle and during most of pregnancy. By contrast, a 4-fold rise in maternal blood acetaldehyde occurred at the end of pregnancy (day 20), continued to increase during lactation (17-fold at day 14) and returned to non-pregnant values after weaning or after pup removal at birth. Both enhanced rate of ethanol oxidation and decreased activity of the low Km aldehyde dehydrogenase in liver mitochondria contributed to the increased acetaldehyde levels. Acetaldehyde was detectable in fetal blood, but only a small fraction of the high maternal values in pregnancy reached the fetus through the umbilical vein. Chronic alcohol administration resulted in decreased fetal size and striking enlargement of the placenta with possible implications for abnormal fetal development. Thus, the high maternal acetaldehyde levels at the end of pregnancy may exert deleterious effects on many maternal organs, including those (such as placenta) which are required for normal fetal development.     

Deficits in trace fear conditioning in a rat model of fetal alcohol exposure: dose–response and timing effects

In humans, prenatal alcohol exposure can result in significant impairments in several types of learning and memory, including declarative and spatial memory. Animal models have been useful for confirming that many of the observed effects are the result of alcohol exposure, and not secondary to poor maternal nutrition or adverse home environments. Wagner and Hunt (2006) reported that rats exposed to ethanol during the neonatal period (postnatal days [PDs] 4–9) exhibited impaired trace fear conditioning when trained as adolescents, but were unaffected in delay fear conditioning. The present series of three experiments represent a more detailed analysis of ethanol-induced deficits in trace conditioning. In Experiment 1, the dose of ethanol given to neonates was varied (3.0, 4.0, or 5.0 g/kg/day). There was a dose-dependent reduction in trace conditioning, with the poorest performance observed in animals treated with the highest dose. In Experiment 2, it was found that the impairment in trace conditioning resulting from neonatal ethanol exposure was dependent on the duration of the trace interval used for training; less learning was evident in ethanol-exposed animals trained with longer trace interval durations. These results confirm other reports of delay-dependent memory deficits. Finally, Experiment 3 determined that ethanol exposure limited to the first half of the neonatal period (PDs 4–6) was more detrimental to later trace conditioning than exposure during the second half (PDs 7–9). These results support the hypothesis that trace-conditioning impairments resulting from early ethanol exposure are due to the drug's teratogenic effects on the developing hippocampus, as the findings parallel those observed in animals with discrete hippocampal lesions. Comparisons between delay and trace fear-conditioning performance in animals exposed to ethanol during the brain growth spurt provide a model system to study both selective learning impairments and possible treatment approaches for humans with fetal alcohol spectrum disorders.     

The role of cortisol in chronic binge alcohol-induced cerebellar injury: Ovine model

Women who drink alcohol during pregnancy are at high risk of giving birth to children with neurodevelopmental disorders. Previous reports from our laboratory have shown that third trimester equivalent binge alcohol exposure at a dose of 1.75 g/kg/day results in significant fetal cerebellar Purkinje cell loss in fetal sheep and that both maternal and fetal adrenocorticotropin (ACTH) and cortisol levels are elevated in response to alcohol treatment. In this study, we hypothesized that repeated elevations in cortisol from chronic binge alcohol are responsible at least in part for fetal neuronal deficits. Animals were divided into four treatment groups: normal control, pair-fed saline control, alcohol and cortisol. The magnitude of elevation in cortisol in response to alcohol was mimicked in the cortisol group by infusing pregnant ewes with hydrocortisone for 6 h on each day of the experiment, and administering saline during the first hour in lieu of alcohol. The experiment was conducted on three consecutive days followed by four days without treatment beginning on gestational day (GD) 109 until GD 132. Peak maternal blood alcohol concentration in the alcohol group was 239 ± 7 mg/dl. The fetal brains were collected and processed for stereological cell counting on GD 133. The estimated total number of fetal cerebellar Purkinje cells, the reference volume and the Purkinje cell density were not altered in response to glucocorticoid infusion in the absence of alcohol. These results suggest that glucocorticoids independently during the third trimester equivalent may not produce fetal cerebellar Purkinje cell loss. However, the elevations in cortisol along with other changes induced by alcohol could together lead to brain injury seen in the fetal alcohol spectrum disorders.     

Vulnerability of macaque cranial nerve neurons to ethanol is time- and site-dependent

The present study tested the hypotheses that vulnerability to ethanol depends upon (1) population-based characteristics of the neuronal progenitors and (2) the maturation of that population by examining the effects of prenatal exposure to ethanol on brainstem nuclei derived from different rhombomeres and from the alar and basal plates. Macaca nemestrina received an ethanol-containing solution 1 day per week during the first 6 (Et6) or 24 (Et24) weeks of gestation. Control animals received an equivalent volume of saline. The treatment regime for some animals included early gastrulation (gestational day [G] 19 or G20), whereas others were treated later (on G21 or G24). Brainstems were cryosectioned and stained with cresyl violet. Stereological methods were used to determine the numbers of neurons in six different nuclei: the abducens, vagal, and hypoglossal motor nuclei and sensory components of the trigeminal brainstem nuclear complex (the principal, oral, and interpolar subnuclei). There were no differences in the numbers of neurons in any of the nuclei between controls and Et6-, or controls and Et24-treated monkeys. In contrast, the number of trigeminal sensory neurons was significantly (P < .05) lower in animals treated on G19/G20 than in control. No differences between controls and monkeys treated on G21/G24 were detected. No motor nuclei exhibited an ethanol-induced change. These data together with data on the trigeminal motor nucleus show that vulnerability to ethanol (1) is greater in sensory nuclei than in motor nuclei and (2) is temporally restricted to the time of gastrulation.     

Impaired odor identification in children with histories of heavy prenatal alcohol exposure

Prenatal alcohol exposure can lead to behavioral and cognitive impairments across multiple domains. Many of the brain regions impacted by prenatal alcohol exposure are also linked with olfactory processing, and odor identification deficits have been documented in certain neurological disorders associated with these brain regions. As odor identification following prenatal alcohol exposure is not well studied, we compared odor identification in children with prenatal exposure to alcohol (AE) to typically developing controls (CON) (N = 16/group). It was hypothesized that children in the AE group would perform more poorly than children in the CON group on the San Diego Odor Identification Test, an identification test of 8 common household odorants. Children exposed to alcohol during prenatal development were significantly impaired in olfactory identification (M = 5.95, SE = 0.37) compared to typically developing controls (M = 7.24, SE = 0.37). These findings confirmed the hypothesis that prenatal exposure to alcohol is associated with odor identification deficits, and suggest that further research is warranted to identify the mechanisms underlying these deficits, the integrity of brain areas that are involved, and to determine whether olfactory performance might contribute to better identification of children at risk for behavioral and cognitive deficits.     

Inflammatory diseases modelling in zebrafish

The ingest of diets with high content of fats and carbohydrates, low or no physical exercise and a stressful routine are part of the everyday lifestyle of most people in the western world. These conditions are triggers for different diseases with complex interactions between the host genetics, the metabolism, the immune system and the microbiota, including inflammatory bowel diseases (IBD), obesity and diabetes. The incidence of these disorders is growing worldwide; therefore, new strategies for its study are needed. Nowadays, the majority of researches are in use of murine models for understand the genetics, physiopathology and interaction between cells and signaling pathways to find therapeutic solutions to these diseases. The zebrafish, a little tropical water fish, shares 70% of our genes and conserves anatomic and physiological characteristics, as well as metabolical pathways, with mammals, and is rising as a new complementary model for the study of metabolic and inflammatory diseases. Its high fecundity, fast development, transparency, versatility and low cost of maintenance makes the zebrafish an interesting option for new researches. In this review, we offer a discussion of the existing genetic and induced zebrafish models of two important Western diseases that have a strong inflammatory component, the IBD and the obesity.     

Proceedings of the 2009 annual meeting of the Fetal Alcohol Spectrum Disorders Study Group

The annual meeting of the Fetal Alcohol Spectrum Disorders Study Group (FASDSG) was held on June 20, 2009 in San Diego, CA, as a satellite of the Research Society on Alcoholism Meeting. The FASDSG membership includes clinical, basic, and social scientists who meet to discuss recent advances and issues in Fetal Alcohol Spectrum Disorders research. The main theme of the meeting was “Epigenetics and Development.” Two keynote speakers, Dr. Randy Jirtle and Dr. Michael Skinner, addressed the role of epigenetics and environmental inputs, including alcohol, during critical stages of development and their potential critical and long-lasting effects. Members of the FASDSG provided new findings through brief “FASt” data reports, and national agency representatives provided updates on activities and funding priorities. Scientific presentations were made by recipients of the Student Research Merit Award and Rosett Award.     

Effects of one- and three-day binge alcohol exposure in neonatal C57BL/6 mice on spatial learning and memory in adolescence and adulthood

Binge-like alcohol exposure during the early postnatal period in rats and mice causes deficits in spatial learning and memory that persist into adulthood. Wozniak et al. (2004) reported that heavy binge alcohol exposure on postnatal day 7 (PD 7) in C57BL/6 (B6) mice produced profound spatial learning deficits in the Morris water maze when tested in adolescence (P30–39); when tested in adulthood, however, the deficits were greatly attenuated. Using a similar PD 7 binge alcohol exposure paradigm in B6 mice, we tested whether a single-day (PD 7 only) alcohol treatment produced place learning deficits in both adolescence and in adulthood, and further tested whether a more extended (3-day, PD 7–9) alcohol exposure would induce more severe and enduring deficits. B6 mice were given either 2 subcutaneous injections of alcohol (2.5 g/kg each) 2 h apart on PD 7 or on PD 7–9, and compared with controls that received saline vehicle injections and controls that received no injections. The alcohol injections on PD 7 produced average peak blood alcohol concentrations of 472 mg/dL and evoked typical patterns of activated caspase-3-positive neurons in the cortex, hippocampal formation, and striatum 6 h after the last injection. Mice were given standard place training or random location training in the Morris water maze either as adolescents (PD 30–39) or adults (PD 70–79). The adolescents acquired the place learning more slowly than adults, and the alcohol treatments produced only modest place acquisition deficits. In contrast, both the PD7 and the PD 7–9 alcohol treatments resulted in large and significant spatial learning impairments in adults. In contrast to the previous findings of Wozniak et al. (2004), these results indicate that binge alcohol exposure in the 3rd trimester equivalent produces significant and enduring deficits in spatial learning in B6 mice.     

Gender differences in ethanol-induced behavioral sensitivity in zebrafish

Gender-related differential sensitivity to ethanol has long been recognized. Our previous studies have demonstrated that the zebrafish, an animal model used currently to study genetics and development related to a variety of human diseases, is also sensitive to pharmacologically relevant concentrations of ethanol. Sensitivity to ethanol in the zebrafish can be easily gauged with a simple nonintrusive behavioral test that measures ethanol-related alterations in schooling by determining the distance between each fish and its nearest neighbor. The purpose of this study was to determine the influence of gender on the strain-specific ethanol sensitivity that we had observed previously. One hundred and sixty zebrafish of the wild-type (WT) and the long fin striped (LFS) strains were equally divided by gender for use in this study. For acute ethanol treatment, the fish were separated by gender and strain and exposed to 0.0, 0.125, 0.25 0.50, or 1.0% (vol/vol) ethanol. In the chronic study, eight fish of each strain and gender were exposed to 0.5% (vol/vol) ethanol for a period of 10 weeks and the swimming behavior tested before treatment and after each week of treatment. Results showed that female WT zebrafish displayed enhanced sensitivity to the effects of chronic ethanol exposure of increased nearest neighbor distances, whereas male and female LFS fish were not significantly affected by chronic ethanol exposure. Results of the acute ethanol study showed a dose-dependent effect in both strains and a gender effect that needs to be further investigated before enhanced female sensitivity to acute ethanol can be verified.     

Perforant pathway lamination in the dentate gyrus is unaffected by prenatal ethanol exposure

The entorhinal (perforant path) projection to the dentate gyrus was labeled with an anterograde horseradish peroxidase method to test whether prenatal exposure to ethanol affected the normal development of afferent lamination. Mean ethanol consumption of the ethanol-consuming dams was 12.7 g/kg +/- 0.3 g per day during days 1-21 of gestation. Adult offspring of normal and pair-fed controls as well as ethanol-exposed rats were analyzed. Computer-assisted image analysis of the entorhinal terminal field organization revealed no permanent changes in the development of the afferent lamination pattern in the dentate gyrus molecular layer in spite of the heavy in utero ethanol exposure.     

Organization of the commissural projection to the dentate gyrus is unaltered by heavy ethanol exposure during gestation

The anterograde horseradish peroxidase method was used to determine if prenatal exposure to ethanol affected the development of the characteristic afferent lamination pattern of the commissural projection to the dentate gyrus. Mean ethanol consumption for the ethanol-consuming dams was 12.7 g/kg +/- 0.3 g per day. Adult offspring of rats that consumed a liquid diet containing 35% ethanol-derived calories during days 1-21 of gestation, and both pair-fed and normal controls were examined. Brain weights and volumes of the ethanol and pair-fed control rats did not differ significantly from normal controls. However, body weights of ethanol-exposed rats were significantly reduced compared to normal controls. Computer-assisted image analysis of the HRP-labeling revealed that in spite of the heavy ethanol exposure there was no evidence of alterations in the spatial distribution of the commissural terminal field.     

Impact of combined prenatal ethanol and prenatal stress exposures on markers of activity-dependent synaptic plasticity in rat dentate gyrus

Prenatal ethanol exposure and prenatal stress can each cause long-lasting deficits in hippocampal synaptic plasticity and disrupt learning and memory processes. However, the mechanisms underlying these perturbations following a learning event are still poorly understood. We examined the effects of prenatal ethanol exposure and prenatal stress exposure, either alone or in combination, on the cytosolic expression of activity-regulated cytoskeletal (ARC) protein and the synaptosomal expression of AMPA-glutamate receptor subunits (GluA1 and GluA2) in dentate gyrus of female adult offspring under baseline conditions and after 2-trial trace conditioning (TTTC). Surprisingly, baseline cytoplasmic ARC expression was significantly elevated in both prenatal treatment groups. In contrast, synaptosomal GluA1 receptor subunit expression was decreased in both prenatal treatment groups. GluA2 subunit expression was elevated in the prenatal stress group. TTTC did not alter ARC levels compared to an unpaired behavioral control (UPC) group in any of the 4 prenatal treatment groups. In contrast, TTTC significantly elevated both synaptosomal GluA1 and GluA2 subunit expression relative to the UPC group in control offspring, an effect that was not observed in any of the other 3 prenatal treatment groups. Given ARC's role in regulating synaptosomal AMPA receptors, these results suggest that prenatal ethanol-induced or prenatal stress exposure-induced increases in baseline ARC levels could contribute to reductions in both baseline and activity-dependent changes in AMPA receptors in a manner that diminishes the role of AMPA receptors in dentate gyrus synaptic plasticity and hippocampal-sensitive learning.     

Neonatal ethanol exposure results in dose-dependent impairments in the acquisition and timing of the conditioned eyeblink response and altered cerebellar interpositus nucleus and hippocampal CA1 unit activity in adult rats

Exposure to ethanol in neonatal rats results in reduced neuronal numbers in the cerebellar cortex and deep nuclei of juvenile and adult animals. This reduction in cell numbers is correlated with impaired delay eyeblink conditioning (EBC), a simple motor learning task in which a neutral conditioned stimulus (CS; tone) is repeatedly paired with a co-terminating unconditioned stimulus (US; periorbital shock). Across training, cell populations in the interpositus (IP) nucleus model the temporal form of the eyeblink-conditioned response (CR). The hippocampus, though not required for delay EBC, also shows learning-dependent increases in CA1 and CA3 unit activity. In the present study, rat pups were exposed to 0, 3, 4, or 5 mg/kg/day of ethanol during postnatal days (PD) 4–9. As adults, CR acquisition and timing were assessed during 6 training sessions of delay EBC with a short (280 ms) interstimulus interval (ISI; time from CS onset to US onset) followed by another 6 sessions with a long (880 ms) ISI. Neuronal activity was recorded in the IP and area CA1 during all 12 sessions. The high-dose rats learned the most slowly and, with the moderate-dose rats, produced the longest CR peak latencies over training to the short ISI. The low dose of alcohol impaired CR performance to the long ISI only. The 3E (3 mg/kg/day of ethanol) and 5E (5 mg/kg/day of ethanol) rats also showed slower-than-normal increases in learning-dependent excitatory unit activity in the IP and CA1. The 4E (4 mg/kg/day of ethanol) rats showed a higher rate of CR production to the long ISI and enhanced IP and CA1 activation when compared to the 3E and 5E rats. The results indicate that binge-like ethanol exposure in neonatal rats induces long-lasting, dose-dependent deficits in CR acquisition and timing and diminishes conditioning-related neuronal excitation in both the cerebellum and hippocampus.     

The influence of the luteal and follicular phases on major pharmacokinetic parameters of blood and breath alcohol kinetics in women

Drink tests involving 14 women were carried out to determine the effects of the menstrual cycle phases on the pharmacokinetics of ethanol. One experiment was carried out in the follicular phase of the cycle and another in the luteal phase, with the estradiol, progesterone, and testosterone levels being determined in both cases. The target concentration was a final blood alcohol concentration (BAC) of approximately 0.08 g%. After drinking was completed, concurrent BAC and breath alcohol concentration (BrAC) measurements were carried out at intervals of 10-20 min. The ethanol elimination rate was determined by calculating a linear function in the part of the slope that was clearly linear. In addition, the c₀ and Widmark factors r were calculated.In 10 of the volunteers, who had a normal increase in progesterone in the luteal phase, the average hourly elimination rate ß₆₀ in the follicular phase amounted to 0.0194 ± 0.0020 g%/h (BAC) and 0.0975 ± 0.0068 mg/L/h (BrAC), and in the luteal phase to 0.0193 ± 0.0031 g%/h (BAC) and 0.1026 ± 0.0101 mg/L/h (BrAC). There was no significant difference. Other pharmacokinetic parameters (c₀ concentrations, Widmark factors r, distribution volumes, maximal BAC, mean absorption rate, time until the peak concentrations were reached) also revealed no significant differences between the blood and breath alcohol levels of the luteal and follicular phases. In addition, no significant correlations were observed between the absolute progesterone level and the respective elimination rates ß₆₀.     

Effect of gestational ethanol exposure on long-term memory formation in newborn chicks.

Fetal alcohol syndrome (FAS), a condition occurring in some children of mothers who have consumed alcohol during pregnancy, is characterized by craniofacial malformations, and physical and mental retardation. It is significant that even children with history of gestational ethanol exposure but relatively unaffected overall IQ performance, often exhibit learning difficulties and behavioral problems, suggestive of impaired memory formation. Hence, the specific aim of this study was to examine memory formation in chicks exposed to ethanol during early gestation toward the understanding of neurobehavioral disturbances in FAS. Chicks were exposed to alcohol on gestational days 1-3 by injection of ethanol into the airspace of freshly fertilized eggs. The effects of prenatal ethanol on physical growth and development, and memory formation were studied. The one-trial passive avoidance learning paradigm in 1-day-old chicks was used to study memory formation in these chicks. It was observed that chick embryos exposed to 10% ethanol on gestational days 1-3 had significant reduction in all body parameters when compared with appropriate controls. Further, ethanol-exposed chick embryos had significantly impaired (P<.05) long-term memory (LTM) formation after training, though short-term or intermediate-term memory formation was unimpaired. Thus, the findings of the current study demonstrate the detrimental effects of ethanol exposure during early pregnancy on developing chick embryos in general and on memory formation in particular. Hence, it is suggested that impairment in LTM could be a fundamental mechanism for learning disorders and neurobehavioral abnormalities observed in FAS.     

Ethanol exposure during the early first trimester equivalent impairs reflexive motor activity and heightens fearfulness in an avian model

Prenatal alcohol exposure is a leading cause of childhood neurodevelopmental disability. The adverse behavioral effects of alcohol exposure during the second and third trimester are well documented; less clear is whether early first trimester-equivalent exposures also alter behavior. We investigated this question using an established chick model of alcohol exposure. In ovo embryos experienced a single, acute ethanol exposure that spanned gastrulation through neuroectoderm induction and early brain patterning (19-22 h incubation). At 7 days posthatch, the chicks were evaluated for reflexive motor function (wingflap extension, righting reflex), fearfulness (tonic immobility [TI]), and fear/social reinstatement (open-field behavior). Chicks exposed to a peak ethanol level of 0.23-0.28% were compared against untreated and saline-treated controls. Birds receiving early ethanol exposure had a normal righting reflex and a significantly reduced wingflap extension in response to a sudden descent. The ethanol-treated chicks also displayed heightened fearfulness, reflected in increased frequency of TI, and they required significantly fewer trials for its induction. In an open-field test, ethanol treatment did not affect latency to move, steps taken, vocalizations, defecations, or escape attempts. The current findings demonstrate that early ethanol exposure can increase fearfulness and impair aspects of motor function. Importantly, the observed dysfunctions resulted from an acute ethanol exposure during the period when the major brain components are induced and patterned. The equivalent period in human development is 3-4 weeks postconception. The current findings emphasize that ethanol exposure during the early first trimester equivalent can produce neurodevelopmental disability in the offspring.     

The Knowledge of Rehabilitation Professionals Concerning Fetal Alcohol Spectrum Disorders

The purpose of this study was to explore rehabilitation professionals’ knowledge regarding signs and symptoms, prevention, and intervention of fetal alcohol spectrum disorders (FASD). Participants were 111 rehabilitation practitioners (e.g., occupational therapy, physical therapy, and speech-language pathology practitioners) recruited through email using a quantitative online survey design with purposive, snowball sampling. Results showed the majority of participants’ demonstrated accurate knowledge of the signs and symptoms of FASD. Since professionals who received formal education on FASD reported significantly higher feelings of preparedness to identify children with FASD and manage/coordinate intervention plans, this study suggests rehabilitation professionals may be better prepared to treat individuals with FASD if they participate in formal training.