David Gilliam

Ethanol teratogenesis in the C57BL/6J, DBA/2J, and A/J inbred mouse strains

Research has shown variations in susceptibility to alcohol-related birth defects in humans. Genetic differences are one reason for this variability. This study compared three inbred mouse strains to determine whether they differ in their susceptibilities to ethanol teratogenesis because previous studies have generated conflicting data. Pregnant C57BL/6J (B6), DBA/2J (D2), and A/J (A) dams were intubated intragastrically with either an acute dose of ethanol (5.8 g/kg) or an isocaloric amount of maltose-dextrine on day 9 of pregnancy. Litters were removed on day 18 of pregnancy and examined for gross, soft-tissue, and skeletal malformations. Results showed that ethanol-exposed B6 litters had a higher percentage of digit (19%), kidney (24%), and skeletal (32%, mostly vertebral) malformations than their maltose-exposed controls (7% or below). Prenatal exposure to ethanol increased skeletal (68%, both rib and vertebral) malformations for A litters when compared to their maltose-exposed controls (4%), but did not increase digit or kidney malformations. Ethanol-exposed D2 litters did not differ from maltose-exposed controls. Maternal blood ethanol levels did not differ among the B6, D2, and A strains. These results provide additional evidence suggesting a genetic component to ethanol teratogenesis.

Ethanol Teratogenesis in Five Inbred Strains of Mice

BACKGROUND Previous studies have demonstrated individual differences in susceptibility to the detrimental effects of prenatal ethanol exposure. Many factors, including genetic differences, have been shown to play a role in susceptibility and resistance, but few studies have investigated the range of genetic variation in rodent models. METHODS We examined ethanol teratogenesis in 5 inbred strains of mice: C57BL/6J (B6), Inbred Short-Sleep, C3H/Ibg, A/Ibg, and 129S6/SvEvTac (129). Pregnant dams were intubated with either 5.8 g/kg ethanol (E) or an isocaloric amount of maltose-dextrin (MD) on day 9 of pregnancy. Dams were sacrificed on day 18 and fetuses were weighed, sexed, and examined for gross morphological malformations. Every other fetus within a litter was then either placed in Bouins fixative for subsequent soft-tissue analyses or eviscerated and placed in ethanol for subsequent skeletal analyses. RESULTS B6 mice exposed to ethanol in utero had fetal weight deficits and digit, kidney, brain ventricle, and vertebral malformations. In contrast, 129 mice showed no teratogenesis. The remaining strains showed varying degrees of teratogenesis. CONCLUSIONS Differences among inbred strains demonstrate genetic variation in the teratogenic effects of ethanol. Identifying susceptible and resistant strains allows future studies to elucidate the genetic architecture underlying prenatal alcohol phenotypes.

No Effect of Prenatal Alcohol Exposure on Activity in Three Inbred Strains of Mice

AIMS Prenatal exposure to alcohol can have adverse effects on the developing fetus. Two of the hallmarks of children exposed to alcohol prenatally are attention deficits and hyperactivity. While hyperactivity has been observed in rats following prenatal ethanol exposure, few studies have examined these effects in mice. The present study investigated the effects of prenatal ethanol exposure on activity in mice from three inbred strains: C57BL/6 (B6), Inbred Long Sleep (ILS) and Inbred Short Sleep (ISS). METHODS On Days 7 through 18 of gestation, mice were intragastrically intubated twice daily with either 3.0 g/kg ethanol (E) or an isocaloric amount of maltose-dextrin (MD); non-intubated control (NIC) litters were also generated. Offspring activity was monitored at 30, 60, 90 and 150 days of age. RESULTS While results showed no effects of prenatal ethanol exposure on any measures of activity, we did observe differences in baseline activity among the strains. ISS mice were more active than B6 and ILS for all activity measures except stereotypy; B6 mice had higher measures of stereotypy than ILS and ISS. Younger mice were more active than older mice. The only sex effects were on measures of stereotypy, where males had higher scores. CONCLUSIONS Mice are an excellent organism to study genetic influences on many phenotypes. However, our study and others have shown few effects of prenatal ethanol exposure on behavior in mice. It appears as if the prenatal period in mice, corresponding to organogenesis, is not a sensitive period for producing behavioral deficits following ethanol exposure. It is likely that the first 2 weeks postnatally, corresponding to the brain growth spurt, are more sensitive for producing behavioral effects.

Dose-related growth deficits in LS but not SS mice prenatally exposed to alcohol.

Genetically based alcohol sensitivity may influence the severity of alcohol-related birth defects. To examine this question, measures of growth and survival were examined in offspring of the alcohol sensitive Long-Sleep (LS) and alcohol-resistant Short-Sleep (SS) mouse lines following prenatal ethanol exposure. Pregnant LS and SS mice received an ethanol dose of either 6 or 8 g/kg/day from days 7 through 18 of pregnancy. Control groups received a maltose-dextran solution made isocaloric to the 8 g/kg/day dose. Ethanol and maltose-dextrin solutions were administered as split doses, 6 h apart, via gavage. Nonintubated lab chow control groups were also included for both mouse lines. Offspring were fostered at birth to lactating mice of an outbred stock. Pregnancy was longer for ethanol-treated LS dams compared to maltose-dextrin and lab chow LS control groups, whereas pregnancy length for ethanol-treated SS dams was similar to SS controls. Prenatal ethanol exposure resulted in dose-related growth deficits in LS but not in SS litters. Line differences in postnatal growth deficits in response to prenatal alcohol exposure suggest maternal or fetal alcohol sensitivity influence alcohol-related birth defects.

LONG-TERM CONSEQUENCES OF PRENATAL COCAINE EXPOSURE ON BIOGENIC AMINES IN THE BRAINS OF MICE : THE ROLE OF SEX

Prenatal cocaine exposure leads to multiple abnormalities in the mature offspring. We explored the effects of gestational exposure to cocaine on neurotransmitter systems of adult mice. The subjects were the mature offspring of mice (a) prenatally fed cocaine between gestational day (G) 8 and G19, (b) pair-fed chow and water, or fed chow and water ad libitum. The forebrains of the mature offspring were assayed for monoamines and amino acids. Cocaine exposure particularly affected the dopaminergic system and in a sex-specific manner. In males dopamine concentrations were decreased and dopamine turnover was increased, whereas in females dopamine concentrations were increased and turnover was decreased. Neither norepinephrine, the serotonergic system, nor neuroactive amino acids (or their precursors) were affected by cocaine. Thus, in utero exposure to cocaine produces long-lasting, specific defects in the dopaminergic system.

Cytoplasmic factors do not contribute to a maternal effect on ethanol teratogenesis.

Both maternal and fetal genetic factors influence variations in response to prenatal ethanol exposure. To assess the effect of maternal genotype on the incidence of ethanol teratogenesis, a reciprocal cross study was conducted in an animal mode using the relatively susceptible C57BL/6J (B6) and the relatively resistant DBA/2J (D2) inbred mice. This mating pattern produced four embryonic genotypes: true-bred B6B6 and D2D2 litters and hybrid B6D2 and D2B6 litters. To examine the role of maternal egg cytoplasm as the source of variation that could account for a maternal effect, B6D2 and D2B6 F1 females were mated back to B6 males, which produced two additional embryonic genotypes: B6D2.B6 and D2B6.B6. Dams were intubated with either 5.8 g/kg of ethanol or an isocaloric amount of maltose–dextrin on day 9 of pregnancy. On day 18 of pregnancy, dams were sacrificed, fetuses were removed, weighed, sexed, and examined for gross morphological malformations. Every other fetus within a litter was prepared for either skeletal or soft tissue analysis. Results showed a higher rate of teratogenesis in the B6D2 group compared to the genetically similar D2B6 group, which indicates an influence of maternal genotype on susceptibility to ethanol teratogenesis. The percentage of affected male and female fetuses did not differ, which suggests that sex-linked factors are not responsible for the maternal effect. The backcross B6D2.B6 and D2B6.B6 litters did not differ significantly for any measure of teratogenesis, suggesting that differences in maternally transmitted cytoplasmic material are not the cause of the maternal effect. Factors that could account for the maternal effect are differences in the maternal uterine environment and genomic imprinting. Separating maternal from fetal-mediated mechanisms responsible for susceptibility to ethahol teratogenesis is needed for identifying mothers and infants at risk.

Genetic and Maternal Effects on Valproic Acid Teratogenesis in C57BL/6J and DBA/2J Mice

Valproic acid (VPA) is used worldwide to treat epilepsy, migraine headaches, and bipolar disorder. However, VPA is teratogenic and in utero exposure can lead to congenital malformations. Using inbred C57BL/6J (B6) and DBA/2J (D2) mice, we asked whether genetic variation could play a role in susceptibility to VPA teratogenesis. Whereas B6 fetuses were more susceptible than D2 fetuses to digit and vertebral malformations, D2 fetuses were more susceptible to rib malformations. In a reciprocal cross between B6 and D2, genetically identical F1 mice carried in a B6 mother had a greater percentage of vertebral malformations following prenatal VPA exposure than F1 mice carried in a D2 mother. This reciprocal F1 difference is known as a maternal effect and shows that maternal genotype/uterine environment is an important mediator of VPA teratogenecity. VPA is a histone deacetylase inhibitor, and it is possible that the differential teratogenesis in B6 and D2 is because of strain differences in histone acetylation. We observed strain differences in acetylation of histones H3 and H4 in both embryo and placenta following in utero VPA exposure, but additional studies are needed to determine the significance of these changes in mediating teratogenesis. Our results provide additional support that genetic factors, both maternal and fetal, play a role in VPA teratogenesis. Lines of mice derived from B6 and D2 will be a useful model for elucidating the genetic architecture underlying susceptibility to VPA teratogenesis.

Quantitative Trait Locus Mapping for Ethanol Teratogenesis in BXD Recombinant Inbred Mice

BACKGROUND Individual differences in susceptibility to the detrimental effects of prenatal ethanol (EtOH) exposure have been demonstrated. Many factors, including genetics, play a role in susceptibility and resistance. We have previously shown that C57BL/6J (B6) mice display a number of morphological malformations following an acute dose of EtOH in utero, while DBA/2J (D2) mice are relatively resistant. Here, we present the results of quantitative trait locus (QTL) mapping for EtOH teratogenesis in recombinant inbred strains derived from a cross between B6 and D2 (BXD RIs). METHODS Pregnant dams were intubated with either maltose-dextrin or 5.8 g/kg EtOH on day 9 of gestation (GD9). On GD 18, dams were sacrificed and fetuses and placentae were removed. Placentae and fetuses were weighed; fetuses were sexed and examined for gross morphological malformations. Fetuses were then either placed in Bouins fixative for subsequent soft-tissue analyses or eviscerated and placed in EtOH for subsequent skeletal examinations. QTL mapping for maternal weight gain (MWG), prenatal mortality, fetal weight (FW) at c-section, placental weight (PW), and several morphological malformations was performed using WebQTL. RESULTS Heritability for our traits ranged from 0.06 for PW to 0.39 for MWG. We found suggestive QTLs mediating all phenotypes and significant QTLs for FW and digit and rib malformations. While most QTL regions are large, several intriguing candidate genes emerged based on polymorphisms between B6 and D2 and gene function. CONCLUSIONS In this first mapping study for EtOH teratogenesis, several QTLs were identified. Future studies will further characterize these regions. Identification of genes and epigenetic modifications mediating susceptibility to the teratogenic effects of alcohol in mice will provide targets to examine in human populations.

Differential effects of MK-801 on cerebrocortical neuronal injury in C57BL/6J, NSA, and ICR Mice

1. Antagonists of the N-methyl-D-aspartate (NMDA) glutamate (Glu) receptor, including [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate], dizocilpine maleate (MK-801), injure pyramidal neurons in the posterior cingulate/retrosplenial (PC/RS) cortex when administered systemically to adult rats and mice. 2. These results have, to our knowledge, only been reported previously in Harlan Sprague Dawley albino rats and International Cancer Research (ICR) mice, an outbred albino strain. 3. Male Non-Swiss Albino (NSA) mice, an albino outbred strain, and male C57BL/6J (B6) mice, a pigmented inbred strain, were injected systemically with 1 mg/kg of MK-801 in the first experiment. This dose of MK-801 reliably produces cytoplasmic vacuoles in neurons in layers III and IV of the PC/RS cortex in 100% of ICR mice treated 4. There was a significant difference in the number of vacuolated neurons in B6 and NSA mice, as assessed by ANOVA. The NSA were not significantly different than previously examined ICR mice, but the B6 had fewer vacuolated neurons than either of the two outbred strains. 5. In the second experiment, male NSA, ICR, and B6 mice were injected systemically with a high dose, 10 mg/kg, of MK-801. This dose has been demonstrated to result in necrosis in the same population of neurons injured by lower doses of MK-801. 6. An ANOVA indicated that there was a significant difference among the three strains of mice, and a Fishers protected t revealed that the B6 mice were significantly different from both the NSA and ICR, but that, with our test, those two strains were indistinguishable. 7. Male ICR, NSA, and B6 mice were tested in the holeboard food search task 5 hours after 1 mg/kg of MK-801. There were significant differences between the strains in performance both pre and posttreatment. The effect of the drug was not statistically significant. 8. These results suggest that there may be a genetically mediated difference in the reaction to NMDA receptor antagonists, a finding which may be important given the NMDA receptor hypofunction hypothesis for the etiology of schizophrenic symptoms.

Maternal effects on ethanol teratogenesis in a cross between A/J and C57BL/6J mice

Genetic factors influence adverse pregnancy outcome in both humans and animal models. Animal research reveals that both the maternal and fetal genetic profiles are important for determining the risk of physical birth defects and prenatal mortality. Using a reciprocal-cross breeding design, we investigated whether the mothers genes may be more important than fetal genes in determining risk for ethanol teratogenesis. Examination of possible synergistic genetic effects on ethanol teratogenesis was made possible by using two mouse strains known to be susceptible to specific malformations. Inbred A/J (A) and C57BL/6J (B6) mice were mated to produce four fetal genotype groups: the true-bred AċA and B6ċB6 genotypes and the genetically identical AċB6 and B6ċA genotypes (the F(1) genotype). Dams were administered either 5.8 g/kg ethanol or an isocaloric amount of maltose-dextrin on day 9 of pregnancy. Fetuses were removed by laparotomy on gestation day 18, weighed, and assessed for digit, vertebral, and kidney malformations. Digit malformations in the genetically identical F(1) ethanol-exposed litters showed a pattern consistent with a maternal genetic effect (AċB6 [2%] and B6ċA [30%]). In contrast, vertebral malformations were similar in all ethanol-exposed litters (AċA [26%], AċB6 [18%], B6ċA [22%], and B6ċB6 [33%]). The percentage of malformations did not differ between male and female fetuses, indicating sex-linked factors are not responsible for the maternal effect. Ethanol exposure decreased litter weights but did not affect litter mortality compared with maltose-exposed controls. This study supports the idea that genes influence malformation risk following in utero alcohol exposure. Specifically, maternal genes influence risk more than fetal genes for some teratogenic outcomes. No evidence supported synergistic genetic effects on ethanol teratogenesis. This research supports the conclusion that uterine environment contributes to determining risk of Fetal Alcohol Spectrum Disorder.