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Ethanol exposure during fetal development can result in behavioral and neurological deficits, including reduced cognitive functions, retarded growth, and craniofacial abnormalities. Adenosine is an endogenous neuromodulator that fine-tunes the release and/or synaptic activities of several neurotransmitters, including glutamate, dopamine, and serotonin. Our aim was to determine whether ethanol exposure during early development affects adenosine receptors, particularly the A1 receptor subtype, in adult rats. Female rats were given water or 15% (vol/vol) ethanol in water prior to mating and throughout gestation and lactation. Sixty-day-old male rat offspring from these dams were randomly selected and assayed for adenosine A1 receptor expression in four brain areas: cortex, cerebellum, hippocampus, and striatum. Our results indicate that ethanol intake by dams decreased body and brain weights of offspring and reduced both A1 receptor mRNA and protein density in cortex and cerebellum. These preliminary findings indicate that ethanol intake by dams during pregnancy and lactation can affect adenosine A1 receptor signalling in the offspring. A pair-fed controlled study is warranted to explore these findings further.

Pregnant mice were fed equivalent daily amounts of a liquid diet containing 25% (kcal) ethanol, or with maltose dextrin substituted isocalorically for ethanol. In addition, the diet contained 20% oil; this was either of two mixtures, one comprised of predominantly n‐6 (18:2n‐6) fatty acids, and the other containing an equivalent amount of n‐6, but supplemented with a source of long chain n‐3 (20:5n‐3, 22:6n‐3) fatty acids. An additional control group was fed lab chow ad libitum. The treatment was implemented from day 7 to 17 of gestation, whereafter all groups were fed lab chow. Ethanol decreased maternal weight gain and pup body and brain weight; it also retarded both sensory and motor development in the pups and impeded reversal learning in a water maze. The n‐3 supplementation lowered maternal blood alcohol concentration, but counteracted only some of the effects of ethanol, by increasing maternal weight gain and pup body weight, and also by enhancing sensory development in the pups. Such effects were additive, in that they were also present in the maltose‐dextrin control group. These findings suggest that n‐3 supplementation may ameliorate some of the effects of ethanol on neurobehavioral development, but the magnitude of the effect appears to be small.

The effects of ethanol and starvation on ketone body production and utilization were investigated. In the first experiment, adult C57BL/6J mice were divided into four groups: (i) control (fed); (ii) starvation (up to 31 h); (iii) ethanol (acute 5 g/kg i.p.); (iv) ethanol (ETOH) + starvation. Plasma ketone body (KB) concentrations in control mice remained constant at approx. 0.37 mM. The levels of KBs in starved mice began to increase at about 7 h and rose to a peak of 2.5 mM at about 24 h, then fell to 1.8 mM at 31 h. The levels in mice treated with ETOH began to rise soon after injection, reached 1.5 mM at 10 h, and returned to control levels by 15 h. Although there was no difference in elevated levels of KBs between two groups of mice treated with ETOH plus starvation and ETOH alone at 7-10 h, the level continued to rise steadily to 2.0 mM through 31 h in the former group. At 10 h post ETOH, mice either fed ad lib. or fasted had increased hepatic capacity to synthesize acetoacetate (AcAc) from palmitate; this effect was prolonged and enhanced by continued fasting for 24 h. In the brain, the rate of AcAc oxidation was twice that for beta-hydroxybutyrate (beta OHB) and glucose. Neither ETOH nor starvation affected energy production from KB and glucose. AcAc was also utilized for fatty acid synthesis and the rate of synthesis was stimulated by ETOH at 10 h after injection. The rate of lipogenesis from beta OHB accounted for less than 10% of that from AcAc. Together these experiments demonstrate that ETOH increases both hepatic ketone production and plasma KB levels for at least 10 h. ETOH alone led to elevated KB levels long before the rise due to starvation. In brain, at 10 h, an increased capacity to utilize AcAc for lipogenesis was found. The results indicate that ETOH through the production of KBs could provide an important source of energy and lipid precursors for the brain of mice.

Prior research had indicated that moderate maternal ethanol consumption during gestation affected the growth of the corpus callosum and anterior commissure in BALB/c mice when measured at day 19 postconception. Our purpose was to assess whether or not this was an enduring effect. Pregnant BALB/cCRBL mice were fed ethanol 10% v/v in the drinking water from days 5 to 26 postconception. Control animals received an isocaloric sucrose solution and were pair-fed to the experimental animals. An additional control group fed laboratory chow ad libitum was included. Using a split-litter design, brain development was assessed on days 26 and 50 postconception and behavioral development of the pups was measured on day 32. The ethanol-treated offspring had lower brain weights at both ages as well as a smaller cross-sectional area of the anterior commissure on day 50, which was significantly related to the smaller brain weight. There was no apparent effect of ethanol on the area of the corpus callosum at either age. Similarly, behavioral development was not affected by the treatment, although eye-opening was delayed in ethanol-treated animals. Measures of maternal behavior indicated that the animals consuming alcohol were more active than those in the control groups. An unexpected finding was that the control group fed sucrose appeared to be adversely affected. The body weight of these pups was lower, as was the area of the corpus callosum at day 50.

High levels of ethanol (EtOH) consumption during pregnancy adversely affect fetal development; however, the effects of lower levels of exposure are less clear. Our objectives were to assess the effects of daily EtOH exposure (3.8 USA standard drinks) on fetal-maternal physiological variables and the fetal brain, particularly white matter. Pregnant ewes received daily intravenous infusions of EtOH (0.75 g/kg maternal body wt over 1 h, 8 fetuses) or saline (8 fetuses) from 95 to 133 days of gestational age (DGA; term ∼145 DGA). Maternal and fetal arterial blood was sampled at 131–133 DGA. At necropsy (134 DGA) fetal brains were collected for analysis. Maternal and fetal plasma EtOH concentrations reached similar maximal concentration (∼0.11 g/dl) and declined at the same rate. EtOH infusions produced mild reductions in fetal arterial oxygenation but there were no changes in maternal oxygenation, maternal and fetal PaCO2, or in fetal mean arterial pressure or heart rate. Following EtOH infusions, plasma lactate levels were elevated in ewes and fetuses, but arterial pH fell only in ewes. Fetal body and brain weights were similar between groups. In three of eight EtOH-exposed fetuses there were small subarachnoid hemorrhages in the cerebrum and cerebellum associated with focal cortical neuronal death and gliosis. Overall, there was no evidence of cystic lesions, inflammation, increased apoptosis, or white matter injury. We conclude that daily EtOH exposure during the third trimester-equivalent of ovine pregnancy has modest physiological effects on the fetus and no gross effects on fetal white matter development.

AbstractThis study assesses the combined effects on brain and behavioral development of ethanol administration and supplementation of the maternal diet with long chain n−3 polyunsaturated fatty acids. From day 7 to 17 of gestation, pregnant mice were fed equivalent daily amounts of isocaloric liquid diets; 20% of the energy was provided by either ethanol or maltose‐dextrin, and a further 20% by either safflower oil (rich in linoleic acid, 18∶2n−6), or a combination of safflower oil with a fish oil concentrate (rich in eicosapentaenoic acid, 20∶5n−3, and docosahexaenoic acid, 22∶6n−3). On day 18 the liquid diets were replaced by lab chow; a fifth group was maintained on lab chow throughout the experiment. Measures on the pups included brain weight and the fatty acid composition of the brain phospholipids on days 22 and 32 post‐conception (birth=day 19), as well as behavioral development. Maternal weight gain during gestation was decreased by ethanol relative to maltose‐dextrin, and increased by fish relative to safflower oil. On day 32, the brain weight of ethanoltreated animals fed fish oil was greater than their safflower oil controls, whereas the reverse was true in the two maltose‐dextrin groups; a similar trend was apparent on day 22. The brain phospholipid content of the longer chain fatty acids (20∶4n−6, 22∶4n−6, 22∶5n−6, 20∶5n−3, 22∶5n−3, 22∶6n−3) on day 22 reflected that of the prenatal diet, with the proportion of n−3 compounds being higher and that of n−6 floer in the fish oil than safflower oil groups. Prenatal dietary effects were absent by day 32, with the exception of lower 22∶5n−6 in fish oil groups. Dietary supplementation with n−3 fatty acids increased the ratio of 20∶3n−6 to 20∶4n−6, which is consistent with a blockade of the activity of Δ‐5 desaturase. On day 22 the incorporation of dietary long chain n−3 fatty acids into the brain phosphatidylcholine fraction was enhanced in the ethanol‐treated animals; by day 32 the animals treated prenatally with ethanol also showed increased levels of long chain n−6 compounds. Behavioral development was retarded by ethanol, but there was no effect of the dietary oils. These results support the hypothesis that effects of ethanol on the developing brain may be modified by the availability of an exogenous supply of long chain fatty acids.

We investigated whether or not moderate ethanol consumption during gestation would interact with the effects of a low-protein diet in affecting brain development in BALB/c mice. The independent variables included fetal body and brain weights and cross-sectional area in midsagittal sections of the corpus callosum (CC) and anterior commissure (CA). Pregnant animals were fed either ethanol 12% v/v or an isocaloric sucrose solution from days 5 to 19 of gestation, when fetal development was assessed. In addition, the animals were fed semisynthetic isocaloric diets containing either 8 or 20% casein. All animals were pair-fed to those in the group receiving ethanol and 20% casein; an additional control group was fed lab chow ad libitum. There was clearly an interactive effect of diet and ethanol consumption on blood alcohol concentrations: those in the low-protein group were significantly higher than in the normal-protein group. Similarly, the effect on body weight in the group receiving low protein plus ethanol was greater than the additive effect of either treatment alone, although this may have been due partly to differences in litter size. Brain weight in this group was also significantly less than in the other three groups, which did not differ from each other. Covariance analysis, adjusting brain weight for body weight, suggested a brain-sparing effect of low protein but not ethanol. Neither treatment affected the incidence of the CC being absent at midline. The low-protein treatment decreased the cross-sectional area of both the CC and CA; the effect on the CC was independent of brain weight. There was no effect of ethanol on either of those measures.

Effects of perinatal exposure to ethanol on growth and cellular development were investigated. Alcohol was administered in liquid diets designed to provide optimal nutrition during pregnancy. Pair-fed and ad lib control groups were included. The 3 groups of females were similar in body weight during gestation and lactation, and offspring weights were similar on gestation Day 21 and at birth. By Day 9 of lactation control pups weighted more than both alcohol and pair-fed pups which were similar in body weight. Weights of brain, heart, liver and kidney were reduced in alcohol pups compared to pair-feds and controls. Decreased liver weight reflected both decreased cell size and decreased protein content, but was primarily due to decreased caloric intake. Decreased heart weight appeared to result from a direct effect of ethanol on heart protein content. Even more marked were the adverse effects of ethanol on kidney protein content and kidney DNA (reflecting a decrease in cell number). In contrast, although both absolute brain weight and DNA content were decreased in ethanol-exposed offspring, relative brain weight was increased. Finally, maternal ethanol consumption significantly increased relative placenta weights as well as placental DNA and protein content.

OBJECTIVE: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. METHODS: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. RESULTS: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. CONCLUSIONS: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.