Model Of Fetal Alcohol Syndrome Research Articles

Growth and Formation of the Tooth Germ in a Rat Model of Fetal Alcohol Syndrome

The purpose of the present study was to elucidate the influence of alcohol ingestion in pregnant rats on the body and teeth of neonatal rats. Twenty two pregnant rats were divided into a normally-housed control group and alcohol-intake group exposed to alcohol during pregnancy, and 223 neonatal rats from these pregnant rats were used for the study. We observed the amount of water intake and the number of pups in mother rats, and body weight, eye-opening, and tooth development (postnatal day 1, 5, 15, and 20) in neonatal rats. The results showed the decreasing number of pups in the alcohol-intake group. Neonatal rats in the alcohol-intake group exhibited a lower body weight, especially on the first neonatal day, with a significant difference within the group. They also showed a delay in the time to eye-opening. Histopathological examination revealed no significant morphological abnormalities in odontogenic cells, but differential growth of the tooth germ and a delay in dental root formation were observed. The present study confirmed the influences of alcohol ingestion in pregnant rats on neonatal rats, including delays in physical development, differential growth of the tooth germ, and tooth eruption time.

Alcohol-induced learning deficits are not prevented through GABA-A3

Fetal Alcohol Syndrome (FAS) is characterized by neurodevelopmental anomalies and life-long learning impairment. In a model for FAS we showed that prenatal treatment with neuroprotective peptides (NAP+SAL) prevented alcohol-induced learning deficits. We evaluated the role of GABA receptor as a potential mechanism for the peptide neuroprotection. The GABA-Aβ3 subunit is important for learning and is increased after alcohol exposure in the adult. We were interested in evaluating whether prenatal alcohol exposure affects GABA-Aβ3 and if the peptides mechanism of action for preventing alcohol-induced learning deficits includes the subunit.

Alterations in P-CREB activity: A pathway for FAS related neurotoxiticity

NEUROTOXITICITY ROBIN ROBERSON, IRENE CAMERONI, LAURA TOSO, DANIEL ABEBE, STEPHANIE BISSELL, CATHERINE SPONG, National Institutes of Health (NIH), Unit on Perinatal and Develomental Neurobiology, NICHD&NIAAA, Bethesda, Maryland, University of Milano-Bicocca, Obstetrics and Gynecology, Monza, Milan, Italy OBJECTIVE: Fetal alcohol syndrome (FAS) is the leading cause of a spectrum of preventable non-genetic learning disorders. In the adult, neurogenesis occurs in the hippocampal dentate gyrus, which is thought to be important for learning and memory. Previously we have shown alterations in the NMDA receptor underlie some of the learning abnormalities in FAS. NMDA exerts it action through CREB. CREB is a transcription factor that controls gene expression via phosphorylation promoting neuronal survival. Using a model for FAS, we measured P-CREB levels in hippocampal subregions to evaluate if alterations in P-CREB underlie the FAS learning abnormalities after in utero alcohol exposure. STUDY DESIGN: Pregnant C57Bl6/J mice were treated on gestational day 8 with alcohol, n = 10 or control (saline) n = 5. Adult males were assessed in the Morris water maze for learning on days 50-56. On day 57, 5 males/ treatment were perfused for immunohistochemistry, and stained with SER 144 P-CREB antibody with relative densities of staining assessed using NIH Image software. Statistical analysis ANOVA, P!.05 significant. RESULTS: The hippocampal areas CA1, dentate and CA3 were quantitated. In the control, P-CREB staining was greatest in dentate granule cells and lowest in CA1. In all of the hippocampal subregions, alcohol exposure in utero resulted in significant downregulation of P-CREB (P!.001).

Adult administration of peptides resulting in learning enhancement is not mediated through NMDA and GABA receptor expression

IS NOT MEDIATED THROUGH NMDA AND GABA RECEPTOR EXPRESSION LAURA TOSO, ROBIN ROBERSON, DANIEL ABEBE, CATHERINE SPONG, National Institutes of Health (NIH), Unit on Perinatal and Develomental Neurobiology, NICHD&NIAAA, Bethesda, Maryland, National Institutes of Health (NIH), Unit on Perinatal and Develomental Neurobiology, Bethesda, Maryland OBJECTIVE: Administration of neurotrophic peptides NAPCSAL to aged mice resulted in significant learning enhancement (Toso 2006). NMDA and GABA receptors are fundamental for learning as they are the major modulators of the Long Term Potentiation, the electrophisiologic mechanism for learning. Also, these receptors have been shown to be involved in NAPCSAL prevention of learning deficit in a mouse model for fetal alcohol syndrome when administered prenatally during development(Toso 2006). Our objective was to test if NMDA and GABA receptors contribute to the learning enhancement induced by the peptides after adult administration. STUDY DESIGN: Aged (14.5 months) males were treated for 10 consecutive days with placebo or D-NAPCD-SAL (20mg, via gavage). At the end of the treatment brains were harvested. Calibrator-normalized relative real time PCR was performed using primers for GABA-Ab3, GABA-Aa5, NR2A and NR2B with GAPDH standardization. Statistical analysis included ANOVA with p!0.05 significant. RESULTS: 5 control brains and 6 brains from animals treated with NAPCSAL were collected. There was no difference in GABA-Ab3, GABAAa5, NR2A and NR2B subunits after adult administration of NAPCSAL as compared to the controls (pO0.05). CONCLUSION: Postnatal treatment with NAPCSAL induced learning enhancement in aged mice with mechanism that does not involve NMDA and GABA receptor expression. Treatment with the peptides does not acutely alter expression, although it may have an effect on the function of these receptors. Alternatively, the mechanism of learning enhancement may differ when administered to a developing fetus vs an adult.

Neuroprotective peptides prevent the alcohol-induced alteration in GABA-A3 which plays a role in cleft lip and palate in fetal alcohol syndrome

Prenatal alcohol exposure affects 1/200 live births in US and results in cranio-facial dysmorphology including cleft lip and palate and long-term learning disabilities. The GABA receptor subunit GABA-Aβ3 is critical during embryogenesis for neural tube and palate development. The GABA-Aβ3 knock-out results in cleft palate and neonatal death that were rescued by introduction of the β3-subunit transgene. We have shown that prenatal alcohol exposure downregulated GABA-Aβ3 in the embryo. In a mouse model for fetal alcohol syndrome (FAS), neuroprotective peptides prevented alcohol-induced fetal death, growth restriction and learning deficits. Our objective was to determine if the peptides' prevention of alcohol-induced complications includes GABA-Aβ3. Timed, pregnant C57BL6/J mice were treated on gestational day 8 with either alcohol (0.03 mL/g) or placebo or alcohol+peptides (NAP+SAL 20μg). Embryos were harvested at 24h (E9) and 10 days (E18) after treatment. Using samples from at least 3 litters per time-point, calibrator-normalized relative real time PCR was performed using primers for GABA-Aβ3 with GAPDH standardization. Statistical analysis included ANOVA and Fisher PLSD. Twenty-four hours after treatment, the alcohol-induced decrease in GABA-Aβ3 was not prevented by the pretreatment with the peptides (P=0.8). However, at E18, the alcohol induced GABA-Aβ3 down regulation was prevented by the peptides (P=0.01) with levels higher than controls (P=0.007). Prenatal treatment with neuroprotective peptides prevented the alcohol-induced decline and resulted in a significant elevation in GABA-Aβ3 ten days after alcohol exposure. Since palate formation continues through gestational day 18, the increase in GABA-Aβ3 expression may be an attempt to overcompensate for alcohol's effects and may play a role in the prevention of cleft lip and palate. These findings suggest that NAP and SAL may be beneficial for prevention of cleft palate in FAS and in other pathologic conditions.

Ethanol impairs Rho GTPase signaling and differentiation of cerebellar granule neurons in a rodent model of fetal alcohol syndrome.

Developmental exposure to ethanol impairs fetal brain development and causes fetal alcohol syndrome. Although the cerebellum is one of the most alcohol-sensitive brain areas, signaling mechanisms underlying the deleterious effects of ethanol on developing cerebellar granule neurons (CGNs) are largely unknown. Here we describe the effects of in vivo ethanol exposure on neurite formation in CGNs and on the activation of Rho GTPases (RhoA and Rac1), regulators of neurite formation. Exposure of 7-day-old rat pups to ethanol for 3 h moderately increased blood alcohol concentration (BAC) ( approximately 40 mM) and inhibited neurite formation and Rac1 activation in CGNs. Longer exposure to ethanol for 5 h resulted in higher BAC ( approximately 80 mM), induced apoptosis, inhibited Rac1, and activated RhoA. Studies demonstrated a regulatory role of Rho GTPases in differentiation of cerebellar neurons, and indicated that ethanol-associated impairment of Rho GTPase signaling might contribute to brain defects observed in fetal alcohol syndrome.

Effect of a single dose of ethanol on developing peripheral nerve of chick embryos

Fetal Alcohol syndrome (FAS) is a condition occurring in some children of mothers who have consumed alcohol during pregnancy. It is characterized by physical and mental growth retardation, craniofacial anomalies and derangements of numerous body systems. A particular distressing feature of FAS is affection of the nervous system, which manifest as motor and sensory derangements and neurobehavioral deficits. However, no specific changes in the brain and spinal cord that could conclusively explain these neuronal defects have been reported. The aim of this study was to investigate the effects of a single high dose of ethanol exposure during early gestation on the peripheral nerve, with a view to understanding the neuronal disorders appearing in FAS. A chick model of FAS was used and embryos were exposed to 5%, 10% and 15% ethanol immediately after hatching. The effects on general growth and development and on the peripheral nerve of ethanol-exposed embryos were examined following the full period of gestation. There was a significant reduction in crown-rump length, head circumference and body weight in ethanol-exposed chicks as compared with appropriate control groups. The degree of growth retardation among the different alcohol exposed groups was dose dependent and significant prenatal embryo mortality was seen in those exposed to 15% ethanol. In embryos that survived the exposure to 15% ethanol, features of myelin degeneration were observed in the peripheral nerve in the majority of cases. No features of myelin degeneration were observed in embryos exposed to 5% and 10% ethanol or in the control groups. This study demonstrates the direct toxic effects of a single high dose of ethanol on developing embryos in general and the peripheral nerves in particular. Accordingly the pathological changes observed in peripheral nerves could account for some of the neurobehavioral deficits observed in FAS.

Prenatal alcohol exposure alters GABA Aα 5 expression: A mechanism of alcohol-induced learning dysfunction

In a model for fetal alcohol syndrome (FAS), we have previously found an alteration in NMDA receptors suggesting mediation, at least in part, of alcohol-related learning deficit. NMDA and GABA receptors interact in a multisynaptic circuit for the regulation of the inhibitory tone through the CNS. The GABA receptor subunit GABA(A)alpha5 is involved in learning and is developmentally regulated, as it is excitatory in the perinatal brain and inhibitory in the adult. We were interested to evaluate alcohol's effect on GABA(A)alpha5 expression to further understand alcohol-induced learning dysfunction. Timed, pregnant C57B16/J mice were treated on gestational day 8 with alcohol (25% alcohol, 0.03 mL/kg i.p.) or control (saline). Embryos and brains were harvested 10 days after treatment, and brains from adult offspring were collected after evaluation in the Morris Water Maze, a well-established test for spatial learning. Gene expression included samples from at least 3 litters per timepoint, and calibrator-normalized relative real-time polymerase chain reaction (PCR) was performed to quantify GABA(A)alpha5 with GAPDH standardization. Statistical analysis included analysis of variance (ANOVA). Prenatal alcohol exposure significantly decreased GABA(A)alpha5 expression in the embryo (P < .02) and fetal brains (P < .01) 10 days after therapy. However, in adult brains GABA(A)alpha5 expression was increased versus controls (P < .01). As previously demonstrated, prenatal alcohol exposure resulted in deficits in adults learning the Morris Water Maze with controls learning faster (P < .05). Prenatal alcohol exposure alters developmental GABA(A)alpha5 expression. This may further explain the long-lasting damage of alcohol on learning skills. Both the alcohol-induced reduction in the GABA(A)alpha5 subunit during development and up-regulation in adult brain may be related to learning deficits resulting in decreased learning potential caused by the developmental defect and an increased inhibition of learning resulting from increased expression as an adult. In combination with our previous findings, these suggest that alcohol-induced learning impairment is likely the result of alterations of both NMDA and GABA expression and function.

Prevention of alcohol-induced learning deficits in fetal alcohol syndrome mediated through NMDA and GABA receptors

Vasoactive intestinal peptide (VIP)-related peptides prevented the learning deficit in the offspring in a model for fetal alcohol syndrome. We evaluated whether the mechanism of the peptide protection included NR2B, NR2A, and GABAAalpha5. Timed, pregnant C57BL6/J mice were injected on gestational day 8 with alcohol (0.03 mL/kg), placebo, or alcohol plus peptides. Embryos were harvested after 6 hours, 24 hours, and on gestational day 18. Some of the litters were allowed to deliver, and the adult brains harvested after the offspring were tested for learning. Calibrator-normalized relative real-time polymerase chain reaction (PCR) was performed using primers for NR2B, NR2A, and GABAAalpha5 with GAPDH standardization. Statistic: analysis of variance (ANOVA) and Fisher PLSD, P < .05 was considered significant. In the embryo, the peptides prevented NR2B rise (P < .001) at 6 hours, NR2B down-regulation (P = .002), and GABAAalpha5 decrease (P < .01) on gestational day 18. In the adult, the peptides prevented NR2B down-regulation (P = .01) and NR2A up-regulation (P < .001). VIP-related peptides prevented alcohol-induced changes in NR2B, NR2A, and GABAAalpha5. This may explain, at least in part, the peptides' prevention of alcohol-induced learning deficits.

Chronic gestational exposure to ethanol causes insulin and insulin‐like growth factor resistance in the developing brain: link to impaired acetylcholine biosynthesis

In an experimental model of fetal alcohol syndrome (FAS), cerebellar hypoplasia was found to be associated with increased apoptosis and impaired insulin-stimulated survival signaling. However, little is known about the effects produced following lower levels of ethanol exposure. This study characterizes the dose-effects of chronic gestational ethanol exposure on cerebellar development and the expression of genes required for insulin and insulin-like growth factor signaling, and also examines upstream mechanisms and downstream consequences of ethanol-impaired insulin and IGF signaling in relation to acetylcholine biosynthesis. Pregnant Long-Evans rats were fed isocaloric liquid diets in which ethanol comprised 0%, 8%, 18%, 26%, or 35.4% of the caloric content beginning on gestation day 6 and continuing throughout pregnancy. After examination of the rat pup cerebella (P0), the results demonstrated ethanol dose-dependent increases in the degree of cerebellar hypoplasia, DNA damage, and neuronal cell loss. In addition, ethanol dose-dependent reductions in brain insulin and choline acetyltransferase (ChAT) expression, and increased expression of acetylcholinesterase were observed. Despite normal receptor expression levels, ligand binding to insulin and IGF receptors was significantly reduced. Further studies linked the ethanol-impaired receptor binding and stimulation of ChAT to reduced membrane cholesterol content. In conclusion, FAS associated cerebellar hypoplasia is correlated with impaired neuronal cell survival and decreased levels of ChAT expression, mediated by pathological alterations in membrane lipid composition that result in impaired ligand-receptor binding.

Perinatal ethanol exposure alters met-enkephalin levels of male and female rats

This study used a rat model of Fetal Alcohol Syndrome to investigate whether combined prenatal and postnatal ethanol exposure affects met-enkephalin levels in the brains of male and female Long–Evans adult rats. Intragastric ethanol was administered to a group of rats (ET) from gestational day (GD) 1 through 22 and from postnatal day (PD) 2 through 10. The control groups consisted of a nontreated control group (NTC) and an intubated control group (IC) that received the intragastric intubation procedure but no exposure to ethanol. We measured met-enkephalin levels in the prefrontal cortex, nucleus accumbens, hypothalamus, central and basolateral nucleus of amygdala and ventral tegmental area. Met-enkephalin levels in the hypothalamus of male and female ET animals were significantly higher than those in either the NTC or IC animals. Met-enkephalin levels in the central nucleus of the amygdala of male and female ET animals were significantly lower than the levels in the NTC animals. Met-enkephalin levels in the nucleus accumbens of ET females were significantly greater than those in the IC females. These results demonstrate that the combination of prenatal and postnatal ethanol exposure affects basal met-enkephalin levels in specific regions in a sex-specific manner. These changes in met-enkephalin levels may explain how early ethanol exposure affects opioid-regulated behaviors such as social play, sexual behavior, and other social behaviors.

Japanese medaka (Oryzias latipes): developmental model for the study of alcohol teratology

Animal models are necessary to investigate the mechanism of alcohol-induced birth defects. We have used Japanese medaka (Oryzias latipes) as a non-mammalian model to elucidate the molecular mechanism(s) of ethanol teratogenesis. Medaka eggs, within 1 hr post-fertilization (hpf) were exposed to waterborne ethanol (0-1000 mM) in hatching solution for 48 hr. Embryo development was observed daily until 10 days post-fertilization (dpf). The concentration of embryonic ethanol was determined enzymatically. Cartilage and bones were stained by Alcian blue and calcein, respectively and skeletal and cardiovascular defects were assessed microscopically. Genetic gender of the embryos was determined by PCR. Levels of two isoenzymes of alcohol dehydrogenase (Adh) mRNAs were determined by semi-quantitative and real-time RT-PCR. The concentration of ethanol required to cause 50% mortality (LC50) in 10 dpf embryos was 568 mM, however, the embryo absorbed only 15-20% of the waterborne ethanol at all ethanol concentrations. The length of the lower jaw and calcification in tail fin cartilaginous structures were reduced by ethanol exposure. Active blood circulation was exhibited at 50+ hpf in embryos treated with 0-100 mM ethanol; active circulation was delayed and blood clots developed in embryos treated with 200-400 mM ethanol. The deleterious effects of ethanol were not gender-specific. Moreover, ethanol treatment was unable to alter the constitutive expression of either Adh5 or Adh8 mRNA in the medaka embryo. Preliminary results suggested that embryogenesis in medaka was significantly affected by ethanol exposure. Phenotypic features normally associated with ethanol exposure were similar to that observed in mammalian models of fetal alcohol syndrome. The results further indicated that medaka embryogenesis might be used as an alternative non-mammalian model for investigating specific alterations in gene expression as a means to understand the molecular mechanism(s) of ethanol-induced birth defects.

Reversal of Neuronal Migration in a Mouse Model of Fetal Alcohol Syndrome by Controlling Second-Messenger Signalings

The brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. Here we show that Ca2+ signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. Acute administration of ethanol reduced the frequency of transient Ca2+ elevations in migrating neurons and cGMP levels and increased cAMP levels. Experimental manipulations of these second-messenger pathways, through stimulating Ca2+ and cGMP signaling or inhibiting cAMP signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. Each second messenger has multiple but distinct downstream targets, including Ca2+/calmodulin-dependent protein kinase II, calcineurin, protein phosphatase 1, Rho GTPase, mitogen-activated protein kinase, and phosphoinositide 3-kinase. These results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways.

Prenatal alcohol exposure alters GABAA5 expression: A mechanism of alcohol-induced learning dysfunction

Prenatal alcohol contributes to long-term learning disabilities, affecting 1/200 US live-births. In a model for fetal alcohol syndrome (FAS), we showed that alteration in NMDA receptors mediates adult alcohol-related learning deficit (Toso L AJOG2005). Alcohol is GABA-agonist and alcohol-induced inhibition of the excitatory NMDA may reduce activation of GABA inhibitory signals, resulting in uncontrolled firing at the synapse. GABA receptor subunit GABAAα5 is involved in spatial learning and is developmentally regulated, being excitatory in the perinatal brain and inhibitory in the adult.

NMDA in adult mouse brain: Mechanism of learning deficits prevention after prenatal alcohol exposure

Fetal Alcohol Syndrome (FAS) is characterized by developmental anomalies and life-long learning impairment.In a model for FAS we showed that prenatal treatment with neuroprotective peptides (NAP+SAL) prior to alcohol exposure prevented alcohol-induced learning deficits (Spong CY JPET 2001). These results were partially explained by their ability to prevent alcohol-induced alterations in the NMDA receptor subunit composition during embryogenesis (Toso L SGI 2005). NMDA are tetrameric complexes whose functionality depends on subunit composition.

N-Methyl-D-aspartate subunit expression during mouse development altered by in utero alcohol exposure

Alcohol-related neurodevelopmental disorders are contributors to long-term learning disabilities. By using a model for fetal alcohol syndrome, we have shown that prenatal alcohol exposure results in adult learning deficits of unknown mechanisms. In the developing hippocampus, the N-methyl-D-aspartate (NMDA) receptor subunit NR2B triggers long-term potentiation, fundamental to learning and memory; this is supplemented by the less plastic NR2A subunit in the adult. To understand the mechanism of learning deficits in FAS, we evaluated NR2B and NR2A expression in embryonic and adult mice. Pregnant C57Bl6/J mice were treated on gestational day 8 with alcohol or control (saline solution). Embryos were harvested at 6 hours, 24 hours, and 10 days, and brains from adult offspring were collected at 3 months (after evaluation for learning deficit). Calibrator-normalized relative real-time polymerase chain reaction was performed for NR2B and NR2A with glyceraldehyde-3-phosphate dehydrogenase standardization. Statistical analysis included analysis of variance. At 6 hours, NR2B expression in the alcohol-exposed embryos was higher than in controls (P < .01). NR2A was not expressed in either group. By 24 hours there was no difference in NR2B (P = .3). However, at 10 days NR2B was lower in alcohol-exposed animals (P = .02). In the adult brains there was a relative decrease in NR2B (P = .03) and an increase in NR2A (P < .01). Prenatal alcohol exposure during development induces NR2B expression deregulation in the embryos that persists until adulthood, when a relative increase in the less modifiable subunit NR2A occurs. This alteration in NMDA receptor subunits may underlie the learning abnormalities in fetal alcohol syndrome.

Novel peptides prevent alcohol-induced spatial learning deficits and proinflammatory cytokine release in a mouse model of fetal alcohol syndrome

Previously, the novel peptides NAPVSIPQ and SALLRSIPA were shown to prevent alcohol-induced fetal death and growth abnormalities in a mouse model of fetal alcohol syndrome. This study evaluated whether these peptides could prevent long-term alcohol-induced learning abnormalities. In addition, because specific cytokines are known to effect long-term potentiation, a model of learning at the molecular level, we studied the effect of these novel peptides on tumor necrosis factor-alpha, interleukin-6, and interferon-gamma levels. We used a well-characterized mouse model of fetal alcohol syndrome. Pregnant mice were injected on day 8 with alcohol (0.03 mL/kg) or placebo. Pretreatment with NAPVSIPQ+SALLRSIPA (20 mug) or placebo was given 30 minutes before alcohol. Embryos were removed after 6 hours, at which time cytokine, tumor necrosis factor-alpha, interleukin-6, and interferon-gamma levels were measured with enzyme-linked immunoassays. To test spatial learning, adult offspring from litters that were treated with alcohol, control, NAPVSIPQ+SALLRSIPA then alcohol, or NAPVSIPQ+SALLRSIPA alone were evaluated for latency to find a hidden platform in the Morris water maze. Alcohol treatment increased tumor necrosis factor-alpha levels versus control levels (50.0 +/- 3.5 pg/mL vs 32.7 +/- 2.4 pg/mL; P < .001). NAPVSIPQ+SALLRSIPA pretreatment prevented this increase (39.9 9 +/- 2.8 pg/mL; P </= .01), with levels similar to control (P=.1). Similarly, alcohol increased interleukin-6 levels versus control levels (22.6 +/- 1.4 pg/mL vs 17.3 +/- 0.6 pg/mL; P < .001), and NAPVSIPQ+SALLRSIPA prevented this increase (19.1 +/- 1.0 pg/mL; P </= .02), with levels similar to control levels (P=.2). Interferon-gamma levels were not different among the 3 groups (alcohol, 14.6 +/- 4.9 pg/mL; control, 17.9 +/- 6.6 pg/mL; alcohol+NAPVSIPQ+SALLRSIPA, 13.6 +/- 4.9 pg/mL; P=.2). In the Morris water maze, alcohol-treated groups did not learn over the 7-day trial compared with the control group (P=.001). Groups that were pretreated with NAPVSIPQ+SALLRSIPA then alcohol learned significantly, which was similar to the control group. Groups that were treated with only NAPVSIPQ+SALLRSIPA learned significantly earlier, with the shortest latency once learning commenced. The peptides, NAPVSIPQ+SALLRSIPA, prevented the alcohol-induced spatial learning deficits and attenuated alcohol-induced proinflammatory cytokine increase in a model of fetal alcohol syndrome. This study demonstrates the peptides' significant in vivo efficacy with long-lasting effects obtained after prenatal administration.

Prevention of alcohol-induced developmental delays and learning abnormalities in a model of fetal alcohol syndrome

Prenatal alcohol exposure results in fetal death and neurobehavioral complications including learning impairment. Previously synthetic peptides derived from activity-dependent neurotrophic factor have been shown to prevent aspects of alcohol-induced damage in pregnancy. The objective of this work was to evaluate whether activity-dependent neurotrophic factor-12 could prevent alcohol-induced damage in a model of fetal alcohol syndrome. Using a well-characterized model, C57Bl6/J mice on gestational day 8 were treated with placebo, alcohol (30% volume/volume alcohol 0.03 mL/kg), alcohol plus activity-dependent neurotrophic factor-12 30 minutes prior to alcohol, or activity-dependent neurotrophic factor-12 alone. Fetal death was assessed on gestational day 18 (25 litters were evaluated: alcohol, n = 5; placebo, n = 9; alcohol plus activity-dependent neurotrophic factor-12, n = 11). Neonatal behavior tests were performed on postnatal days 1 through 21 with the offspring of 12 dams (alcohol, n = 16; placebo, n = 46; alcohol plus activity-dependent neurotrophic factor-12, n = 23; and activity-dependent neurotrophic factor-12, n = 35). Adult males were tested in the Morris water maze for learning assessment and with the hole punch activity test for exploratory activity. Statistical analysis included Kruskal-Wallis and analysis of variance. Fetal death was greater in alcohol (67% +/- 13%) vs placebo (8.4% +/- 3%, P < .001). Pretreatment with activity-dependent neurotrophic factor-12 prevented the alcohol-induced fetal death (2.2% +/- 8.1%) with levels similar to control (P = .12). Alcohol exposure caused a delay in achieving developmental milestones, with alcohol achieving milestones later than all other groups (all P < .001). Pretreatment with activity-dependent neurotrophic factor-12 prevented the alcohol-induced milestone delays. In the Morris water maze, the placebo learned, decreasing their latency to find the hidden platform over 70% (P < .01). Alcohol plus activity-dependent neurotrophic factor-12 also significantly learned, with a learning curve not different from placebo (all P > .5) and significantly better than alcohol on days 4, 6, and 7 (all P < .05). Alcohol exposure resulted in significantly less time in hole punch activity (P < .02) than control. Activity-dependent neurotrophic factor-12 pretreatment prevented the alcohol-induced decline, with levels the same as control (P = .1). The novel peptide activity-dependent neurotrophic factor-12 prevents alcohol-induced fetal death and developmental and learning abnormalities in a model of fetal alcohol syndrome. This demonstrates that a single treatment with a peptide is efficacious and may be of value in the prevention of alcohol-induced damage.

Short-Term Memory Impairment and Reduced Hippocampal c-Fos Expression in an Animal Model of Fetal Alcohol Syndrome

Previous work in our laboratory has shown that exposure to ethanol during the brain growth spurt impairs spatial short-term memory in rats on the delayed matching-to-place (DMP) version of the Morris water maze. The objectives of this study were to ascertain whether this impairment could: 1) be prevented by increasing the length of encoding time and 2) be related to hippocampal c-Fos expression. Using an artificial rearing model, male Long-Evans rats were fed 6.5 g/Kg/day of ethanol from postnatal days 6-9, with controls fed an isocaloric amount of maltose dextrin. As adults, rats in each treatment condition were trained and subsequently tested on either the DMP version of the Morris water maze, or on a random platform version (RAN) that incorporated the same performance requirements, but disallowed spatial learning. Brains were processed for c-Fos expression. Ethanol-exposed rats showed longer search trials during training and took longer to learn the DMP task. When the delay between search and recall trials was increased from 60 sec to 120 min, the performance of ethanol-exposed rats was impaired compared with that of controls after a 10 sec, but not after a 45 sec, encoding time. Brain c-Fos expression was increased in hippocampus, prefrontal cortex and visual cortex in rats trained on the DMP compared to the RAN task. Furthermore, in the DMP-trained rats, hippocampal c-Fos expression was lower in ethanol-exposed rats. These results suggest that the short-term memory impairment of ethanol-exposed rats 1) can be improved slightly by an increase in encoding time and 2) is related to a decrease in c-Fos expression in the hippocampus.

Ethanol inhibits insulin expression and actions in the developing brain

Ethanol-induced cerebellar hypoplasia is associated with inhibition of insulin-stimulated survival signaling. The present work explores the mechanisms of impaired insulin signaling in a rat model of fetal alcohol syndrome. Real-time quantitative RT-PCR demonstrated reduced expression of the insulin gene in cerebella of ethanol-exposed pups. Although receptor expression was unaffected, insulin and insulin-like growth factor (IGF-I) receptor tyrosine kinase (RTK) activities were reduced by ethanol exposure, and these abnormalities were associated with increased PTP1b activity. In addition, glucose transporter molecule expression and steady-state levels of ATP were reduced in ethanol-exposed cerebellar tissue. Cultured cerebellar granule neurons from ethanol-exposed pups had reduced expression of genes encoding insulin, IGF-II, and the IGF-I and IGF-II receptors, and impaired insulin- and IGF-I-stimulated glucose uptake and ATP production. The results demonstrate that ethanol inhibits insulin-mediated actions in the developing brain by reducing local insulin production and insulin RTK activation, leading to inhibition of glucose transport and ATP production.