Fetal alcohol syndrome is the most common nongenetic cause of mental retardation and occurs in 0.2 to 1.5 per 1,000 live births in the United States.1 The syndrome includes growth restriction, craniofacial dysmorphology, neurodevelopmental anomalies, and life-long compromises in learning and memory. Often, alcohol exposure during pregnancy does not result in all of the features of fetal alcohol syndrome but is associated with less severe findings known as fetal alcohol spectrum disorder, which includes a range of developmental outcomes, including learning deficits and neurobehavioral abnormalities.2 The mechanism by which prenatal alcohol exposure affects brain development is multifactorial.
N-methyl-d-aspartate (NMDA) receptors are ionotropic glutamate receptors critically involved in brain development and synaptic plasticity, including those associated with learning and memory. Alcohol is a known NMDA antagonist and inhibits NMDA receptor–dependent long-term potentiation. Long-term potentiation is considered the cellular equivalent of long-lasting memory in paradigms of cognition and learning and is initiated by NMDA receptor activation. Previous studies have suggested that ethanol exposure decreases the total number and function of NMDA receptors in the hippocampus and other brain regions.3–5 This alteration of NMDA receptor pharmacology could be related to modifications of NMDA receptor subunit composition. N-methyl-d-aspartate receptors comprising NR1/NR2A and/or NR1/NR2B subunits are the most sensitive to acute ethanol exposure.5,6 Whereas NR2B is present during embryonic development and is the more plastic subunit facilitating learning, NR2A expression commences only after birth and is the more rigid subunit.
Furthermore, NMDA and γ-aminobutyric acid type A (GABAA) receptors have been shown to mediate alcohol-induced neuronal loss and apoptosis during postnatal stages of development.7 The GABA receptor subclass GABAA plays a regulative role in long-term potentiation by means of inhibition of glutamate transmission. In addition to its toxic effect on neurons, alcohol has also been shown to affect glial cells; in particular, astrocytes.8
Vasoactive intestinal peptide (VIP) is a neuropeptide with neurotrophic actions that influence mitosis, neuronal survival, and neurodifferentiation and plays an important role in the regulation of embryonic growth and development.9 Vasoactive intestinal peptide stimulation of high-affinity receptors on astrocytes results in the release of neurotrophic factors, including activity-dependent neuroprotective protein and activity-dependent neurotrophic factor, which have demonstrated neuroprotective proprieties.10–12 In both humans and mice, activity-dependent neuroprotective protein is expressed predominantly in the cerebellum, hippocampus, and cerebral cortex, and has an important role in brain function.12 NAPVSIPQ, a sequence contained in activity-dependent neuroprotective protein, and SALLRSIPA, derived from the related activity-dependent neurotrophic factor, demonstrated potent protective effects against oxidative stress associated with alcohol exposure, electrical blockade induced by tetrodotoxin, and ethanol inhibition of L1 cell adhesion.13–15
Previous studies have demonstrated that NAPVSIPQ and SALLRSIPA administered prenatally prevent alcohol-induced damage, including fetal death, growth abnormalities, and learning deficits in adult offspring.13,16,17 They have also been found to prevent alcohol-induced changes in NR2A, NR2B, and GABAAα5 during development and adulthood.18 Because pregnant women do not often admit consuming alcohol during pregnancy and because treatment during pregnancy may alter normal and abnormal development, prenatal therapy may be of limited value.
The objective of this study was to evaluate whether treatment with the peptides prevents alcohol-induced learning deficits and whether the mechanism of the peptides involves the NMDA and GABAA receptors in young adult mice exposed prenatally to alcohol. Because glial cells are a target of ethanol toxicity during brain development and release of neurotrophic factors that are important for neuronal development and synaptic plasticity,8 VIP, activity-dependent neuroprotective protein, and glial fibrillary acidic protein (GFAP) (an astrocyte marker) expression was also evaluated.
MATERIALS AND METHODS
C57Bl6/J female mice (The Jackson Laboratory, Bar Harbor, ME) were kept under a 12-hour-light/12-hour-dark regimen, with food and water available at all times. The mice received humane animal care in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Animal and Care Use Committee. Six- to 8-week-old female mice were mated with C57Bl/6J male mice for 4–6 hours to minimize time variability of conception. The presence of a vaginal plug was considered day 0 of pregnancy.
A well-described model for fetal alcohol syndrome was used.19 On gestational day 8, pregnant mice were injected intraperitoneally with 25% ethyl alcohol in saline solution at 0.03 ml/g (alcohol) or saline solution alone (placebo). Because the animals receiving alcohol were incapacitated for approximately 6 hours after injection, food and water were withheld in both groups for 6 hours. After delivery, animals were weaned on postnatal day 20, and male offspring were ear-tagged. Male offspring were used to eliminate any potential hormonal effects encountered in female mice at this age. The all-D amino acid configurations of the peptides (D-NAPVSIPQ and D-SALLRSIPA) were used that allow for absorption after oral administration.15 On postnatal day 40, male offspring exposed to alcohol in utero were treated by means of gavage with 40 micrograms of D-NAPVSIPQ and 40 micrograms of D-SALLRSIPA (n=20) or placebo (n=13) (0.2-ml dose), and control offspring were gavaged with placebo daily (n=46). All treatments were coded to the ear tag to keep the group assignment blinded. The animals were treated for 10 consecutive days and the tests for learning and memory were started at day 3 of treatment, allowing a 3-hour latency between treatment (10:00 am) and testing (13:00 pm). D-NAPVSIPQ was diluted in 50 microliters of dimethyl sulfoxide and diluted in 2.5 ml of filtered Dulbecco's phosphate-buffered saline solution; D-SALLRSIPA was dissolved and diluted in filtered Dulbecco's phosphate-buffered saline solution. Animals were fasted 1 hour before treatment to avoid interference with drug absorption. Food and water were replaced immediately after treatment.
Learning and memory were evaluated with the Morris watermaze and the T-maze continuous alternation task.20,21 The watermaze evaluates spatial learning, a measure of cognitive function. The apparatus consists of a circular pool with a water level of approximately 30 cm, maintained at 24–26°C (Columbus Instruments, Columbus, OH). Nontoxic tempura paint is added to make the water opaque and to blend with the color of the pool wall. External cues (eg, arrow, star, circle, and rectangle) are located around the pool as references of location for the mice. Each animal underwent two consecutive trials daily of finding the hidden platform using the visual cues external to the water maze. Two trials were completed daily, with the average time required to reach the platform recorded over 7 consecutive days. The latency in finding the hidden platform for each trial was recorded and the average of the two trials was calculated for each of the 7 days. Two separate independent trials were performed and the data combined.
The T-maze is a task that measures the exploratory behavior and the spatial memory capacity in rodents. The apparatus consists of a wooden T-shaped platform (length of start and goal stems, 75 cm; width, 10 cm; height, 20 cm). The recording session consisted of a forced trial and 14 subsequent choice trials. After 5 seconds of confinement in the start box, the door was lifted and the mice were allowed to explore the start arm and one of the goal arms. Entry to the other goal arm was blocked. This was the first forced trial. The mice explored the area available to them and eventually reentered the start arm and moved down to the start box. When they entered the start box, they were confined there for 5 seconds by putting the start box door in place. During confinement, the door blocking one of the goal arms was removed. The start box door was then released and the mice engaged in the second trial. However, this time they could choose between the two goal arms (first free choice). After the mouse had chosen and entered one goal arm halfway down, the other arm was blocked by a door. The mice readily left the explored goal arm and moved down to the start box again. They were confined there for 5 seconds again and the testing cycle was repeated. The overall alternation rate during 14 free-choice trials was calculated and analyzed.
After testing, the animals were killed by carbon dioxide asphyxiation and brain tissues were removed quickly. Hippocampus, cerebellum, and cortex were collected separately, frozen on liquid nitrogen, and kept at −80°C until homogenization. For RNA extraction and protein isolation, the samples were ground to a fine powder with a mortar and processed with Nucleospin RNA/protein kit (Machery-Nagel, Düren, Germany). A 5-microliter aliquot was taken for spectrophotometric determination of RNA content. Using 5 micrograms of total RNA, the reverse-transcriptase reaction was performed (Applied Biosystems, Foster City, CA) in a final volume of 150 microliters.
Calibrator-normalized real-time polymerase chain reaction (PCR) was performed using samples from at least 3 liters per treatment. For real-time PCR, the NR2A and NR2B primer pair was synthesized by IDT (Integrated DNA Technologies, Coralville, IA). The GABAAα5, GABAAβ3, and VIP primer pair were designed and synthesized by TIB Molbiol (Adelphia, NJ), the activity-dependent neuroprotective protein primer pair was synthesized by Biosynthesis (Louisville, TX), and the GFAP primer pair was synthesized by Superarray (Frederick, MD). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. The GAPDH gene, a housekeeping gene located on mouse chromosome 6, is used frequently as a normalized gene because of its stability in cells. With the use of the FastStart DNA Master SYBR Green 1 dye-base detection (Roche, Indianapolis, IN), VIP, activity-dependent neuroprotective protein, GFAP, NR2A, NR2B, GABAAα5, GABAAβ3, and GAPDH expression was measured by real-time PCR using the LightCycler with relative quantification software (Roche) and melting point analysis to assess the specificity of the amplified genes. To further eliminate the risk of cross contamination, LightCycler Uracil-DNA Glycosylase (Roche Diagnostic Corp) was added to the master mix in all PCR experiments. The presence and purity of target gene sequence expression in the real-time PCR reaction were confirmed by gel electrophoresis. Samples were run in duplicate and relative quantification was performed with calibrator-normalized data with efficiency correction. Results are presented as the normalized ratios of VIP, activity-dependent neuroprotective protein, GFAP, NR2A, NR2B, GABAAα5, and GABAAβ3 to GAPDH expression.
Statistical analysis was performed with analysis of variance for overall comparison between the treatment groups, and post hoc Fisher's analysis for comparison between pairs, with P<.05 considered significant (StatView 5.0.1, SAS Institute, Inc., Cary, NC).
Treatment of young adult mice with D-NAPVSIPQ and D-SALLRSIPA reversed the alcohol-induced learning deficits in the watermaze and in the T-maze. In the watermaze, animals exposed prenatally to alcohol did not appreciably learn, compared with control (alcohol compared with control, P<.05 on days 7, 8, 9, and 10), whereas mice exposed in utero to alcohol and treated with D-NAPVSIPQ and D-SALLRSIPA as young adults for 10 days learned similar to the control animals and significantly better than animals exposed to alcohol in utero and treated with placebo (alcohol compared with alcohol and D-NAPVSIPQ plus D-SALLRSIPA, P<.05 on days 7, 8, 9 and 10; alcohol and D-NAPVSIPQ plus D-SALLRSIPA compared with control, P>.05 on days 7, 8, 9, and 10) (Fig. 1A). In the T-maze, where a higher rate of alternation is desirable, demonstrating learning and exploration behavior, animals exposed to alcohol in utero had a lower alternation rate compared with the control animals. The animals exposed prenatally to alcohol and treated with D-NAPVSIPQ and D-SALLRSIPA in young adulthood performed similar to control and significantly better then those exposed prenatally to alcohol and treated with placebo as young adults (alcohol compared with control, P<.05; alcohol compared with alcohol and D-NAPVSIPQ plus D-SALLRSIPA, P<.05; alcohol and D-NAPVSIPQ plus D-SALLRSIPA compared with control, P=.09) (Fig. 1B).
PCR analysis showed a down-regulation of NR2A and NR2B expression in the hippocampus, and concomitantly an up-regulation of NR2A expression in the cortex and NR2B expression in the cortex and cerebellum after prenatal alcohol exposure (NR2A and NR2B alcohol compared with control, all P<.05) (Fig. 2). Young adult treatment with D-NAPVSIPQ and D-SALLRSIPA reversed the down-regulation of NR2A and NR2B in the hippocampus, the up-regulation of NR2A in the cortex, and NR2B in the cortex and cerebellum (alcohol compared with alcohol and D-NAPVSIPQ plus D-SALLRSIPA, all P<.05) (Fig. 2). There was no difference in NR2A expression in the cerebellum (alcohol compared with control, P=.19) (Fig. 2A).
There was no difference in GABAAβ3 expression in the animals exposed to alcohol in utero and in controls in the hippocampus and in the cortex (alcohol compared with control, all P>.05). Treatment with D-NAPVSIPQ and D-SALLRSIPA did not reverse the alcohol-induced down-regulation of GABAAβ3 in the cerebellum after prenatal alcohol exposure (alcohol compared with control, P<.05; alcohol compared with alcohol and D-NAPVSIPQ plus D-SALLRSIPA, P=.51). No significant differences were found in GABAAα5 expression in the animals exposed to alcohol in utero and in the controls (alcohol compared with control, P>.05).
Activity-dependent neuroprotective protein, VIP, and GFAP expression were evaluated in young adult brains after in utero alcohol exposure. Prenatal alcohol exposure induced a down-regulation of VIP in the hippocampus (alcohol compared with control, P=.033) that was not reversed by treatment with D-NAPVSIPQ and D-SALLRSIPA (alcohol compared with alcohol and D-NAPVSIPQ plus D-SALLRSIPA, P=.883). There was no difference in the expression of VIP in cerebral cortex and cerebellum after prenatal alcohol exposure (alcohol compared with control; all P>.05) (Fig. 3A). No significant differences were found in the expression of activity-dependent neuroprotective protein in the hippocampus and cerebellum after prenatal alcohol exposure (alcohol compared with control; all P>.05), whereas prenatal alcohol exposure induced an up-regulation of activity-dependent neuroprotective protein in the cortex (alcohol compared with control, P=.016) that was reversed by young adult treatment with D-NAPVSIPQ and D-SALLRSIPA (alcohol compared with alcohol and D-NAPVSIPQ plus D-SALLRSIPA, P=.006) (Fig. 3B). There was no difference in GFAP (an astrocyte marker) expression between animals exposed to alcohol in utero and control animals (alcohol compared with control, P>.05).
We found that treatment with D-NAPVSIPQ and D-SALLRSIPA reversed the alcohol-induced learning deficit in young adult mice after prenatal alcohol exposure as well as the NR2A and NR2B changes in hippocampus and cortex. The alteration of NR2A and NR2B after prenatal alcohol exposure may have effects on normal brain development and plasticity.
NAPVSIPQ and SALLRSIPA have been shown to be neuroprotective in vivo against diverse neuronal insults, including excitotoxicity, oxidative stress, closed head injury, and ischemic brain injury.22–24 In a model of fetal alcohol syndrome that was based on chronic alcohol exposure, treatment with SALLRSIPA reversed alcohol-induced brain growth compromise and anatomical alterations.16 Intranasal administration of SALLRSIPA reversed learning deficit induced by a blocker of choline-uptake,25 and daily injections of NAPVSIPQ to newborn Apo-lipoprotein E–deficient mice, a model for Alzheimer disease, accelerated the acquisition of developmental reflexes and long-term memory deficits.12 In a mouse model for Down syndrome, prenatal treatment with NAPVSIPQ and SALLRSIPA reversed developmental deficits in newborn pups.26 In normal animals, NAPVSIPQ and SALLRSIPA have been shown to enhance learning.27 However, despite the well-documented neurotrophic and neuroprotective actions of NAPVSIPQ and SALLRSIPA in numerous abnormalities, the molecular mechanisms and signaling pathways that mediate these effects are not well understood, and recent studies of hippocampal cultures found that activity-dependent neurotrophic factor alters glutamate release and NMDA receptors.28
In this study, we confirm previous studies that found a modification in the NMDA receptors in the young adult mouse brain after prenatal alcohol exposure.3,18,29 We have shown previously that, during embryogenesis, prenatal alcohol administration down-regulates NR2B and GABAAα5. In adult brains exposed to alcohol in utero, NR2B down-regulation persists, whereas there is an up-regulation in NR2A.18
In particular, the NR2B subunit is the most critical for long-term generation of potentiation and most sensitive to the effects of ethanol. Therefore, in our study, the down-regulation of the hippocampal NR2B and the up-regulation of NR2B expression in the cortex likely affect long-term potentiation. Indeed, transgenic overexpression of NR2B by different strategies improved spatial memory in mice, and both hippocampal and extrahippocampal NR2B-containing NMDA receptors critically contribute to spatial performance, suggesting an important role for NR2B in the adult brain.30
Administration of D-NAPVSIPQ and D-SALLRSIPA to young adult mice reverses the NMDA alterations and may, at least in part, explain the concomitant hippocampus-dependent learning deficit and cortical memory formation in this model of fetal alcohol syndrome. Given the role of NMDA receptors in learning, this may clarify the mechanism of prevention of alcohol-induced learning deficits by the peptides in young adult mice with fetal alcohol syndrome and identify a therapy for prenatal alcohol-induced learning deficits. Further studies are necessary to understand how alcohol exposure can affect the expression of NMDA receptor subunits, for example, by affecting the actions of neurotrophic factors or other intracellular signaling pathways.
Unlike other studies, we did not find a significant difference in the GABAA subunits studied between animals exposed to alcohol prenatally and controls,7,18 perhaps because of the developmental period during which ethanol exposure takes place and the age at which the affected offspring were studied. Glial cells are a target of ethanol toxicity during brain development because ethanol affects glial development and function (eg, the release of neurotrophic factors that are important for neuronal development and synaptic plasticity).8 Messenger RNA levels of VIP are decreased in a mouse model of fetal alcohol syndrome.31 GFAP is an astrocyte marker, and it has been found that ethanol decreases the levels of GFAP and its gene expression on astrocytes in primary culture. Adult treatment with D-NAPVSIPQ and D-SALLRSIPA reversed the activity-dependent neuroprotective protein up-regulation in the cortex but did not reverse the VIP down-regulation in the hippocampus, probably because the peptides are downstream of VIP. We observed no change in expression of activity-dependent neuroprotective protein or GFAP in hippocampus. Vasoactive intestinal peptide stimulates the release of activity-dependent neuroprotective protein and activity-dependent neurotrophic factor from glial cells during developmental and adult neurogenesis. In fetal alcohol syndrome, VIP expression is altered and remains down-regulated through adulthood.31 Chronic alcohol use also alters VIP levels. Our interest in measuring VIP levels was to determine whether the peptides affect VIP levels themselves or work downstream only. Our findings suggest that the peptides do not affect the VIP levels but work downstream, as do the parent proteins, activity-dependent neurotrophic factor and activity-dependent neuroprotective protein.
Prenatal alcohol exposure produces changes that may interfere with the establishment of effective thresholds for plasticity in adulthood. These findings identify a novel treatment administered to the grown animal that prevents the long-term sequelae of alcohol exposure in utero and suggest a mechanism for neuroprotection. Implications of these finding may allow an intervention for children identified first during school with fetal alcohol syndrome or fetal alcohol spectrum disorder to enhance their learning ability, and may remove the concerns for administering therapies in utero.
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