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N-Methyl-D-aspartate subunit expression during mouse development altered by in utero alcohol exposure
American Journal of Obstetrics and Gynecology (2005) 193, 1534–9
www.ajog.org
N-Methyl-D-aspartate subunit expression during mouse development altered by in utero alcohol exposure Laura Toso, MD,a,b,* Sarah H. Poggi, MD,a,c Daniel Abebe,a Robin Roberson,a Veronica Dunlap,a Jane Park,a Catherine Y. Spong, MDa,c Unit on Perinatal and Developmental Neurobiology, NICHD,a National Institute of Alcohol Abuse and Alcoholism,b National Institutes of Health, Bethesda, MD; Department of Obstetrics and Gynecology, Georgetown University Hospital, Washington, DCc Received for publication November 16, 2004; accepted February 18, 2005
KEY WORDS Fetal alcohol syndrome Alcohol N-methyl-D-aspartate receptors Mouse development Learning
Objective: 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. Study design: 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). Calibratornormalized relative real-time polymerase chain reaction was performed for NR2B and NR2A with glyceraldehyde-3-phosphate dehydrogenase standardization. Statistical analysis included analysis of variance. Results: 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). Conclusion: 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. Ó 2005 Mosby, Inc. All rights reserved.
Supported by the DIR of National Institutes of Child Health and Human Development. * Reprint requests: Laura Toso, MD, Unit on Perinatal and Developmental Neurobiology, NICHD, NIH, Building 9, R 1W125, 9 Memorial Dr, Bethesda MD 20892-0925. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2005 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.02.105
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Figure 1 NR2B (10^2) messenger RNA expression in the embryos 6 hours, 24 hours, and 10 days after alcohol treatment (standardized to GAPDH). The initial rise at 6 hours in alcohol-treated embryos progressively lowers after 24 hours and decreases 10 days later. Bar represent mean G SD.
Fetal alcohol syndrome (FAS) is the most common nongenetic cause of mental retardation, occurring in 0.5 births per 1000 births each year in the United States.1 The diagnostic criteria for FAS include the presence of a characteristic set of facial features, as well as craniofacial dismorphology, microcephaly, growth restriction, and central nervous system (CNS) anomalies or dysfunction. In addition, FAS is 1 category of the fetal alcohol spectrum disorders (FASD) that include a broader range of outcomes associated with prenatal alcohol exposure, characterized by the absence of FAS facial features but with sequelae of fetal injury, including mental and learning impairments and structural brain defects, as shown by magnetic resonance imaging.2,3 Glutamatergic transmission in the CNS toward the N-methyl-D-aspartate (NMDA) receptors (NMDAR) has been strongly suggested to be 1 of the pathways damaged in FASD. NMDARs are tetrameric complexes assembled from the ubiquitous NR1 subunit, which is an essential component of all NMDAR complexes, along with various combinations of NR2 or NR3 subunits. NR1 is encoded by a single gene, whereas NR2 and NR3 subunits are encoded by 4 (NR2A-B-C-D) and 2 (NR3AB) distinct genes, respectively. The functional proprieties of the receptor complex depend on the specific subunit composition and the stoichiometry in which subunits combine to form the channel.4 Alcohol is a known NMDA-antagonist: in vitro studies have found that an ethanol intoxication episode can cause millions of
neurons to commit suicide in the developing CNS through a NMDA-mediated apoptotic neurodegenerative response, particularly in systems that play critical roles in learning, memory, sensory information processing, and cognitive function.5 Multiple mechanisms have been shown to contribute to ethanol-induced alteration in NMDARs function, including an overall decrease in NMDA receptor density and differential regulation of the expression of NMDA receptor subunits.6,7 The NR2B subunit has been shown to be the most sensitive to ethanol, and it has been postulated that a relative increase in NR2A expression in respect to NR2B subunits after chronic ethanol exposure can cause the functional changes in the NMDA ion channel.7 Molecular studies in the mouse have found that NMDAR-dependent long-term potentiation (LTP) is essential for spatial learning and memory8-10 and that a switch in the subunit composition in NMDARs from NR2B to NR2A subunits lowers the receptor susceptibility to LTP and makes the synapse less plastic.4 In a well-known mouse model for FAS, we have previously shown that alcohol induces fetal death, growth restriction at birth, and learning deficits in adulthood.11 The purpose of our study is to evaluate whether the expression of NMDARs NR2A and NR2B subunits is affected by prenatal alcohol exposure during development and whether the alcohol-induced learning deficits are due, at least in part, to a change in these subunits in the composition of the NMDAR.
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Figure 2 NR2A (A) and NR2B (B) expression in adult brains (standardized to GAPDH). In utero-exposed animals show a relative decrease in NR2B and a relative increase in the less plastic subunit NR2A. Bars represent mean G SD.
Material and methods Animals C57Bl6/J female mice (Harlan Sprague-Dawley, Inc, Indianapolis, IN) were kept in 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 (NIH) guidelines for care and use of experimental animals. The protocol was approved by National Institute of Child Health and Human Development (NICHD) Animal Care and Use Committee. Six-week-old females (21-24 g) were mated with C57-B16J males for 4 hours. The presence of vaginal plug was considered day 0 of pregnancy.
Treatment A well-described model for FAS was used.12 Animals were randomly injected intraperitoneally on gestational day 8 with 25% ethyl alcohol in saline (alcohol) solution or vehicle alone (saline solution) at 0.030 mL/g of body weight at 7 AM. Because the animals receiving alcohol were incapacitated for approximately 6 hours after injection, food and water were withheld in both groups for 6 hours.
Tissue collection and processing At 6 hours, 24 hours, and 10 days after injection, embryonic and decidua/trophoblast at 6 and 24 hours
or placental tissues at 10 days were explanted by using microdissection from the uterus and placed in phosphate-buffered saline solution (PBS). Each gestational time point included at least 3 samples with each sample representing 3 to 5 litters (a gestation typically includes 8-10 embryos). For adult tissues, 3 brains per treatment, each from a different litter, were collected separately. For RNA extraction, samples were homogenized by using disposable micropestles to avoid cross contamination. The samples were processed with SV Total RNA Isolation System (Promega, Madison, WI). A 5-mL aliquot was taken for spectrophotometric determination of RNA content. The remaining sample was stored at 80(C. With the use of 5 mg of total RNA, the reverse transcriptase (RT) reaction was performed (Perkin-Elmer Corp, Branchburg, NJ) in a final volume of 150 mL. Each RNA sample was run in duplicate. Negative controls included RT reactions omitting RNA or RT.
RT-Polymerase chain reaction NR2B (GenBank Accession Number NM_008171) primer pair was designed using Primer3 software and synthesized by IDT (Integrated DNA Technologies, Coralville, Iowa). NR2B primer sequence was 50 -CCG CAG CAC TAT TAGA GAA CA-30 (sense) and 50 -ATC CAT GTG TAG CCG TAG CC-50 (antisense). NR2A (GenBank Accession Number NM_008170) primer pair was designed by TIB Molbiol (Adelphia,
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Figure 3 NR2B messenger RNA expression in the decidua/trophoblast at 6 hours and 24 hours and placenta 10 days later after alcohol treatment (standardized to GAPDH). Bar represent mean G SD.
NJ) and synthesized by IDT (Integrated DNA Technologies). NR2A primer sequence was 50 -GGA AGT TGG ACG CTT TCA TC-30 (sense) and 50 -TCT TCC ATC TCA CCG TCA CC-30 (antisense). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as an internal standard with the primer pair synthesized by IDT. GAPDH primer sequence was 50 -TGC ACC ACC AAC TGC TTA (sense) and 50 GGA TGC AGG GAT GAT GTT C (antisense). With the use of the FastStart DNA Master SYBR Green 1 dye-base detection (Roche, Indianapolis, Ind), NR2A or NR2B and GAPDH expression were measured by real-time polymerase chain reaction (PCR) using the LightCycler with relative quantification software (Roche) and melting point analysis to assess the specificity of the amplified genes. Optimization for NR2A, NR2B, and GAPDH-specific primers was performed with separate runs by varying magnesium chloride and primer concentrations, amount of template, and annealing temperature, with a magnesium chloride concentration of 2 mmol/L for NR2A, 3 mmol/L for NR2B, and of 2.5 mmol/L for GAPDH, primer concentrations of 0.5 mmol/L for NR2B and NR2A and 0.3 mmol/L for GAPDH, 2 mL of the RT product for all the primers and annealing temperatures of 55(C for NR2B, 52(C for NR2A, and of 60(C for GAPDH considered optimal. The presence and purity
of target gene sequence expression in the real-time PCR were confirmed by gel electrophoresis. Samples were run in duplicate and relative quantification was performed by using calibrator-normalized data with efficiency correction. Results are presented as the normalized ratio of NR2A-to-GAPDH and NR2B-to-GAPDH expression. Real-time PCR comparisons were made at all time points (6 hours, 24 hours, 10 days, and 3 months) and in each tissue type (embryo, deciduas or placentas, and brains) between the alcohol and control groups. Analysis of variance was performed for overall comparisons at each time point with post hoc Fisher analysis performed to compare between pairs, with P ! .05 considered significant (StatView v5.0.1, SAS, Cary, NC).
Results At 6 hours, NR2B expression in the alcohol-exposed embryos was elevated in comparison with controls (P = .006, Figure 1). At 24 hours there was no difference in NR2B expression between the groups (P = .3); however, at 10 days there was a lower expression of NR2B in alcohol-exposed animals (P = .02, Figure 1) that persisted until adulthood in the brains (P = .03, Figure 2). NR2A was not expressed during development (6 hours, 24 hours, and 10 days).
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Figure 4 NR2B messenger RNA expression comparison between the control embryos and control at 6 hours and 24 hours in the decidua/trophoblast and 10 days later in the placenta after alcohol injection shows a relative increase over time of NR2B in the embryo, and a concomitant decrease in the decidua/trophoblast and placenta. Bars represent mean G SD.
In the adult brains, concomitant with the decrease in NR2B associated with alcohol exposure there was a relative increase in NR2A (P = .002, Figure 2). NR2B was not differentially expressed in the decidua at any time point (6 hours, P O .99; 10 days, P = .09, Figure 3). Interestingly, in the control animals NR2B expression in the decidua/trophoblast or placenta resulted to be decreasing over time, whereas in the embryo, it increased. NR2B is more expressed in the decidua/trophoblast respect to the embryos at 6 hours (P = .05) and no difference is reported at 24 hours (P = .4); conversely, at 10 days, NR2B is more expressed in the embryo respect to the placenta (P ! .001, Figure 4).
Comment Acute in utero alcohol exposure alters NR2B expression in the embryo during development and results in longterm alterations in the NR2A and NR2B subunits in the adult brain. The shift from NR2B to NR2A in the adult brain after exposure to alcohol may underlie some of the alcohol-induced learning impairment associated with in utero exposure.
Six-hours after alcohol, NR2B expression is significantly higher; this rise may be a compensatory response to the effects of alcohol in which the embryo attempts to compensate for the reduction in the NMDA functionality through hyperactivation of this subunit to maintain an homeostatic synaptic plasticity. This hypothesis is supported by the evidence that NR2B is the most sensitive NMDAR subunit to ethanol and is fundamental for the generation of LTP. After 24 hours, the embryo is not able to maintain the compensatory response, as shown in Figure 1, and by 10 days the expression of NR2B is decreased. This is the first study that addresses the effects of alcohol on NR2B composition during development13 (MEDLINE, 1966-September 2004 Keywords: NMDA, development, fetus, NR2A, NR2B, all languages). Because the deciduas and placenta have an integral role in development and directly influence fetal growth, changes in gene expression may affect the development of the fetus. However, no changes in NR2B expression were identified suggesting that the deleterious effects of alcohol toward the maternal-fetal unit are confined to the fetus. Interestingly, although NR2B expression increased during gestation in the embryo, it decreases in the maternal tissues. This may
Toso et al imply that the maternal role for embryonic development is greatest when the embryo is unable to express specific genes, and the role declines once the embryo is able to take over (Figure 4), similar to the maternal and fetal regulation of vasosactive intestinal peptide.14 No expression for NR2A subunit was found during development. NMDARs are known to go through an age-dependent maturation process along with the synapse itself that originates by changes in the subunit composition. During development extrasynaptic receptors that mainly contain the NR2B subunit, which is more susceptible to LTP, thus more adaptable for learning, are abundant to develop the brain circuits. As the brain matures, they are replaced by synaptic NMDAR, which contain mainly the NR2A subunits, which are less susceptible to LTP. This key step in the transition between ‘‘developmental’’ and ‘‘adult’’ forms of the NMDAR results in the stabilization of ‘‘mature’’ NMDAR subtypes at synapses and coincides with a lowered susceptibility to LTP that closes a critical period for the refinement of connections in the braindperhaps by making synapses ‘‘less plastic.’’4 Thus, it is not surprising that NR2A was not expressed during fetal development. Adult mice brains exposed to alcohol prenatally experience significant downregulation in NR2B and upregulation in NR2A expression. The initial acute rise during development in NR2B ultimately results in suppression of this key element in learning and memory at a crucial time point for brain circuit maturation and results in a switch in the NMDAR composition with the less modifiable subunit NR2A. These findings are concurrent with phenotypic evidence of learning deficits. In conclusion, prenatal acute alcohol exposure modifies NMDARs subunit composition during development; these changes are long-lasting and may underlie learning deficits in adulthood.
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References 1. Stratton K, Howe C, Battaglia F, editors. Fetal alcohol syndrome: diagnosis, epidemiology, prevention and treatment. Washington (DC): National Academy Press; 1996. 2. O’Leary CM. Fetal alcohol syndrome: diagnosis, epidemiology, and developmental outcomes. J Pediatr Child Health 2004;40:2-7. 3. Mattson SN, Riley EP, Sowell ER, Jernigan TL, Sobel DF, Jones KL. A decrease in the size of the basal ganglia in children with fetal alcohol syndrome. Alcohol Clin Exp Res 1996;20:1088-93. 4. Pe´rez-Otanˇo I, Ehlers D. Learning form NMDA receptor trafficking: clues to the development and maturation of glutamatergic synapses. Neurosignals 2004;3:175-89. 5. Faber NB, Olney JW. Drugs of abuse that cause developing neurons to commit suicide. Dev Brain Res 2003;147:37-45. 6. Savage DD, Queen SA, Sanchez CF, Paxton LL, Manhoney JC, Goodlett CR, et al. Prenatal ethanol exposure during the last third of gestation in rat reduces hippocampal NMDA antagonist binding site density in 45-day-old-rats. Alcohol 1991;9:37-41. 7. Wernicke C, Samochowies J, Shmidt LG, Winterer G, Smolka M, Kucharska-Mazur J, et al. Polymorphisms in the N-methyl-Daspartate receptor 1 and 2B subunits are associates with alchoholism-raltes traits. Biol Psychiatry 2003;54:922-8. 8. Giese KP, Peters M, Vernon J. Modulation of excitability as a learning and memory mechanism: a molecular genetic prospective. Physiol Behav 2001;73:803-10. 9. Morris RGM, Frey U. Hippocampal synaptic plasticity: role in spatial learning or the automatic recording of attended experience? Phil Trans R Soc Lond 1997;352:1489-503. 10. Quinlan EM, Lebel D, Brosh I, Barkal E. A molecular mechanism for stabilization of learning-induced synaptic modifications. Neuron 2004;41:185-92. 11. Spong C, Abebe D, Gozes I, Brenneman D, Hill J. Prevention of fetal demise and growth restriction in a mouse model of fetal alcohol syndrome. J Pharm Exp Ther 2001;297:774-9. 12. Webster WS, Walsh DA, Lipson AH, McEwen SE. Teratogenensis after acute alcohol exposure in inbred and outbred mice. Neurobehav Toxicol 1980;2:227-34. 13. Honse Y, Nixon KM, Browning MD, Leslie SW. Cell surface expression of NR1 splice variants and NR2 subunits is modified by prenatal ethanol exposure. Neuroscience 2003;122:689-98. 14. Spong CY, Lee SJ, McCune SK, Gibney G, Abebe DT, Alvero R, et al. Maternal regulation of embryonic growth: the role of vasoactive intestinal peptide. Endocrinology 1999;140:917-24.
www.ajog.org
N-Methyl-D-aspartate subunit expression during mouse development altered by in utero alcohol exposure Laura Toso, MD,a,b,* Sarah H. Poggi, MD,a,c Daniel Abebe,a Robin Roberson,a Veronica Dunlap,a Jane Park,a Catherine Y. Spong, MDa,c Unit on Perinatal and Developmental Neurobiology, NICHD,a National Institute of Alcohol Abuse and Alcoholism,b National Institutes of Health, Bethesda, MD; Department of Obstetrics and Gynecology, Georgetown University Hospital, Washington, DCc Received for publication November 16, 2004; accepted February 18, 2005
KEY WORDS Fetal alcohol syndrome Alcohol N-methyl-D-aspartate receptors Mouse development Learning
Objective: 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. Study design: 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). Calibratornormalized relative real-time polymerase chain reaction was performed for NR2B and NR2A with glyceraldehyde-3-phosphate dehydrogenase standardization. Statistical analysis included analysis of variance. Results: 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). Conclusion: 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. Ó 2005 Mosby, Inc. All rights reserved.
Supported by the DIR of National Institutes of Child Health and Human Development. * Reprint requests: Laura Toso, MD, Unit on Perinatal and Developmental Neurobiology, NICHD, NIH, Building 9, R 1W125, 9 Memorial Dr, Bethesda MD 20892-0925. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2005 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.02.105
Toso et al
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Figure 1 NR2B (10^2) messenger RNA expression in the embryos 6 hours, 24 hours, and 10 days after alcohol treatment (standardized to GAPDH). The initial rise at 6 hours in alcohol-treated embryos progressively lowers after 24 hours and decreases 10 days later. Bar represent mean G SD.
Fetal alcohol syndrome (FAS) is the most common nongenetic cause of mental retardation, occurring in 0.5 births per 1000 births each year in the United States.1 The diagnostic criteria for FAS include the presence of a characteristic set of facial features, as well as craniofacial dismorphology, microcephaly, growth restriction, and central nervous system (CNS) anomalies or dysfunction. In addition, FAS is 1 category of the fetal alcohol spectrum disorders (FASD) that include a broader range of outcomes associated with prenatal alcohol exposure, characterized by the absence of FAS facial features but with sequelae of fetal injury, including mental and learning impairments and structural brain defects, as shown by magnetic resonance imaging.2,3 Glutamatergic transmission in the CNS toward the N-methyl-D-aspartate (NMDA) receptors (NMDAR) has been strongly suggested to be 1 of the pathways damaged in FASD. NMDARs are tetrameric complexes assembled from the ubiquitous NR1 subunit, which is an essential component of all NMDAR complexes, along with various combinations of NR2 or NR3 subunits. NR1 is encoded by a single gene, whereas NR2 and NR3 subunits are encoded by 4 (NR2A-B-C-D) and 2 (NR3AB) distinct genes, respectively. The functional proprieties of the receptor complex depend on the specific subunit composition and the stoichiometry in which subunits combine to form the channel.4 Alcohol is a known NMDA-antagonist: in vitro studies have found that an ethanol intoxication episode can cause millions of
neurons to commit suicide in the developing CNS through a NMDA-mediated apoptotic neurodegenerative response, particularly in systems that play critical roles in learning, memory, sensory information processing, and cognitive function.5 Multiple mechanisms have been shown to contribute to ethanol-induced alteration in NMDARs function, including an overall decrease in NMDA receptor density and differential regulation of the expression of NMDA receptor subunits.6,7 The NR2B subunit has been shown to be the most sensitive to ethanol, and it has been postulated that a relative increase in NR2A expression in respect to NR2B subunits after chronic ethanol exposure can cause the functional changes in the NMDA ion channel.7 Molecular studies in the mouse have found that NMDAR-dependent long-term potentiation (LTP) is essential for spatial learning and memory8-10 and that a switch in the subunit composition in NMDARs from NR2B to NR2A subunits lowers the receptor susceptibility to LTP and makes the synapse less plastic.4 In a well-known mouse model for FAS, we have previously shown that alcohol induces fetal death, growth restriction at birth, and learning deficits in adulthood.11 The purpose of our study is to evaluate whether the expression of NMDARs NR2A and NR2B subunits is affected by prenatal alcohol exposure during development and whether the alcohol-induced learning deficits are due, at least in part, to a change in these subunits in the composition of the NMDAR.
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Figure 2 NR2A (A) and NR2B (B) expression in adult brains (standardized to GAPDH). In utero-exposed animals show a relative decrease in NR2B and a relative increase in the less plastic subunit NR2A. Bars represent mean G SD.
Material and methods Animals C57Bl6/J female mice (Harlan Sprague-Dawley, Inc, Indianapolis, IN) were kept in 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 (NIH) guidelines for care and use of experimental animals. The protocol was approved by National Institute of Child Health and Human Development (NICHD) Animal Care and Use Committee. Six-week-old females (21-24 g) were mated with C57-B16J males for 4 hours. The presence of vaginal plug was considered day 0 of pregnancy.
Treatment A well-described model for FAS was used.12 Animals were randomly injected intraperitoneally on gestational day 8 with 25% ethyl alcohol in saline (alcohol) solution or vehicle alone (saline solution) at 0.030 mL/g of body weight at 7 AM. Because the animals receiving alcohol were incapacitated for approximately 6 hours after injection, food and water were withheld in both groups for 6 hours.
Tissue collection and processing At 6 hours, 24 hours, and 10 days after injection, embryonic and decidua/trophoblast at 6 and 24 hours
or placental tissues at 10 days were explanted by using microdissection from the uterus and placed in phosphate-buffered saline solution (PBS). Each gestational time point included at least 3 samples with each sample representing 3 to 5 litters (a gestation typically includes 8-10 embryos). For adult tissues, 3 brains per treatment, each from a different litter, were collected separately. For RNA extraction, samples were homogenized by using disposable micropestles to avoid cross contamination. The samples were processed with SV Total RNA Isolation System (Promega, Madison, WI). A 5-mL aliquot was taken for spectrophotometric determination of RNA content. The remaining sample was stored at 80(C. With the use of 5 mg of total RNA, the reverse transcriptase (RT) reaction was performed (Perkin-Elmer Corp, Branchburg, NJ) in a final volume of 150 mL. Each RNA sample was run in duplicate. Negative controls included RT reactions omitting RNA or RT.
RT-Polymerase chain reaction NR2B (GenBank Accession Number NM_008171) primer pair was designed using Primer3 software and synthesized by IDT (Integrated DNA Technologies, Coralville, Iowa). NR2B primer sequence was 50 -CCG CAG CAC TAT TAGA GAA CA-30 (sense) and 50 -ATC CAT GTG TAG CCG TAG CC-50 (antisense). NR2A (GenBank Accession Number NM_008170) primer pair was designed by TIB Molbiol (Adelphia,
Toso et al
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Figure 3 NR2B messenger RNA expression in the decidua/trophoblast at 6 hours and 24 hours and placenta 10 days later after alcohol treatment (standardized to GAPDH). Bar represent mean G SD.
NJ) and synthesized by IDT (Integrated DNA Technologies). NR2A primer sequence was 50 -GGA AGT TGG ACG CTT TCA TC-30 (sense) and 50 -TCT TCC ATC TCA CCG TCA CC-30 (antisense). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as an internal standard with the primer pair synthesized by IDT. GAPDH primer sequence was 50 -TGC ACC ACC AAC TGC TTA (sense) and 50 GGA TGC AGG GAT GAT GTT C (antisense). With the use of the FastStart DNA Master SYBR Green 1 dye-base detection (Roche, Indianapolis, Ind), NR2A or NR2B and GAPDH expression were measured by real-time polymerase chain reaction (PCR) using the LightCycler with relative quantification software (Roche) and melting point analysis to assess the specificity of the amplified genes. Optimization for NR2A, NR2B, and GAPDH-specific primers was performed with separate runs by varying magnesium chloride and primer concentrations, amount of template, and annealing temperature, with a magnesium chloride concentration of 2 mmol/L for NR2A, 3 mmol/L for NR2B, and of 2.5 mmol/L for GAPDH, primer concentrations of 0.5 mmol/L for NR2B and NR2A and 0.3 mmol/L for GAPDH, 2 mL of the RT product for all the primers and annealing temperatures of 55(C for NR2B, 52(C for NR2A, and of 60(C for GAPDH considered optimal. The presence and purity
of target gene sequence expression in the real-time PCR were confirmed by gel electrophoresis. Samples were run in duplicate and relative quantification was performed by using calibrator-normalized data with efficiency correction. Results are presented as the normalized ratio of NR2A-to-GAPDH and NR2B-to-GAPDH expression. Real-time PCR comparisons were made at all time points (6 hours, 24 hours, 10 days, and 3 months) and in each tissue type (embryo, deciduas or placentas, and brains) between the alcohol and control groups. Analysis of variance was performed for overall comparisons at each time point with post hoc Fisher analysis performed to compare between pairs, with P ! .05 considered significant (StatView v5.0.1, SAS, Cary, NC).
Results At 6 hours, NR2B expression in the alcohol-exposed embryos was elevated in comparison with controls (P = .006, Figure 1). At 24 hours there was no difference in NR2B expression between the groups (P = .3); however, at 10 days there was a lower expression of NR2B in alcohol-exposed animals (P = .02, Figure 1) that persisted until adulthood in the brains (P = .03, Figure 2). NR2A was not expressed during development (6 hours, 24 hours, and 10 days).
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Figure 4 NR2B messenger RNA expression comparison between the control embryos and control at 6 hours and 24 hours in the decidua/trophoblast and 10 days later in the placenta after alcohol injection shows a relative increase over time of NR2B in the embryo, and a concomitant decrease in the decidua/trophoblast and placenta. Bars represent mean G SD.
In the adult brains, concomitant with the decrease in NR2B associated with alcohol exposure there was a relative increase in NR2A (P = .002, Figure 2). NR2B was not differentially expressed in the decidua at any time point (6 hours, P O .99; 10 days, P = .09, Figure 3). Interestingly, in the control animals NR2B expression in the decidua/trophoblast or placenta resulted to be decreasing over time, whereas in the embryo, it increased. NR2B is more expressed in the decidua/trophoblast respect to the embryos at 6 hours (P = .05) and no difference is reported at 24 hours (P = .4); conversely, at 10 days, NR2B is more expressed in the embryo respect to the placenta (P ! .001, Figure 4).
Comment Acute in utero alcohol exposure alters NR2B expression in the embryo during development and results in longterm alterations in the NR2A and NR2B subunits in the adult brain. The shift from NR2B to NR2A in the adult brain after exposure to alcohol may underlie some of the alcohol-induced learning impairment associated with in utero exposure.
Six-hours after alcohol, NR2B expression is significantly higher; this rise may be a compensatory response to the effects of alcohol in which the embryo attempts to compensate for the reduction in the NMDA functionality through hyperactivation of this subunit to maintain an homeostatic synaptic plasticity. This hypothesis is supported by the evidence that NR2B is the most sensitive NMDAR subunit to ethanol and is fundamental for the generation of LTP. After 24 hours, the embryo is not able to maintain the compensatory response, as shown in Figure 1, and by 10 days the expression of NR2B is decreased. This is the first study that addresses the effects of alcohol on NR2B composition during development13 (MEDLINE, 1966-September 2004 Keywords: NMDA, development, fetus, NR2A, NR2B, all languages). Because the deciduas and placenta have an integral role in development and directly influence fetal growth, changes in gene expression may affect the development of the fetus. However, no changes in NR2B expression were identified suggesting that the deleterious effects of alcohol toward the maternal-fetal unit are confined to the fetus. Interestingly, although NR2B expression increased during gestation in the embryo, it decreases in the maternal tissues. This may
Toso et al imply that the maternal role for embryonic development is greatest when the embryo is unable to express specific genes, and the role declines once the embryo is able to take over (Figure 4), similar to the maternal and fetal regulation of vasosactive intestinal peptide.14 No expression for NR2A subunit was found during development. NMDARs are known to go through an age-dependent maturation process along with the synapse itself that originates by changes in the subunit composition. During development extrasynaptic receptors that mainly contain the NR2B subunit, which is more susceptible to LTP, thus more adaptable for learning, are abundant to develop the brain circuits. As the brain matures, they are replaced by synaptic NMDAR, which contain mainly the NR2A subunits, which are less susceptible to LTP. This key step in the transition between ‘‘developmental’’ and ‘‘adult’’ forms of the NMDAR results in the stabilization of ‘‘mature’’ NMDAR subtypes at synapses and coincides with a lowered susceptibility to LTP that closes a critical period for the refinement of connections in the braindperhaps by making synapses ‘‘less plastic.’’4 Thus, it is not surprising that NR2A was not expressed during fetal development. Adult mice brains exposed to alcohol prenatally experience significant downregulation in NR2B and upregulation in NR2A expression. The initial acute rise during development in NR2B ultimately results in suppression of this key element in learning and memory at a crucial time point for brain circuit maturation and results in a switch in the NMDAR composition with the less modifiable subunit NR2A. These findings are concurrent with phenotypic evidence of learning deficits. In conclusion, prenatal acute alcohol exposure modifies NMDARs subunit composition during development; these changes are long-lasting and may underlie learning deficits in adulthood.
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