DEVELOPMENTAL GENETICS 12:293-298 (1991)
Effect of Prenatal Ethanol Exposure on Postnatal Neural Gene Expression in the Rat CHRISTIAN C.G. NAUS AND JOHN F. BECHBERGER Department of Anatomy, The University of Western Ontario, London, Ontario, Canada To examine the effects of ethABSTRACT anol exposure on neural development, pregnant rats were fed a liquid diet in which 37.5% of the total caloric content was ethanol-derived. The developmental appearance of the messenger RNAs coding for preprosornatostatin, glial fibrillary acidic protein, and proteolipid protein was examined by Northern blotting of total cellular RNA obtained from forebrain and hindbrain at various times after birth. In general, there was a delay in the developmental pattern of appearance of these rnRNAs which was most noticeable at the early postnatal times. These results suggest that the previously reported delay in neural maturation is reflected at the level of the gene expression.
Key words: Neural development, messenger RNA, somatostatin, glial fibrillary acid protein, proteolipid protein
INTRODUCTION The development of the central nervous system (CNS) is a complex interaction of various processes including cell division, migration, differentiation, axonal outgrowth, synaptogenesis, and cell death (Cowan et al., 1984). The CNS develops over a protracted period of time, from early fetal into postnatal stages. Each stage of neural development imposes particular functional demands on the developing cell populations, these demands being reflected by proteins synthesized by those cells. The temporal and spatial regulation of gene expression is therefore a critical component of development. Since the development of the nervous system may be viewed a s a continuum of developmental processes involving diverse cell types and neural systems, any teratogen might be expected to display a spectrum of effects, depending upon time of exposure. In the case of ethanol, prenatal exposure may lead to the condition known as fetal alcohol syndrome, characterized by intrauterine growth retardation, microcephaly, craniofacia1 and eye abnormalities, and mental deficiency (Colangelo and Jones, 1982). Ethanol may exert its effects on neural development a t various levels, and little is
0 1991 WILEY-LISS, INC.
presently known concerning the specific teratological mechanism(s) involved (West, 1986). While it is generally accepted that genes are regulated a t the level of mRNA transcription, a toxic agent such as ethanol might exert its effect at any stage in mRNA or protein processing, resulting in quantitative or even qualitative changes in the functional gene product. This study examines the effects of prenatal exposure to ethanol on postnatal neural gene expression. The effect of prenatal ethanol exposure on the developmental pattern of appearance of various neuronal and glial mRNAs, including somatostatin (SS), glial fibrillary acidic protein (GFAP), and proteolipid protein (PLP), was examined during postnatal development of the rat brain. These specific gene products were chosen since they represent specific markers of terminal differentiation for astrocytes, oligodendrocytes, and a class of neurons. The expression of these genes was followed by using Northern (RNA) blotting to monitor relative levels of specific mRNAs. In general, prenatal ethanol exposure induces a noticeable delay in the accumulation pattern of these specific mRNAs, suggesting a delay in the normal pattern of gene expression.
MATERIALS AND METHODS Ethanol Exposure Twenty timed-pregnant female Sprague Dawley rats were purchased (Charles River, Que.) and maintained on protein-enriched control or ethanol Liquidiet (diets F1264, F1265, Bioserv, Frenchtown, NJ) from gestational day (GD) 7 to GD 20. For the ethanol diet, 37.5% of the total caloric content was derived from ethanol. Controls were pair-fed relative to the ethanol animals, with a diet containing isocaloric amounts of dextrins and maltose. Liquidiet was then replaced with conventional rat chow and water on GD 20. Maternal blood alcohol concentrations (BACs) were determined from
Received for publication July 16, 1990; accepted January 28, 1991. Address reprint requests to Christian C.G. Naus, Dept. of Anatomy, The University of Western Ontario, London, Ontario, Canada N6A 5C1.
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venous tail bleeds on GD 18 at 9:00 A.M. and on GD 19 at 5 3 0 A.M. Serum ethanol levels were detected by measuring NADH produced by ethanol oxidation by alcohol dehydrogenase in the presence of NAD (Sigma Diagnostics, No. 332-UV). Pregnant rats were weighed on GD 14 and GD 19 to determine weight gain over the course of ethanol administration. After birth, both control and ethanoltreated pups were cross-fostered to lactating dams, and litters were culled to 10 pups per dam. Their weights were monitored at 5 day intervals from birth to GD 25.
RNA Extraction and Northern Blotting Total RNA was obtained from the forebrain or hindbrain (i.e., rostra1 or caudal to the midbraindiencephalic junction, respectively) of 3 to 6 ethanolexposed and control rats from different litters at each of postnatal days 0, 5, 10, 15, 20, 30, and 60 by extraction with phenol-chloroform-isoamyl alcohol (Schibler et al., 1980). For detection of various mRNAs by Northern blot hybridization analysis, 10 pg aliquots of total RNA, as determined by measurements of optical density at 260 nm, were dissolved in 50% formamide containing MOPS (20 mM 3-N-morpholinopropanesulphonic acid) and denatured with 3.7% formaldehyde at 55°C for 15 min. The reaction was stopped on ice and 1 p.1 loading buffer added (0.4% bromphenol blue, 1mM EDTA, 50% glycerol). Samples were subjected to a denaturing agarose gel electrophoresis for 3 hours (Rave et al., 1979) and transferred overnight to nitrocellulose (Thomas, 1980). After baking in a vacuum oven at 80°C for 3 hours, the blots were prehybridized for 3 hours at 42°C in a solution containing 50% formamide, 5 x Denhardt’s solution, 5 x PIPES (750 mM NaC1, 25 mM 1,4-piperazinediethylene sulphonic acid, 25 mM EDTA, pH 6.81, 0.2% sodium dodecyl sulphate (SDS), and 100 p.g/ml herring sperm DNA. The cDNAs used in this study included those for preprosomatostatin (Goodman et al., 19831, GFAP (Lewis et al., 1984), and PLP (Milner et al., 1985). These cDNAs were radiolabeled by random priming extension (Feinberg and Vogelstein, 1983). Hybridization was performed overnight at 42°C in a solution containing 50% formamide, 5 x Denhardt’s, 5 x PIPES, 0.2% SDS. Following hybridization, membranes were washed at room temperature for 45 min in 2 x standard saline citrate (SSC), 0.2% SDS, followed by 45 min in 0.5 x SSC, 0.2% SDS a t 37°C and 0.15 x SSC, 0.2% SDS at 65°C. Autoradiographs were obtained by exposing Kodak XAR film to the hybridized membranes, and the resultant images were subjected to laser densitometry (Ultrascan XL, LKB) to obtain relative levels of mRNA. The same blots were hybridized with a radiolabeled cDNA for U l b snRNA to allow for subsequent normalization of densitometric values to the level of this RNA.
RESULTS In general, both the control and the alcohol-fed pregnant rats gained weight during the experimental feeding. However, while control animals increased their weight a n average of 12% between GD 14 and GD 19, the alcohol-fed group gained a n average of 7%. It was obvious th a t the BAC fluctuated during the day, and only early morning readings indicated that high levels of alcohol were present in the blood. The average BAC at 5 3 0 A.M. was 202 mg%, while at 9:00 A.M. the average BAC had dropped to 42 mg%. Delivery resulted in a number of viable pups per litter, and the cross-fostering of control and ethanol pups was uneventful. However, the alcohol-fed mothers had smaller litter sizes than the control mothers (8 vs. 12). There was a tendency for mothers with higher BACs to have smaller litter sizes. Although newborn weights from both groups were similar, the ethanol-treated pups subsequently gained less weight than the control pups. To examine the effect of ethanol on specific gene expression, the proportion of mRNA coding for various neural proteins was determined in equivalent amounts of total RNA isolated from control and ethanol-treated pups a t various times after birth. Northern blot analysis revealed a similar pattern in all the mRNAs examincd (Fig. 1). It was evident that there was a delay in the temporal pattern of appearance of the mRNAs for SS (Fig. lA), GFAP (Fig. lB), and PLP (Fig. lC,D). In control animals, the 0.7 kb SS mRNA is readily detected in hindbrain (Fig. 1A) and forebrain (not shown) a t birth. As we have previously reported (Naus et al., 1988), there is a decrease in the level of SS mRNA during postnatal development of the brain, such that neonatal levels are 2-3-fold higher than that in the adult (Fig. 2A). While this same decrease is evident in the pattern of mRNA prevalence in the animals which had been prenatally exposed to ethanol, there is a delay or lag in this pattern (Fig. 2A). Initially, SS mRNA is more abundant in the control animals (Pl-P5). However, by P15, the level of SS mRNA in the controls is lower than that in the ethanol-treated animals. Although the levels are similar by P30, the level of SS mRNA remains noticeably higher in the adult hindbrain. The postnatal appearance of the 2.7 kb GFAP mRNA is depicted in Figure 1B. Again, both control and ethanol-treated animals display a similar developmental profile, with the level of GFAP mRNA increasing with time, confirming the previous report by Lewis and Cowan (1985). As with SS mRNA, there is a delay in the pattern of appearance of GFAP mRNA in the ethanol-treated animals. This is particularly evident in the data obtained from densitometric analysis (Fig. 2B). As previously noted by Milner et al. (1985), the developmental pattern of PLP mRNA is characterized by a dramatic increase after the first postnatal week (Fig.
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E Fig. 1. Northern blot analysis to determine the relative levels of specific mRNAs a t various postnatal times in control and ethanoltreated rats. There is a consistent delay in the normal pattern of mRNA appearance in the hindbrain for SS (A) and GFAP (B) and for PLP in forebrain (C) and hindbrain (D). Hybridization of the same
Northern blot with radiolabeled cDNA for U l b snRNA indicates equivalent levels during development, and no observable effect of ethanol exposure (E). See text for details. P1-postnatal day 1; C-control; E-ethanol-treated.
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Fig. 2. Relative levels of specific mRNAs in control and ethanol-treated rats at various times after birth. Data were obtained from densitometric analysis of the Northern blots in Figure 1.Values were normalized to the level of U l b snRNA in each lane. In each case, there is a delay in the pattern of appearance of these specific mRNAs.
1C,D). The postnatal appearance of PLP mRNA differs dramatically between forebrain (Fig. 1C) and hindbrain (Fig. 1D). While the 2.3 kb PLP mRNA is detectable in both regions soon after birth, much higher levels are evident a t an earlier time in the hindbrain. In both forebrain and hindbrain, there is a lag in the developmental pattern of appearance of PLP mRNA, this delay being most evident in the forebrain (Fig. 2C,D).
DISCUSSION Variable effects of ethanol on RNA synthesis and subsequent protein synthesis in the developing brain have been reported. In reviewing the literature, Druse (1986) suggested t h a t while there may be some changes soon after ethanol exposure, these are transient and disappear with subsequent development. The inconsistencies reported in the literature probably reflect variations in the methods of ethanol administration, dose, duration, and subsequent BAC. From in vitro translation of subcellular fractions isolated from the brain of ethanol-treated animals, it has been suggested that
protein synthesis is compromised due to ethanol effects on ribosomes (Tewari et al., 1980) and tRNA (Fleming et al., 1975). Effects of prenatal ethanol exposure on RNA levels in the brain generally suggest a decrease in transcription. Two weeks of prenatal ethanol exposure resulted in a decrease of total brain RNA from 100 mg/g wet weight of brain tissue to 78 mg/g in newborn pups, while DNA decreased from 96 mg/g to 55 mg/g (Rawat, 1980). The loss of DNA probably reflects ethanol-induced inhibition of mitosis (Miller, 1986) or cell death. These findings suggests that the amount of RNAkell may actually increase in response to ethanol treatment. Specific effects of ethanol on gene transcription have not been extensively studied. Milner et al. (1987), using chronic ethanol vapour exposure of pregnant rats from GD 7 to GD 21, demonstrated a delay in the expression patterns of the genes for the myelin proteins PLP and myelin-associated glycoprotein (MAG), as well as tubulin. The present study also demonstrates a delay in
ETHANOL EFFECTS ON NEURAL GENE EXPRESSION the developmental pattern of PLP mRNA, although the delay is not as dramatic a s that reported by Milner et al. (1987). In addition, we have noted that prenatal ethanol exposure also produces a delay in the pattern of expression of additional glial and neuronal genes, including GFAP and somatostatin. The ethanol exposure paradigm used in the present study is much different than the ethanol vapour inhalation method used by Milner et al. (1987) since a protein-enriched liquid diet was used. Our results, in conjunction with these, probably reflect a dosetduration response since the administration of ethanol via vapour inhalation produces chronic exposure with BAC of 100-200 mg% (Rogers et al., 1979), while the liquid diet would result in diurnal variation of the BAC depending on feeding behaviour. Due to circadian rhythyms, alcohol consumption varied throughout the day in the present study, resulting in the BAC varying between 50 and 200 mg%. Prenatal ethanol exposure has been reported to cause a delay in the maturation of the nervous system (Clarren and Smith, 1978; Colangelo and Jones, 1982). Our data suggest that this ethanol-induced delay in neural development reported by many investigators is a result of a general effect on the process of transcription, reflected in the pattern of appearance of various mRNAs. In particular, a delay in the maturation of astrocytes may underlie the observed disruption in migration of cortical neurons (Miller, 1986), since astrocytes play a major role in the process of neuronal migration (Rakic, 1981). Likewise, the lag in myelination reported following neonatal ethanol exposure (Jacobsen et al., 1979) could reflect a delay in the expression of genes coding for myelin proteins such a s PLP. While somatostatin levels have been shown to incrase in adult Balbtc mice after chronic alcohol exposure (Fuhrmann et al., 1988), no studies have examined the effect of prenatal ethanol exposure on this neuropeptide. The observed delay in the expression of mRNA for somatostatin suggests there may be a delay in the differentiation of these neurons following prenatal ethanol exposure. The present results thus suggest that a n early manifestation of the maturational delay in the nervous system induced by ethanol exposure may be seen at the level of gene expression in the different neural cell types.
ACKNOWLEDGMENTS The authors are grateful to Dr. F. Miller for her generous provision of the PLP cDNA, to Dr. N. Cowan for the GFAP cDNA, to G. Schultz for U l b cDNA, and to Dr. J. Habener for the SS cDNA. We are also grateful to Mrs. L. Wood and Mr. D. Belliveau for technical assistance. This research was supported by grants from the Hospital for Sick Children Foundation (grant 8805) and the Ministry of Community and Social Services, Ontario, Canada, administered by the Research
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and Program Evaluation Unit, in cooperation with the Ontario Mental Health Foundation, funded from the “Interprovincial” Lottery Research Program. C.C.G.N. is a Scholar of the Medical Research Council of Canada.
REFERENCES Clarren SK, Smith DW (1978): The fetal alcohol syndrome. N Engl J Med 298:1063-1067. Colangelo W, Jones DG (1982): The fetal alcohol syndrome: a review and assessment of the syndrome and its neurological sequelae. Prog Neurobiol 19:271-314. Cowan WM, Fawcett J , OLeary D, Stanfield B (1984): Regressive events in neurogenesis. Science 255:1258-1265. Druse MJ (1986): Effects of perinatal alcohol exposure on neurotransmitters, membranes and proteins. In West J R (ed): “Alcohol and Brain Development.” New York: Oxford University Press, pp 343372. Feinberg AP, Vogelstein B (1983):A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13. Fleming E, Tewari S, Noble E (1975): Effects of chronic ethanol ingestion on brain amino acyl-tRNA synthetases and tRNA. J Neurochem 24:553-560. Fuhrmann G, Strosser MT, Besnard F, Kempf E, Kempf J , Ebel A (1988): Genotypic differences in age and chronic alcohol exposure effects on somatostatin levels in hippocampus and striatum in mice. Neurochem Res 11525-636. Goodman RH, Aran DC, Bernard AR (1983): Rat preprosomatostatin: structure and processing by microsomal membranes. J Biol Chem 258:5570-5573. Jacobsen S, Rich J , Tovsky NJ (1979): Delayed myelination and lamination in the cerebral cortex of the albino rat as a result of fetal alcohol syndrome. Curr Alcohol 6:123-133. Lewis SA, Cowan NJ (1985): Temporal expression of mouse glia fibrillary acid protein mRNA studied by a rapid in situ hybridization procedure. J Neurochem 45:913-919. Lewis SA, Balcarek JM, Krek V, Shelansi ML, Cowan N J (1984): Sequence of a cDNA clone encoding mouse glial fibrillary acid protein: structural conservation of intermediate filaments. Proc Natl Acad Sci USA 81:2743-2746. Miller MW (1986): Effects of alcohol on the generation and migration of cerebral cortical neurons. Science 233:1308-1311. Milner RJ, Lai C, Nave K-A, Lenoir D, Ogata J , Sutcliffe J G (1985): Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein. Cell 42:931-939. Milner RJ, Randolph L, Bahr D, Cappello M, Lenoir D, Miller F, Bloom FE (1987): Molecular biological approaches to the brain and their application to the study of alcoholism. In Goedde HW, Agarwal DP (eds): “Genetics and Alcoholism.” New York: Alan R. Liss, Inc., pp 291-302. Naus CCG, Miller FD, Morrison JH, Bloom FE (1988): Immunohistochemical and in situ hybridization analysis of the development of the rat somatostatin-containing neocortical neuronal system. J Comp Neurol 269:448-463. Rakic P (1981): Neuron-glial interaction during brain development. Trends Neurosci 4:184-187. Rave N, Crjvenjakov R, Boedtker H (1979): Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucleic Acids Res 6:3559-3567. Rawat AK (1980): Biochemical aspects of neuroteratogenic effects of alcohol. Neurobehav Toxic01 2:259-265. Rogers J , Wiener SC, Bloom FE (1979): Long-term ethanol administration methods for rats: advantages of inhalation over intubation or liquid diets. Behav Neural Biol 27:466-486. Schibler K, Tosei M, Pittet A-C, Fabiani L, Wellames P (1980):Tissuespecific expression of mouse a-amylase genes. J Mol Biol 142:93116. Tewari S, Sweeney F, Fleming E (1980): Ethanol-induced changes in properties of rat brain ribosomes. Neurochem Res 5:1025-1035.
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Thomas PS (1980):Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5215.
West J R (1986):“Alcohol and Brain Development.” New York: Oxford Univ Press.
Effect of Prenatal Ethanol Exposure on Postnatal Neural Gene Expression in the Rat CHRISTIAN C.G. NAUS AND JOHN F. BECHBERGER Department of Anatomy, The University of Western Ontario, London, Ontario, Canada To examine the effects of ethABSTRACT anol exposure on neural development, pregnant rats were fed a liquid diet in which 37.5% of the total caloric content was ethanol-derived. The developmental appearance of the messenger RNAs coding for preprosornatostatin, glial fibrillary acidic protein, and proteolipid protein was examined by Northern blotting of total cellular RNA obtained from forebrain and hindbrain at various times after birth. In general, there was a delay in the developmental pattern of appearance of these rnRNAs which was most noticeable at the early postnatal times. These results suggest that the previously reported delay in neural maturation is reflected at the level of the gene expression.
Key words: Neural development, messenger RNA, somatostatin, glial fibrillary acid protein, proteolipid protein
INTRODUCTION The development of the central nervous system (CNS) is a complex interaction of various processes including cell division, migration, differentiation, axonal outgrowth, synaptogenesis, and cell death (Cowan et al., 1984). The CNS develops over a protracted period of time, from early fetal into postnatal stages. Each stage of neural development imposes particular functional demands on the developing cell populations, these demands being reflected by proteins synthesized by those cells. The temporal and spatial regulation of gene expression is therefore a critical component of development. Since the development of the nervous system may be viewed a s a continuum of developmental processes involving diverse cell types and neural systems, any teratogen might be expected to display a spectrum of effects, depending upon time of exposure. In the case of ethanol, prenatal exposure may lead to the condition known as fetal alcohol syndrome, characterized by intrauterine growth retardation, microcephaly, craniofacia1 and eye abnormalities, and mental deficiency (Colangelo and Jones, 1982). Ethanol may exert its effects on neural development a t various levels, and little is
0 1991 WILEY-LISS, INC.
presently known concerning the specific teratological mechanism(s) involved (West, 1986). While it is generally accepted that genes are regulated a t the level of mRNA transcription, a toxic agent such as ethanol might exert its effect at any stage in mRNA or protein processing, resulting in quantitative or even qualitative changes in the functional gene product. This study examines the effects of prenatal exposure to ethanol on postnatal neural gene expression. The effect of prenatal ethanol exposure on the developmental pattern of appearance of various neuronal and glial mRNAs, including somatostatin (SS), glial fibrillary acidic protein (GFAP), and proteolipid protein (PLP), was examined during postnatal development of the rat brain. These specific gene products were chosen since they represent specific markers of terminal differentiation for astrocytes, oligodendrocytes, and a class of neurons. The expression of these genes was followed by using Northern (RNA) blotting to monitor relative levels of specific mRNAs. In general, prenatal ethanol exposure induces a noticeable delay in the accumulation pattern of these specific mRNAs, suggesting a delay in the normal pattern of gene expression.
MATERIALS AND METHODS Ethanol Exposure Twenty timed-pregnant female Sprague Dawley rats were purchased (Charles River, Que.) and maintained on protein-enriched control or ethanol Liquidiet (diets F1264, F1265, Bioserv, Frenchtown, NJ) from gestational day (GD) 7 to GD 20. For the ethanol diet, 37.5% of the total caloric content was derived from ethanol. Controls were pair-fed relative to the ethanol animals, with a diet containing isocaloric amounts of dextrins and maltose. Liquidiet was then replaced with conventional rat chow and water on GD 20. Maternal blood alcohol concentrations (BACs) were determined from
Received for publication July 16, 1990; accepted January 28, 1991. Address reprint requests to Christian C.G. Naus, Dept. of Anatomy, The University of Western Ontario, London, Ontario, Canada N6A 5C1.
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venous tail bleeds on GD 18 at 9:00 A.M. and on GD 19 at 5 3 0 A.M. Serum ethanol levels were detected by measuring NADH produced by ethanol oxidation by alcohol dehydrogenase in the presence of NAD (Sigma Diagnostics, No. 332-UV). Pregnant rats were weighed on GD 14 and GD 19 to determine weight gain over the course of ethanol administration. After birth, both control and ethanoltreated pups were cross-fostered to lactating dams, and litters were culled to 10 pups per dam. Their weights were monitored at 5 day intervals from birth to GD 25.
RNA Extraction and Northern Blotting Total RNA was obtained from the forebrain or hindbrain (i.e., rostra1 or caudal to the midbraindiencephalic junction, respectively) of 3 to 6 ethanolexposed and control rats from different litters at each of postnatal days 0, 5, 10, 15, 20, 30, and 60 by extraction with phenol-chloroform-isoamyl alcohol (Schibler et al., 1980). For detection of various mRNAs by Northern blot hybridization analysis, 10 pg aliquots of total RNA, as determined by measurements of optical density at 260 nm, were dissolved in 50% formamide containing MOPS (20 mM 3-N-morpholinopropanesulphonic acid) and denatured with 3.7% formaldehyde at 55°C for 15 min. The reaction was stopped on ice and 1 p.1 loading buffer added (0.4% bromphenol blue, 1mM EDTA, 50% glycerol). Samples were subjected to a denaturing agarose gel electrophoresis for 3 hours (Rave et al., 1979) and transferred overnight to nitrocellulose (Thomas, 1980). After baking in a vacuum oven at 80°C for 3 hours, the blots were prehybridized for 3 hours at 42°C in a solution containing 50% formamide, 5 x Denhardt’s solution, 5 x PIPES (750 mM NaC1, 25 mM 1,4-piperazinediethylene sulphonic acid, 25 mM EDTA, pH 6.81, 0.2% sodium dodecyl sulphate (SDS), and 100 p.g/ml herring sperm DNA. The cDNAs used in this study included those for preprosomatostatin (Goodman et al., 19831, GFAP (Lewis et al., 1984), and PLP (Milner et al., 1985). These cDNAs were radiolabeled by random priming extension (Feinberg and Vogelstein, 1983). Hybridization was performed overnight at 42°C in a solution containing 50% formamide, 5 x Denhardt’s, 5 x PIPES, 0.2% SDS. Following hybridization, membranes were washed at room temperature for 45 min in 2 x standard saline citrate (SSC), 0.2% SDS, followed by 45 min in 0.5 x SSC, 0.2% SDS a t 37°C and 0.15 x SSC, 0.2% SDS at 65°C. Autoradiographs were obtained by exposing Kodak XAR film to the hybridized membranes, and the resultant images were subjected to laser densitometry (Ultrascan XL, LKB) to obtain relative levels of mRNA. The same blots were hybridized with a radiolabeled cDNA for U l b snRNA to allow for subsequent normalization of densitometric values to the level of this RNA.
RESULTS In general, both the control and the alcohol-fed pregnant rats gained weight during the experimental feeding. However, while control animals increased their weight a n average of 12% between GD 14 and GD 19, the alcohol-fed group gained a n average of 7%. It was obvious th a t the BAC fluctuated during the day, and only early morning readings indicated that high levels of alcohol were present in the blood. The average BAC at 5 3 0 A.M. was 202 mg%, while at 9:00 A.M. the average BAC had dropped to 42 mg%. Delivery resulted in a number of viable pups per litter, and the cross-fostering of control and ethanol pups was uneventful. However, the alcohol-fed mothers had smaller litter sizes than the control mothers (8 vs. 12). There was a tendency for mothers with higher BACs to have smaller litter sizes. Although newborn weights from both groups were similar, the ethanol-treated pups subsequently gained less weight than the control pups. To examine the effect of ethanol on specific gene expression, the proportion of mRNA coding for various neural proteins was determined in equivalent amounts of total RNA isolated from control and ethanol-treated pups a t various times after birth. Northern blot analysis revealed a similar pattern in all the mRNAs examincd (Fig. 1). It was evident that there was a delay in the temporal pattern of appearance of the mRNAs for SS (Fig. lA), GFAP (Fig. lB), and PLP (Fig. lC,D). In control animals, the 0.7 kb SS mRNA is readily detected in hindbrain (Fig. 1A) and forebrain (not shown) a t birth. As we have previously reported (Naus et al., 1988), there is a decrease in the level of SS mRNA during postnatal development of the brain, such that neonatal levels are 2-3-fold higher than that in the adult (Fig. 2A). While this same decrease is evident in the pattern of mRNA prevalence in the animals which had been prenatally exposed to ethanol, there is a delay or lag in this pattern (Fig. 2A). Initially, SS mRNA is more abundant in the control animals (Pl-P5). However, by P15, the level of SS mRNA in the controls is lower than that in the ethanol-treated animals. Although the levels are similar by P30, the level of SS mRNA remains noticeably higher in the adult hindbrain. The postnatal appearance of the 2.7 kb GFAP mRNA is depicted in Figure 1B. Again, both control and ethanol-treated animals display a similar developmental profile, with the level of GFAP mRNA increasing with time, confirming the previous report by Lewis and Cowan (1985). As with SS mRNA, there is a delay in the pattern of appearance of GFAP mRNA in the ethanol-treated animals. This is particularly evident in the data obtained from densitometric analysis (Fig. 2B). As previously noted by Milner et al. (1985), the developmental pattern of PLP mRNA is characterized by a dramatic increase after the first postnatal week (Fig.
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E Fig. 1. Northern blot analysis to determine the relative levels of specific mRNAs a t various postnatal times in control and ethanoltreated rats. There is a consistent delay in the normal pattern of mRNA appearance in the hindbrain for SS (A) and GFAP (B) and for PLP in forebrain (C) and hindbrain (D). Hybridization of the same
Northern blot with radiolabeled cDNA for U l b snRNA indicates equivalent levels during development, and no observable effect of ethanol exposure (E). See text for details. P1-postnatal day 1; C-control; E-ethanol-treated.
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Fig. 2. Relative levels of specific mRNAs in control and ethanol-treated rats at various times after birth. Data were obtained from densitometric analysis of the Northern blots in Figure 1.Values were normalized to the level of U l b snRNA in each lane. In each case, there is a delay in the pattern of appearance of these specific mRNAs.
1C,D). The postnatal appearance of PLP mRNA differs dramatically between forebrain (Fig. 1C) and hindbrain (Fig. 1D). While the 2.3 kb PLP mRNA is detectable in both regions soon after birth, much higher levels are evident a t an earlier time in the hindbrain. In both forebrain and hindbrain, there is a lag in the developmental pattern of appearance of PLP mRNA, this delay being most evident in the forebrain (Fig. 2C,D).
DISCUSSION Variable effects of ethanol on RNA synthesis and subsequent protein synthesis in the developing brain have been reported. In reviewing the literature, Druse (1986) suggested t h a t while there may be some changes soon after ethanol exposure, these are transient and disappear with subsequent development. The inconsistencies reported in the literature probably reflect variations in the methods of ethanol administration, dose, duration, and subsequent BAC. From in vitro translation of subcellular fractions isolated from the brain of ethanol-treated animals, it has been suggested that
protein synthesis is compromised due to ethanol effects on ribosomes (Tewari et al., 1980) and tRNA (Fleming et al., 1975). Effects of prenatal ethanol exposure on RNA levels in the brain generally suggest a decrease in transcription. Two weeks of prenatal ethanol exposure resulted in a decrease of total brain RNA from 100 mg/g wet weight of brain tissue to 78 mg/g in newborn pups, while DNA decreased from 96 mg/g to 55 mg/g (Rawat, 1980). The loss of DNA probably reflects ethanol-induced inhibition of mitosis (Miller, 1986) or cell death. These findings suggests that the amount of RNAkell may actually increase in response to ethanol treatment. Specific effects of ethanol on gene transcription have not been extensively studied. Milner et al. (1987), using chronic ethanol vapour exposure of pregnant rats from GD 7 to GD 21, demonstrated a delay in the expression patterns of the genes for the myelin proteins PLP and myelin-associated glycoprotein (MAG), as well as tubulin. The present study also demonstrates a delay in
ETHANOL EFFECTS ON NEURAL GENE EXPRESSION the developmental pattern of PLP mRNA, although the delay is not as dramatic a s that reported by Milner et al. (1987). In addition, we have noted that prenatal ethanol exposure also produces a delay in the pattern of expression of additional glial and neuronal genes, including GFAP and somatostatin. The ethanol exposure paradigm used in the present study is much different than the ethanol vapour inhalation method used by Milner et al. (1987) since a protein-enriched liquid diet was used. Our results, in conjunction with these, probably reflect a dosetduration response since the administration of ethanol via vapour inhalation produces chronic exposure with BAC of 100-200 mg% (Rogers et al., 1979), while the liquid diet would result in diurnal variation of the BAC depending on feeding behaviour. Due to circadian rhythyms, alcohol consumption varied throughout the day in the present study, resulting in the BAC varying between 50 and 200 mg%. Prenatal ethanol exposure has been reported to cause a delay in the maturation of the nervous system (Clarren and Smith, 1978; Colangelo and Jones, 1982). Our data suggest that this ethanol-induced delay in neural development reported by many investigators is a result of a general effect on the process of transcription, reflected in the pattern of appearance of various mRNAs. In particular, a delay in the maturation of astrocytes may underlie the observed disruption in migration of cortical neurons (Miller, 1986), since astrocytes play a major role in the process of neuronal migration (Rakic, 1981). Likewise, the lag in myelination reported following neonatal ethanol exposure (Jacobsen et al., 1979) could reflect a delay in the expression of genes coding for myelin proteins such a s PLP. While somatostatin levels have been shown to incrase in adult Balbtc mice after chronic alcohol exposure (Fuhrmann et al., 1988), no studies have examined the effect of prenatal ethanol exposure on this neuropeptide. The observed delay in the expression of mRNA for somatostatin suggests there may be a delay in the differentiation of these neurons following prenatal ethanol exposure. The present results thus suggest that a n early manifestation of the maturational delay in the nervous system induced by ethanol exposure may be seen at the level of gene expression in the different neural cell types.
ACKNOWLEDGMENTS The authors are grateful to Dr. F. Miller for her generous provision of the PLP cDNA, to Dr. N. Cowan for the GFAP cDNA, to G. Schultz for U l b cDNA, and to Dr. J. Habener for the SS cDNA. We are also grateful to Mrs. L. Wood and Mr. D. Belliveau for technical assistance. This research was supported by grants from the Hospital for Sick Children Foundation (grant 8805) and the Ministry of Community and Social Services, Ontario, Canada, administered by the Research
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and Program Evaluation Unit, in cooperation with the Ontario Mental Health Foundation, funded from the “Interprovincial” Lottery Research Program. C.C.G.N. is a Scholar of the Medical Research Council of Canada.
REFERENCES Clarren SK, Smith DW (1978): The fetal alcohol syndrome. N Engl J Med 298:1063-1067. Colangelo W, Jones DG (1982): The fetal alcohol syndrome: a review and assessment of the syndrome and its neurological sequelae. Prog Neurobiol 19:271-314. Cowan WM, Fawcett J , OLeary D, Stanfield B (1984): Regressive events in neurogenesis. Science 255:1258-1265. Druse MJ (1986): Effects of perinatal alcohol exposure on neurotransmitters, membranes and proteins. In West J R (ed): “Alcohol and Brain Development.” New York: Oxford University Press, pp 343372. Feinberg AP, Vogelstein B (1983):A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13. Fleming E, Tewari S, Noble E (1975): Effects of chronic ethanol ingestion on brain amino acyl-tRNA synthetases and tRNA. J Neurochem 24:553-560. Fuhrmann G, Strosser MT, Besnard F, Kempf E, Kempf J , Ebel A (1988): Genotypic differences in age and chronic alcohol exposure effects on somatostatin levels in hippocampus and striatum in mice. Neurochem Res 11525-636. Goodman RH, Aran DC, Bernard AR (1983): Rat preprosomatostatin: structure and processing by microsomal membranes. J Biol Chem 258:5570-5573. Jacobsen S, Rich J , Tovsky NJ (1979): Delayed myelination and lamination in the cerebral cortex of the albino rat as a result of fetal alcohol syndrome. Curr Alcohol 6:123-133. Lewis SA, Cowan NJ (1985): Temporal expression of mouse glia fibrillary acid protein mRNA studied by a rapid in situ hybridization procedure. J Neurochem 45:913-919. Lewis SA, Balcarek JM, Krek V, Shelansi ML, Cowan N J (1984): Sequence of a cDNA clone encoding mouse glial fibrillary acid protein: structural conservation of intermediate filaments. Proc Natl Acad Sci USA 81:2743-2746. Miller MW (1986): Effects of alcohol on the generation and migration of cerebral cortical neurons. Science 233:1308-1311. Milner RJ, Lai C, Nave K-A, Lenoir D, Ogata J , Sutcliffe J G (1985): Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein. Cell 42:931-939. Milner RJ, Randolph L, Bahr D, Cappello M, Lenoir D, Miller F, Bloom FE (1987): Molecular biological approaches to the brain and their application to the study of alcoholism. In Goedde HW, Agarwal DP (eds): “Genetics and Alcoholism.” New York: Alan R. Liss, Inc., pp 291-302. Naus CCG, Miller FD, Morrison JH, Bloom FE (1988): Immunohistochemical and in situ hybridization analysis of the development of the rat somatostatin-containing neocortical neuronal system. J Comp Neurol 269:448-463. Rakic P (1981): Neuron-glial interaction during brain development. Trends Neurosci 4:184-187. Rave N, Crjvenjakov R, Boedtker H (1979): Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucleic Acids Res 6:3559-3567. Rawat AK (1980): Biochemical aspects of neuroteratogenic effects of alcohol. Neurobehav Toxic01 2:259-265. Rogers J , Wiener SC, Bloom FE (1979): Long-term ethanol administration methods for rats: advantages of inhalation over intubation or liquid diets. Behav Neural Biol 27:466-486. Schibler K, Tosei M, Pittet A-C, Fabiani L, Wellames P (1980):Tissuespecific expression of mouse a-amylase genes. J Mol Biol 142:93116. Tewari S, Sweeney F, Fleming E (1980): Ethanol-induced changes in properties of rat brain ribosomes. Neurochem Res 5:1025-1035.
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Thomas PS (1980):Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5215.
West J R (1986):“Alcohol and Brain Development.” New York: Oxford Univ Press.
