Maternal Ethanol Ingestion Effects on Fetal Rat Brain Vitamin A as a Model for Fetal Alcohol Syndrome

Fetal embryo, head, and brain tissue from different gestational age were analyzed for retinol content, nuclear retinoic acid receptor and cytosolic retinoic acid binding protein levels after maternal ethanol ingestion and compared with fetal levels in control diet pregnancies. Retinol levels in fetal embryo and brain of ethanol-ingesting pregnancies were 2- to 3-fold higher than fetal embryo and brain of ethanol-ingesting pregnancies were 2-to 3-fold higher than fetal embryo and retinol of control pregnancies. Nuclear retinoic acid receptor was lower in 10-day embryo of ethanol pregnancies and apparently unaffected in fetal head and brain by maternal ethanol consumption at other days of gestation. In fetal head there was a significant overall ethanol effect on cytosolic retinoic acid binding protein, with increased levelsin fetal tissue from ethanol-consuming pregnancies. These observations of altered embryo, fetal head, and fetal brain retinol and receptor protein levels support the hypothesis of a possible role of vitamin A in fetal alcohol syndrome.     

Hypothesis: Prenatal Ethanol-Induced Birth Defects and Retinoic Acid

A hypothesis is presented to explain the biochemical basis of ethanol-induced birth defects. Prenatal ethanol exposure causes central nervous system and limb abnormalities in humans and in animals. Retinoic acid and didehydroretinoic acid are known to play an important role in the central nervous system and limb developments. Ethanol is known to inhibit the formation of retinoic acid from retinol and deplete hepatic retinoid levels. It is hypothesized that ethanol reduces the levels of retinoic acid in the developing embryo either by inhibiting conversion of retinol to retinoic acid and/or by depleting the level of retinol, thereby causing central nervous system and limb abnormalities.     

Effects of Ethanol on Biotransformation of All-Trans-Retinol and All-Trans-Retinal to AILTrans-Retinoic Acid in Rat Conceptal Cytosol

Enzymatic catalysis of the oxidations of ethanol, all-trans-retinol (t-retinol) and all-trans-retinal (t-retinal) were demonstrated in the cy-tosolic fractions of rat conceptal homogenates at day 12 of gestation. Products of the retinoid oxidation reactions were identified with HPLC by comparing elution times with those of authentic standard retinoids. NAD-dependent oxidations of each of the three substrates were demonstrable with assay conditions used t-retinol and t-retinal each were converted to readily detectable quantities of all-trans-retinoic acid (t-RA). At 1.0 mM or higher concentrations, ethanol effectively inhibited the synthesis of t-RA from both t-retinol and t-retinal when adult hepatic cytosol was used as enzyme source. Approximately 70% and 40% inhibitions, respectively, were observed at 10 mM ethanol concentrations. By contrast, for the reactions catalyzed by rat conceptal cytosol (RCC) under the same experimental conditions, ethanol failed to inhibit significantly the conversion of either t-retinol or t-retinal to t-RA at concentrations up to 1,000 mM. For the RCC-catalyzed conversion of t-retinal to t-RA, increasing concentrations of ethanol (0 to 1.0 M) resulted in linear increases rather than decreases in quantities of t-RA generated. At a 2.0 M concentration of ethanol, the quantity of t-RA increased by >50%. Significant inhibition of t-RA generation from t-retinal occurred only at extremely high (>4.0 M) concentrations. The results indicated that ethanol was a very ineffective inhibitor of RCC-catalyzed synthesis of t-RA from either t-retinol or t-retinal. This contrasted strongly with effective inhibitory effects with adult hepatic cytosol as enzyme source. The results supported the concept that competitive inhibition of conversion of t-retinol to t-RA in conceptal tissues is not a significant factor in ethanol-elicited embryotoxicity and dysmorphogenesis, at least in rodents. Mechanisms for the eth-anol-induced increases in conversion of t-retinal to t-RA remain to be elucidated.     

A Retinoic Acid Receptor Antagonist Suppresses Brain Retinoic Acid Receptor Overexpression and Reverses a Working Memory Deficit Induced by Chronic Ethanol Consumption in Mice

BACKGROUND: Chronic ethanol consumption induces disorders in the biosynthesis of retinoic acid, an active derivative of vitamin A. Recent evidence suggests that an alteration in the retinoic acid signaling pathway leads to impairments in learning and memory in adult mice. We have previously shown that chronic ethanol consumption in mice produces an increased expression of the brain retinoic acid receptor beta (RARbeta) mRNA. These results prompted us to examine whether suppressing the overexpression of retinoid receptors in alcohol-treated mice by RAR antagonist administration would reverse their cognitive impairment. METHODS: After 10 months of ethanol consumption (12% v/v in drinking water), C57BL/6 mice were submitted to a working memory task in a T-maze. Then, mice of the control and the ethanol-treated groups received an RARbeta antagonist (CD2665 0.6 mg/kg) for 22 days. The behavioral effect of CD2665 administration was evaluated on a spontaneous alternation task and the neurochemical effect was measured by quantifying the mRNA expression of RARalpha, RARbeta, retinoid X receptor (RXRbeta/gamma) and tissue transglutaminase (tTG; a retinoic acid-target gene). RESULTS: Mice submitted to ethanol treatment exhibited a progressive decrease in spontaneous alternation rates over successive trials. Moreover, these mice displayed an increased expression of brain RARbeta and RXRbeta/gamma mRNA, together with an increased level of tTG mRNA and enzymatic activity. The administration of CD2665 to alcohol-treated mice totally reversed the working memory deficit and suppressed the overexpression of brain RARbeta, RXRbeta/gamma and tTG mRNA, whereas the same treatment in control mice decreased only the RARbeta mRNA level without affecting memory performance. CONCLUSION: These data point to the potential role of the retinoid signaling pathway in memory processes and suggest that the overexpression of brain RARbeta and RXRbeta/gamma could be responsible, at least in part, for some memory impairments observed during chronic ethanol consumption.     

Prenatal Ethanol Exposure Selectively Reduces the mRNA Encoding α-1 Thyroid Hormone Receptor in Fetal Rat Brain

Some of the developmental defects characteristic of congenital or experimental hypothyroidism are also observed in children or experimental animals prenatally exposed to ethanol, suggesting that a subset of neurological defects attributable to ethanol exposure are produced by interfering with thyroid hormone action. In this article, we tested whether an ethanol treatment regimen known to produce neurological damage in rats can alter the expression of the mRNAs encoding the thyroid hormone receptor isoforms (TR α-1, TR α-2, and TR α-1) in the fetal rat brain neocortex and hippocampus. Rats were fed an ethanol-containing diet beginning on gestational day (G) 6 and continuing until sacrifice on G15, G17, or G21; controls included animals pair-fed a liquid control diet or fed lab chow. Ethanol selectively reduced the expression of TR a-1 mRNA in the neocortex and hippocampus on G21, compared with pair-fed and control fetuses. In contrast, pair-feeding selectively reduced TR a-2 mRNA in both neocortex and hippocampus on G21, and increased TR 0–1 mRNA on G17. These data support the hypothesis that ethanol may interfere with thyroid hormone action during fetal brain development In addition, these data indicate that ethanol and pair-feeding exert independent effects on thyroid hormone receptor expression in the developing brain.     

Acute Prenatal Ethanol Exposure and Luteinizing Hormone-Releasing Hormone Messenger RNA Expression in the Fetal Mouse Brain

Ethanol exposure during critical periods of development results in alterations of central nervous system morphology and function. In this study, the effects of acute ethanol exposure on the number of neurons expressing luteinizing hormone-releasing hormone (LHRH) messenger RNA (mRNA) has been analyzed. Also, the expression of LHRH mRNA in the diagonal band of Broca/preoptic area (DBB/POA) was determined. Pregnant C57BL/6J mice were intubated with two doses of a 25% solution of ethanol or water (2.9 g/kg body weight) 4 hr apart on gestation day 7 (G7), G10, or G11. Animals were killed on G18, and in situ hybridization was utilized to detect neurons expressing LHRH mRNA. The number of neurons expressing LHRH mRNA was determined along their migration route from the rostrum into the forebrain. Ethanol exposure on G7 did not significantly change the number of neurons expressing LHRH mRNA on G18 compared with that in control animals. However, the number of neurons expressing LHRH mRNA in the nasal septum area in animals exposed to ethanol on G10 or G11 was significantly less than the number in control animals (p < 0.05). Prenatal ethanol exposure on any of the aforementioned treatment days did not alter the expression of LHRH mRNA at the level of the DBB/POA on G18 in ethanol-treated animals compared with control animals. Also, neuron-specific enolase mRNA expression at the level of the DBB/POA was not altered by prenatal ethanol exposure. Therefore, ethanol exposure on the aforementioned treatment days did not differentially affect LHRH mRNA expression compared with neuron-specific enolase mRNA expression at the level of the DBB/POA. These data suggest that acute ethanol exposure may have specific effects on a subset of the LHRH neuron population, depending on the day of ethanol exposure during gestation and the neuroanatomical region in which the LHRH neurons are located.     

Ethanol Impairs Activation of Retinoic Acid Receptors in Cerebellar Granule Cells in a Rodent Model of Fetal Alcohol Spectrum Disorders

Background: Ethanol is the main addictive and neurotoxic constituent of alcohol. Ethanol exposure during embryonic development causes dysfunction of the central nervous system (CNS) and leads to fetal alcohol spectrum disorders. The cerebellum is one of the CNS regions that are particularly vulnerable to ethanol toxic effects. Retinoic acid (RA) is a physiologically active metabolite of vitamin A that is locally synthesized in the cerebellum. Studies have shown that RA is required for neuronal development, but it remains unknown if ethanol impairs RA signaling and thus induces neuronal malformations. In this study, we tested the hypothesis that ethanol impairs the expression and activation of RA receptors in cerebellum and in cerebellar granule cells. Methods: The cerebellum of ethanol unexposed and exposed pups was used to study the expression of retinoic acid receptors (RARs or RXRs) by immunohistochemistry and by Western blot analysis. We also studied the effect of ethanol on expression of RA receptors in the cerebellar granule cells. Activation of RA receptors (DNA‐binding activities) in response to high‐dose ethanol was determined by electrophoretic mobility shift and supershift assays. Results: Findings from these studies demonstrated that ethanol exposure reduced the expression of RARα/γ while it increased the expression of RXRα/γ in the cerebellum and in cerebellar granule neurons. Immuno‐histological studies further strengthened the expression pattern of RA receptors in response to ethanol. The DNA‐binding activity of RARs was reduced, while DNA‐binding activity of RXRs was increased in response to ethanol exposure. Conclusion: For the first time, our studies have demonstrated that high‐dose ethanol affects the expression and activation of RA receptors, which could impair the signaling events and induce harmful effects on the survival and differentiation of cerebellar granule cells. Taken together, these findings could provide insight into the treatment options for brain defects caused by excessive ethanol exposure, such as in Fetal Alcohol Spectrum Disorders.     

Ethanol Reduces Expression of the Nerve Growth Factor Receptor, But Not Nerve Growth Factor Protein Levels in the Neonatal Rat Cerebellum

The cerebellum is especially vulnerable to ethanol's neurotoxic effects during development, and ethanol exposure during the brain growth spurt will deplete cerebellar neurons. The mechanisms underlying this neuronal cell loss remain elusive. Nerve growth factor (NGF) is a neurotrophin that promotes cell survival in various brain areas, and there is evidence that NGF may play a role in the developing cerebellum. This study examined whether ethanol exposure of the neonatal rat cerebellum altered the levels of either NGF or the expression of p75 and trkA, which are two components of the NGF receptor. Ethanol exposure had no effect on NGF levels in the neonatal cerebellum, as determined by an NGF-specific ELISA. Immunohistochemical labeling techniques indicated that both the p75 and trkA NGF receptors were expressed on Purkinje cell dendrites in the developing cerebellum, with posterior lobules expressing higher levels of p75 and trkA NGF receptor, compared with anterior lobules. Ethanol exposure of neonatal rats reduced the expression of both p75 and trkA NGF receptors on the Purkinje cell dendrites. These results suggest that ethanol could interfere with neurotrophic support of Purkinje cells by reducing the levels of available NGF receptor.     

The Interaction of Ethanol and Vitamin A as a Potential Mechanism for the Pathogenesis of Fetal Alcohol Syndrome

The mechanism of the fetal embryopathology resulting from ethanol ingestion during pregnancy is not established. This review summarizes recent research on the interaction of ethanol and vitamin A in models that explore if an interaction between these two compounds might potentially be the mechanism for fetal alcohol syndrome. The rationale for this hypothesis includes the known facts that: (1) in adults, ethanol ingestion alters vitamin A metabolism and tissue distribution; (2) there are many phenotypic similarities between fetal alcohol syndrome and malformations of both vitamin A toxicity and deficiency; and (3) the vitamin A metabolite, retinoic acid (RA), is a potent mediator in embryogenesis and differentiation. One interaction that could possibly alter fetal development is that the synthesis of RA from retinol, catalyzed by alcohol dehydrogenase, might be competitively inhibited by ethanol leading to RA deficiency. Controversy over this hypothesis continues. Another model demonstrates in vivo effects of pregnant rat mother's ethanol consumption on retinol, retinyl ester, RA content, RA receptor (RAR) binding, and the levels of RAR expression in developing fetal organs. The variable responses in this model still need clarification, and specific defects resulting from specific RAR changes have not yet been identified. In a quail embryo model, ethanol treatment mimics vitamin A deficiency, and RA appears to prevent the adverse effects of ethanol. Finally, RA and ethanol reverse or block each other's effects in studies on isolated neuroblastoma cells. Taken together, these experiments show definite interactions between ethanol and vitamin A. Further studies are needed to determine if any of these mechanisms significantly contribute to prenatal ethanol consumption embryopathy; but, clearly this hypothesis is gaining experimental support.     

Chronic Prenatal Ethanol Exposure Alters the Normal Ontogeny of Choline Acetyltransferase Activity in the Rat Septohippocampal System

In animal models of fetal alcohol syndrome (FAS), the hippocampus has been shown to be especially sensitive to the effects of prenatal ethanol exposure, exhibiting neuronal loss and alterations in neuritic process elaboration. We have characterized the influence of chronic prenatal ethanol treatment (CPET) on the postnatal expression of choline acetyltransferase (ChAT) In the hippocampus and the septal area that contains neurons that provide the primary cholinergic innervation to the hippocampus. On gestation days 1–22, pregnant rats were either fed an ethanol-containing liquid diet, pair-fed a calorically equivalent sucrose-containing diet, or given rat chow ad libitum. In Chow control animals, the ontogenetic progression of ChAT activity in the septal area and hippocampus was characterized by a significant period of upregulation during the 2nd and 3rd postnatal weeks, exhibiting an approximate 5-fold increase (septal area) and 7-fold increase (hippocampus) by postnatal day 21 (P21). At P14, ethanol exposure reduced septal and hippocampal ChAT activity levels, compared with those of pair-fed offspring. ChAT activity reached control levels by P21 in ethanol-exposed pups, suggesting that the earlier decline in activity may reflect a delay in the ontogenetic upregulation. In addition, there was a trend toward increased septal and hippocampal ChAT activities at P1 and P7 in both liquid diet groups. This liquid diet-stimulated increase may mask the effects of ethanol on early postnatal ChAT expression in the septohippocampal system. The results suggest that prenatal ethanol exposure may influence factors that regulate the developmental expression of ChAT in the septohippocampal system. In addition, we found a close relationship between the effects of CPET on litter birthweight and subsequent postnatal body and brain weight measures, but not between litter birthweight and ChAT activity from individual animals. This may imply that the influence of ethanol exposure on ChAT activity is independent of its effects on postnatal brain or body growth. Such an observation of neurochemical dysfunction in the absence of morphological indices frequently used to diagnose alcohol-related deficits could have potential clinical significance, both in the diagnostic and therapeutic realms.     

Effect of Prenatal Exposure to Ethanol on the Cell Cycle Kinetics and Growth Fraction in the Proliferative Zones of Fetal Rat Cerebral Cortex

Prenatal exposure to ethanol produces profound changes in the number of neurons in the mature cortex. These changes in neuronal number may reflect ethanol-induced disturbances in early developmental processes, that is in the proliferation of neuronal precursors. Hence, the present study examined the effect of ethanol on cell proliferation in the two neocortical proliferative zones, the ventricular zone (VZ) and subventricular zone (SZ). From gestational day 5 to 21, pregnant rats were fed an ethanol diet (6.7% v/v), pair-fed an isocaloric control diet, or fed chow and water. Pregnant rats were given a series of one to nine injections of bromodeoxyuridine (BrdU). After immunohistochemical processing, the ratio of cells in each proliferative zone that were labeled with BrdU to the total population was determined. The portion of the population that was cycling (growth fraction), the total length of the cell cycle, and the length of the S-phase of the cell cycle were calculated for VZ and SZ cells. Exposure to moderate levels of ethanol has markedly different effects upon the two neocortical proliferative zones. In the VZ, the length of the total cell cycle was significantly greater in ethanol-treated rats than in controls; however, the growth fraction and the length of the S-phase were unaffected by ethanol. In contrast, in the SZ, the growth fraction was significantly greater in ethanol-treated rats, but ethanol had no effect on the length of the total cell cycle or of the S-phase. These differences may underlie the ethanol-induced abnormalities in neuronal generation.     

Expression Patterns of Class I and Class IV Alcohol Dehydrogenase Genes in Developing Epithelia Suggest a Role for Alcohol Dehydrogenase in Local Retinoic Acid Synthesis

Vitamin A (retinol) regulates embryonic development and adult epithelial function via metabolism to retinoic acid, a pleiotrophic regulator of gene expression. Retinoic acid is synthesized locally and functions in an autocrine or paracrine fashion, but the enzymes involved remain obscure. Alcohol dehydrogenase (ADH) isozymes capable of metabolizing retinol include class I and class IV ADHs, with class III ADH unable to perform this function. ADHs also metabolize ethanol, and high levels of ethanol inhibit retinol metabolism, suggesting a possible mode of action for some of the medical complications of alcoholism. To explore whether any ADH isozymes are linked to retinoic acid synthesis, herein we have examined the expression patterns of all known classes of ADH in mouse embryonic and adult tissues, and also measured retinoic acid levels. Using in situ hybridization, class I ADH mRNA was localized in the embryo to the epithelia of the genitourinary tract, intestinal tract, adrenal gland, liver, conjunctival sac, epidermis, nasal epithelium, and lung, plus in the adult to epithelia within the testis, epididymis, uterus, kidney, intestine, adrenal cortex, and liver. Class IV ADH mRNA was localized in the embryo to the adrenal gland and nasal epithelium, plus in the adult to the epithelia of the esophagus, stomach, testis, epididymis, epidermis, and adrenal cortex. Class III ADH mRNA, in contrast, was present at low levels and not highly localized in the embryonic and adult tissues examined. We detected significant retinoic acid levels in the fetal kidney, fetal/adult intestine and adrenal gland, as well as the adult liver, lung, testis, epididymis, and uterus—all sites of class I and/or class IV ADH gene expression. These findings indicate that the expression patterns of class I ADH and class IV ADH, but not class III ADH, are consistent with a function in local retinoic acid synthesis needed for the development and maintenance of many specialized epithelial tissues.     

Fetal Alcohol Exposure Increases Mammary Tumor Susceptibility and Alters Tumor Phenotype in Rats

Background: Altered fetal programming because of a suboptimal in utero environment has been shown to increase susceptibility to many diseases later in life. This study examined the effect of alcohol exposure in utero on N‐nitroso‐N‐methylurea (NMU)‐induced mammary cancer risk during adulthood. Methods: Study 1: Pregnant Sprague Dawley rats were fed a liquid diet containing 6.7% ethanol (alcohol‐fed), an isocaloric liquid diet (pair‐fed), or rat chow ad libitum (ad lib‐fed) from day 11 to 21 of gestation. At birth, female pups were cross‐fostered to ad lib‐fed control dams. Adult offspring were given an I.P. injection of NMU at a dose of 50 mg/kg body weight. Mammary glands were palpated for tumors twice a week, and rats were euthanized at 23 weeks postinjection. Study 2: To investigate the role of estradiol (E2), animals were exposed to the same in utero treatments but were not given NMU. Serum was collected during the preovulatory phase of the estrous cycle. Results: At 16 weeks postinjection, overall tumor multiplicity was greater in the offspring from the alcohol‐fed group compared to the control groups, indicating a decrease in tumor latency. At study termination, 70% of all animals possessed tumors. Alcohol‐exposed animals developed more malignant tumors and more estrogen receptor‐α–negative tumors relative to the control groups. In addition, IGF‐binding protein‐5 (IGFBP‐5) mRNA and protein were decreased in tumors of alcohol‐exposed animals. Study 2 showed that alcohol‐fed animals had significantly increased circulating E2 when compared to either control group. Conclusions: These data indicate that alcohol exposure in utero increases susceptibility to mammary tumorigenesis in adulthood and suggest that alterations in the IGF and E2 systems may play a role in the underlying mechanism.     

A Review of the Neuroanatomical Findings in Children with Fetal Alcohol Syndrome or Prenatal Exposure to Alcohol

Human and animal studies have clearly demonstrated that alcohol is both a physical and behavioral teratogen and that heavy prenatal alcohol exposure can lead to a distinct pattern of birth defects termed the fetal alcohol syndrome. Underlying the behavioral and cognitive anomalies seen in fetal alcohol syndrome are alterations in brain structure and/or function. This paper reviews the literature examining brain anomalies attributable to prenatal alcohol exposure, beginning with a survey of autopsy studies and leading up to current findings using magnetic resonance imaging and positron emission tomography studies. Autopsy reports clearly illustrate the wide and devastating influence alcohol has on the developing brain, although for the most part no specific pattern of brain malformation has been identified. More recent magnetic resonance imaging studies, particularly when combined with quantitative analysis, have indicated that specific brain areas–such as the basal ganglia, the corpus callo-sum, and parts of the cerebellum–might be especially susceptible to alcohol's teratogenic effects. Further studies using functional brain imaging techniques may provide even more information about the unique effects prenatal alcohol exposure has on the developing brain. Discovering specific areas of the brain that are affected by alcohol may allow clinicians and researchers to look for patterns of vulnerable regions in the brain, thereby helping in the future detection of children who are prenatally exposed to alcohol.     

Cell Cycle Kinetics in Fetal Rat Cerebral Cortex: Effects of Prenatal Treatment with Ethanol Assessed by a Cumulative Labeling Technique with Flow Cytometry

The effects of ethanol on the cell cycle kinetics of cortical precursor cells during the period of cortical neuronogenesis [between gestational day (G) 12 and G21] was systematically examined. Samples of dissociated cortical cells were harvested from the cerebral cortices of 13-, 15-, 17-, 19-, and 21-day-old fetuses. The fetuses were obtained from pregnant rats: (a) fed a liquid diet containing 6.7% (v/v) ethanol (Et) ad libitum, (b) pair-fed an Isocaloric liquid control diet (Ct), or (c) fed chow and water (Ch) ad libitum. Before harvesting the cells, the fetuses were administered a series of 1–5 injections of bromodeoxyuridine (BrdU). The proportion of cells that incorporated the BrdU was assessed. Using these raw data, the S-phase length (T_B, total cell cycle length (T_c), and the growth fraction (GF; the fraction of the total population that was actively cycling) were determined with a cumulative labeling procedure. The T₃ was –8–9 hr, regardless of either the date of the injection or the dietary treatment of the dam. On the other hand, the T_c for the Ct- and Ch-treated rats increased over the gestational period. That is, the T_c was shortest on G13 and longest on G21. The T_c for Et-treated rats, however, did not change between G13 and G21. For the Ch- and Ct-treated groups, the GF decreased > 15-fold between G13 and G21. The decline (5-fold) for the Et-treated group over the same period, however, was not as great as it was for the Ct-treated fetuses. Thus, by G17 (and thereafter), the GF for Et-treated fetuses was significantly greater than it was for the Ct-treated group. Ethanol treatment has opposite effects on the two cortical germinal zones; it stimulates the proliferation of subventricular cells, whereas it inhibits the proliferation of ventricular cells). Thus, the ethanol-induced changes in the T_c and the GF reflect the combined effects of ethanol on the two cortical proliferative zones.     

Effects of Prenatal Exposure to Ethanol on the Number of Axons in the Pyramidal Tract of the Rat

We examined the effect of gestational ethanol exposure on the number of axons in the caudal pyramidal tract. Between gestational day (G)6 and G21, inclusive, pregnant rats were fed a liquid ethanol-containing diet (Et), an isocaloric liquid control diet (Ct), or a diet of chow and water (Ch). On postnatal day 30, the offspring of these rats were killed and their caudal medullas were processed for electron microscopy. The overall size of the pyramidal tract and the space occupied by the axons was smaller in the Et-treated rats than in the Ct-treated rats. The myelinated axons were smaller and the myelin was thinner in the Et-treated rats than in the Ct-treated rats. These decreases produced an ethanol-induced increase in the density of axons in the pyramidal tract. In particular, the density of myelinated axons (but not nonmyelinated axons) was greater in Et-treated rats. The net result was that the estimated number of axons in the pyramidal tracts of the Et-treated rats was not significantly different than the number in the Ch-and Ct-treated rats. The present data demonstrate that ethanol does not affect the absolute number of axons in the pyramidal tract. As a result of the ethanol-induced microencephaly, however, the data translate into a relative increase in the number of pyramidal tract axons. This relative increase matches the ethanol-induced increase in the density of corticospinal projection neurons that may result from the retention of a developmentally exuberant projection.