Purkinje cell deficits in nonhuman primates following weekly exposure to ethanol during gestation

TERATOLOGY 53:230-236 (1996)

Purkinje Cell Deficits in Nonhuman Primates Following Weekly Exposure to Ethanol During Gestation DANIEL J. BONTHIUS, NANCY E. BONTHIUS, RUTH M.A. NAPPER, SUSAN J. ASTLEY, STERLING K. CLARREN, AND JAMES R. WEST Departments of Pediatrics and Neurology, University of Iowa College of Medicine, Iowa City, Iowa 52242 (D.J.B.); College of Pharmacy, University of Iowa, Iowa City, Iowa 52242 (N.E.B.); Department of Anatomy, University of Otago, Dunedin, New Zealand (R .M.A.N.); Department of Pediatrics, University of Washington, Seattle, Washington 98195 (S.JA., S.K.C.); Department of Human Anatomy and Medical Neurobiology, Texas A&M University, College Station, Texas 77843 (J.R.W.)

ABSTRACT The most serious features of fetal alcohol syndrome (FAS) are mental retardation and other behavioral problems resulting from alcohol-induced damage to the developing central nervous system (CNS). The mechanism by which alcohol induces its neuroteratogenic effects is unknown. One hypothesis is that gestational alcohol exposure results in a reduction in neuronal number. This study demonstrates that gestational exposure to ethanol in a non-human primate species induces permanent dose-related deficits in the number of cerebellar Purkinje cells. Ethanol was administered via nasogastric tube once per week to 15 gravid pigtailed macaques (Macaca nemistrina) in one of the following doses: 0.0 (intubated controls), 1.2, 1.8, 2.5, 3.3, and 4.1 glkgldose. Offspring were reared with parental surrogates and were sacri- ficed at 6 months of age; 8-bm-thick parasagittal sections were cut through the paraffin-embedded cerebellar vermis. Purkinje cells were quantified, the length of the Purkinie cell line was determined stereologically, and Purkinje cell linear frequency was calculated. The number of Purkinie cells and their linear frequencies were significantly reduced in the alcohol-treated subiects, and the deficits were dose-dependent. The groups receiving 2.5 g/kg/dose and above were most severely affected and had an average deficit in Purkinie cell number of 1 1.8%, relative to controls. Alcohol had no effect on the length of the Purkinje cell line. The findings suggest that alcohol-induced reduction in neuronal number may be an important factor underlying the CNS dysfunction in FAS. o 1996 WiIey-Liss, Inc.

abnormalities resulting from damage to the developing central nervous system (CNS) (Clarren, '86; Streiss- guth et al., '80).

Despite the fact that alcohol abuse is one of the leading known causes of mental retardation in the west- ern world (Abel and Sokol, '91), there is a paucity of human data available concerning the neuroanatomical changes resulting from fetal alcohol exposure. Because FAS was only recently recognized as a distinct clinical entity, and because the syndrome is not usually fatal, few fetuses or infants with recognized FAS have un- dergone autopsy. Those few autopsied cases that have been published generally describe extreme CNS damage (Clarren, '86; Claren et al., '78). However, many of these cases were accompanied by confounding compli- cations, such as severe congenital heart defects and by maternal abuse of other drugs, making it difficult to determine whether the observed CNS anomalies were due to alcohol directly or to the confounding factors.

The mechanism by which fetal alcohol exposure induces postnatal CNS dysfunction is unknown. One hypothesis is that alcohol exposure results in a reduction in neuronal number-either through interference with neuronal production or through induction of neuronal death-and that the resulting neuronal deficits underlie or contribute to the CNS dysfunction. Through the use of animal models, deficits in neuronal number following developmental alcohol exposure have been demonstrated in the hippocampus (Barnes and Walker, ,801, somatosensory cortex (Miller and Potempa, 'go), cerebellum (Hamre and West, '93), retina (Clarren et al., '90) and olfactory bulb (Bonthius et al., '92). Fur- thermore, ethanol-induced neuronal loss has been dem-

Ethanol abuse during pregnancy can have adverse effects on the developing fetus and, in severe cases, results in fetal alcohol syndrome (FAS) (Jones and Smith, '73; Jones et al., '73). The most serious features of FAS are mental retardation and other behavioral

Received August 8, 1995; accepted February 2,1996. Address reprint requests to Dr. James R. West, Department of Human Anatomy and Medical Neurobiology, Texas A&M University, 228 Reynolds Medical Building, College Station, TX 77843.

0 1996 WILEY-LISS, INC.

ETHANOL-INDUCED PURKINJE CELL DEFICITS IN MONKEYS 231

onstrated in regions known to influence specific behaviors, and these behaviors have been shown to be abnormal in animals with these neuronal deficits. For example, spatial memory deficits have been correlated with loss of CAI pyramidal cells in the hippocampus (Goodlett et al., '92). Such results support the hypothesis that neuronal loss associated with ethanol exposure is functionally important.

Nearly all the evidence demonstrating neuronal deficits following developmental alcohol exposure has been derived from rodent models of FAS. The question arises whether the alcohol-induced neuronal deficits in the brains of rats and mice are relevant to the human condition.

Several inherent constraints limit the widespread use of nonhuman primates in biomedical research. An- imals are expensive and difficult to acquire; they have long gestational periods, and pre- and postnatal care is complicated. Nevertheless, primate models offer advantages of evaluating the effects of gestational alcohol exposure on the fetus (Riley and Meyer, '84). Among these advantages are the important facts that nonhuman primates are more similar to humans develop- mentally, ontogenetically, neuroanatomically and be- haviorally than are rodents (Bourne, '75; Golub, '79; Hendrickx and Sawyer, '75).

The principal goal of this study was to determine whether prenatal exposure to ethanol can induce permanent neuronal deficits in a primate species. The neuronal population examined in this study is the cerebellar Purkinje cells, a group of neurons known to be exquisitely vulnerable to alcohol-induced cell death in developing rats (Bonthius and West, '90).

The exposure paradigm in the present study was de- signed to mimic common drinking patterns in humans. Consumption of ethanol once per week has been found to be the most frequent intermittent pattern of drinking by American women (Wilsnack et al., '84). Since the pattern of ethanol exposure during development influences the risk and severity of brain damage (Bonthius and West, '90; Webster et al., ,831, it is important to model conditions similar to which a human fetus is likely to be exposed. Therefore, the gravid monkeys in this study were exposed to intoxicating levels of ethanol on a weekly basis.

METHODS Subjects

The cerebellar tissue evaluated in this study was derived from 15 animals generated as part of a larger project at the University of Washington, the methods and results of which have been described previously (Clarren et al., '88, '90). Ethanol was administered via nasogastric tube once per week to gravid pigtailed macaques (M. nernestrina). The ethanol doses were 1.2 (n=l ) , 1.8 (n=4), 2.5 (n=2), 3.3 (n=3), and4.1 (n=1) g/kg/dose. Animals that were exposed to the lower doses (1.2 and 1.8 g/kg) of ethanol were nasogastrically

intubated weekly throughout gestation (weeks 1-24), while animals exposed to the higher doses (2.5-4.1 gkg) received the ethanol starting with the fifth week of gestation. Delay in administration of the higher doses was necessary because earlier work showed that the higher doses produced insufficient viable offspring in the time allotted for the experiments when administered throughout gestation (Clarren et al., '88). A control group of gravid females (n=4) received a weekly oral sucrose solution that was isocaloric and isovolumetric to the highest ethanol dose.

Maternal peak plasma ethanol concentrations Blood samples were taken from each pregnant fe-

male once every 8 weeks (three times per pregnancy), and plasma ethanol concentrations were determined as previously described (Clarren et al., '87). The three se- rum ethanol concentrations for each animal were averaged to provide a mean maternal peak plasma ethanol concentration (MPPEC). The complete MPPEC data for each of these animals have been published previously (Clarren et al., '88).

Tissue preparation The offspring were reared independently of their

mothers, were housed with parental surrogates (dia- pers) and had daily social experience with groups of like-aged peers. After participating in extensive physical and behavioral evaluations, the offspring were sac- rificed at approximately six months of age (329-380 days postconception). Following sedation by intramus- cular injections of ketamine, an intravenous line was placed in the saphenous vein through which chloral hydrate was infused. When a deep level of anesthesia was reached, cranial nerves and spinal cord were sev- ered, and the brain was carefully removed. The cerebellar peduncles were cut, separating the cerebellum from the brainstem. The cerebellum was wrapped in gauze and placed in 10% neutral buffered formalin to await further processing.

A 2-mm-thick sagittal slice was taken from each cerebellar vermis. The cuts were made parallel to and 1 mm to either side of the midline so that the entire midcerebellum was included. The 2-mm-thick slices were dehydrated through a series of alcohols, cleared in xylene and embedded in paraffin. Each slice was ori- ented to allow sectioning in the parasagittal plane.

The blocks were cut as 8-micrometer-thick sections on a rotary microtome, and a 1-in-5 series of sections was saved. The sections were floated in a 37°C water bath for 10 sec, mounted onto gelatin-coated slides and allowed to dry overnight at room temperature. The sections were stained with a combination of safranin 0 and cresyl violet, dehydrated, cleared in xylene, and coverslipped with Permount.

Cell counting and estimation of line length From the set of sections saved, three sections from

each subject which were free of folds and tears were

232 D.J. BONTHIUS ET AL.

Fig. 1. Photomicrographs of cerebellar cortex from 6-month-old pigtailed macaques. A From a control animal exposed to no alcohol during gestation. The arrow points to a typical Purkinje cell body. Purkinje cells with clearly discernible nuclear membranes were counted in three representative sections per animal. Note that the Purkinje cells in this control section are spaced quite closely together along the length of the Purkinje cell line. B: From an animal exposed

to weekly doses of 3.3 gkg of alcohol during gestation. The alcohol exposure has resulted in a diminishment of Purkinje cell number. The deficit in Purkinje cells manifests itself as large gaps in the Purkinje cell line in which few Purkinje cells exist. Arrow, points to the only surviving Purkinje eel1 in this segment of cortex. ml, molmlar layer; P1, Purkinje cell layer; gl, granule cell layer. Bar = 25 km.

randomly chosen for analysis. In each analyzed section (Fig. l), Purkinje cells with clearly discernible nuclear membranes were counted manually (Bonthius and West, '90, '91).

In order to determine the length of the Purkinje cell line, a magnified ( x 125) image of the section was pro- jected onto a square grid consisting of parallel lines separated by a distance of 1 cm. The intersections that the Purkinje cell layer made with each of the two sets of parallel lines making up the grid were counted. The

number of intersections (I) provided a direct and unbi- ased estimate of the length (L) of the Purkinje cell line according to the formula (Harvey and Napper, '88; Wei- bel, '79):

L = I x 6 4 x D x 1/M

where, in this case, D is 1 cm and M is 125. The linear frequency of Purkinje cells was derived by dividing the number of Purkinje cells by the length of the Purkinje cell line.


TABLE 1. Maternal peak plasma ethanol concentrations (MPPECs)'

Weekly alcohol dose Gestational weeks MPPEC (g/kg/dose) n of exposure (mg/dl) 0.0 4 1-24 1.2 1 1-24 140.5 2 3.5 1.8 4 1-24 208.4 f 29.1 2.5 2 5-24 262.3 f 23.4 3.3 3 5-24 431.6 * 74.4 4.1 1 5-24 548.3 f 87.8

'All measures represent means * S.D.

Nuclear diameters In order to correct for potential changes in nuclear

fragment number due to differences in nuclear size, the distribution of nuclear fragments was estimated from random, systematically chosen samples of Purkinje cell populations. The long and short axes of each sampled nucleus were measured under oil immersion with a gradicule eyepiece. The axes of 400 nuclear fragments per treatment group were measured. The geometric means of the axial measurements were calculated, pooled, and plotted as nuclear fragment diameter frequency histograms.

Statistical analyses Cell counts were made from three sections from each

animal. For the statistical analyses, the values from each of the three sections were averaged. The number of Purkinje cells per section, Purkinje cell line length, and Purkinje cell linear frequency were analyzed sep- arately by simple linear regression (Wonnacott and Wonnacott, '77). The nuclear diameter frequency histograms were analyzed by chi-square test.

RESULTS Table 1 demonstrates that the MPPECs ranged from

140.5 mg/dl in the animal exposed to the lowest alcohol dose (1.2 gkg) to 548.3 mg/dl in the animal exposed to the highest alcohol dose (4.1 gkg). As expected, as the dose of alcohol increased, so did the MPPEC rise in a stepwise fashion.

The cerebellums of two subjects (one each in the 3.3- and 4.1-gkg groups) were mechanically damaged during processing; several small lobules were broken and lost. As a result, it was not possible to determine accurately the total number of Purkinje cells per section nor the Purkinje cell line length for these two subjects. It was possible, however, to determine the Purkinje cell linear frequency on the remaining undamaged tissue from these two subjects. Therefore, the data regarding total Purkinje cell number and line length were derived from 13 subjects, while the data regarding Pur- kinje cell linear frequency were derived from 15 subjects. The mean number of Purkinje cells per section, estimated Purkinje cell line length and calculated Pur-

TABLE 2. Purkinje cell number, Purkinje cell line length, and Purkinje cell linear frequency for

individual animals'

Weekly Purkinje cell ethanol Purkinje cell linear

Subject dose Purkinje line length frequency no. (g/kg) cell no. (mm) (cells/mm)

1 0.0 2202 368 6.00 2 0.0 2371 422 5.62 3 0.0 2346 404 5.82 4 0.0 2467 399 6.18 5 1.2 2421 416 5.83 6 1.8 2084 376 5.45 7 1.8 2156 398 5.42 8 1.8 2457 397 6.19 9 1.8 2279 442 5.13

10 2.5 2103 377 5.58 11 2.5 1995 373 5.34 122 3.3 1582 279 5.68 13 3.3 2189 416 5.27 14 3.3 1996 378 5.29 15' 4.1 1576 301 5.20

'All measures represent the mean values from three analyzed sections. 'Subjects in which the cerebellum was mechanically damaged during processing, resulting in the loss of several small lobules. In these subjects, only the Purkinje cell linear frequency could be determined accurately and was included in the statistical analyses.

kinje cell linear frequency for each of the animals is shown in Table 2.

Alcohol exposure during gestation induced dose-dependent Purkinje cell deficits. The regression analysis indicated a significant effect of ethanol dose on Pur- kinje cell number [F(l,ll) = 8.164; P<0.051. As shown in Figure 2A and in Table 2, the subjects exposed to the highest doses (2.5 and 3.3 gkg) were the most severely affected and had an average deficit in Purkinje cell number of 11.8%, relative to controls. A strong inverse correlation existed between the ethanol dose and Pur- kinje cell number (r = -0.653).

The regression analysis also indicated a significant effect of ethanol dose on Purkinje cell linear frequency [F(1,13) = 9.59; P<O.Ol l . As shown in Figure 2B, as the dose of ethanol increased, the linear frequency of Pur- kinje cells tended to decrease linearly.

The alcohol-induced deficits in Purkinje cell number and linear frequency manifested themselves as gaps in the Purkinje cell line. Whereas control animals had a relatively frequent and uniform presence of Purkinje cells throughout the Purkinje cell line, the alcohol-exposed animals had segments of cortex in which few Purkinje cells existed (Fig. 1). Although the location of these gaps was not systematically analyzed, they seemed to occur in a random pattern, unrelated to any particular rostrocaudal position, cerebellar lobule, or depth within the folia.

The ethanol treatment had no significant effect on the length of the Purkinje cell line (Fig. 2C), indicating

234 D.J. BONTHIUS ET AL.

25 r

24

21

20

0 0 a

0 0

I 1 1 I I I 0 1.2 1.8 2.5 3.3 4.1

Alcohol Dose

6.0 1 0

5.5

5.0

1 I I I I I 0 1.2 1.8 2.5 3.3 4.1

Alcohol Dose cg 450

3.g 400 0 r=-0.150 pzO.63

0 0 a

0

0 1.2 1.8 2.5 3.3 4.1

c 3

Z E 0- .s 350 e

a

3 a Alcohol Dose

Fig. 2. Effect of gestational alcohol exposure on the number of Pur- kinje cells, Purkinje cell linear frequency and length of the Purkinje cell line. A: Dose-response effect of alcohol on the number of Purkinje cells per sagittal section. Gestational alcohol exposure induced dose- dependent Purkinje cell deficits, with the animals exposed to the highest doses (2.5 and 3.3 g/kg) being most severely affected. B Dose-

response effect of alcohol on Purkinje cell linear frequency. As the dose of alcohol increased, the linear frequency of Purkinje cells tended to decrease. C: Relationship of alcohol dose to length of the Purkinje cell line. Increasing doses of alcohol had no significant effect on the length of the Purkinje cell line.

that the alcohol-induced reduction in Purkinje cell number was not merely due to a reduced length of cortex along which the cells were counted.

Examination of nuclear fragment diameter frequency histograms indicated no significant differences in modal diameter or in the distribution of diameters among the treatment groups (Fig. 3).

DISCUSSION The most important findings of this study are that

ethanol exposure during gestation in monkeys pro- duces dose-dependent cerebellar Purkinje cell deficits and a corresponding decline in Purkinje cell linear frequency. Other than a multidisciplinary report that in-

cluded some counts of retinal ganglion cells (Clarren et al., '901, this is the first study to report quantitative evidence of dose-dependent neuronal deficits in the nonhuman primate brain as a consequence of fetal ethanol exposure. These findings of diminished Purkinje cell number and linear frequency support the hypothesis that deficits in neuronal number may underlie or contribute to the cognitive and behavioral problems associated with FAS. Indeed, as described in an earlier report (Clarren, Astley and Bowden, '88), many of the alcohol-exposed animals in this study exhibited substantial motor and cognitive deficits and delays.

A second important finding of this study is that neuronal deficits occurred following patterns of alcohol ex-


Iln, Alcohol Dose

rn 0

rn

0

0

5 - 7 7 - 8 8 - 9 9 - 1 0 1 0 - 1 1 1 1 - 1 2 1 2 - 1 3 1 3 - 1 4 1 4 - 1 8

Nucleer Fragment Dlameter (mlcrometerr)

Fig. 3. Nuclear fragment diameter frequency histogram for the Purkinje cell population of 6-month-old pigtailed macaques. The axes of 400 nuclear fragments per treatment group were measured, and the

posure and blood alcohol concentrations commonly seen in humans. "he alcohol in this study was administered once per week. Epidemiological studies have demonstrated that women who drink during pregnancy tend to binge drink-often once per week (Stephens, '85; Little and Streissguth, '78). The findings of the present study demonstrate, as have several rodent studies (Bonthius and West, '90, '911, that binge alcohol consumption during pregnancy can induce substantial and permanent brain injury in the fetus.

The mechanism underlying fetal ethanol-induced damage in general, and Purkinje cell depletion in particular, remains unknown (West et al., '94). Purkinje cells are the earliest generated cells of the cerebellar cortex (Altman and Bayer, '78). In the monkey, Pur- kinje cell neurogenesis is completed during the first half of pregnancy (Verbitskaya, '69). Since the ethanol exposure in this study occurred throughout all or most of gestation, it encompassed a broad period of brain development including eras preceeding, during and following Purkinje cell neurogenesis. Therefore, the ethanol may have interfered with any of a host of developmental processes upon which the Purkinje cell population depends for generation, differentiation and survival. Ethanol has been shown to interfere with cell

0.0

1.2

1.8

2.5

3.3

4.1

geometric means of the axial measurements were pooled and plotted. The alcohol treatments had no significant effect on the distribution of the nuclear diameters.

proliferation and migration (Borges and Lewis, '83; Miller, '86). However, studies in the rat have shown that Purkinje cells are most vulnerable to alcohol-induced cell death during the period of cellular differentiation-after they have completed the periods of neurogenesis and migration, but before they have reached full maturity (Bonthius and West, '90; Hamre and West, '93; Marcussen et al., '94). Exposure to alcohol for a single day has been shown to be sufficient to kill a substantial number of Purkinje cells during this period of vulnerability (Goodlett et al., '92). In the rat, an altricial species, this period of maximum vulnerability occurs in the early postnatal period (Marcussen et al., '94). The monkey, however, is a precocial species, and its brain is substantially more mature at the time of birth than is the rat's (Dobbing and Sands, '79). There- fore, if maximum vulnerability of the Purkinje cell to alcohol occurs a t the same developmental stage of Pur- kinje cell differentiation in the monkey as it does in the rat, then the monkey Purkinje cell would be expected to be most vulnerable during the final third of pregnancy.

Most of the alcohol-exposed animals in this study, including those exposed to the highest doses of alcohol (22.5 gkgldose), had no evidence of external dysmor-

236 D. J. BONTHIUS ET AL.

phology (for details, see Clarren, Astley and Bowden, 1988). Yet, many of these animals clearly had alcohol- induced neuronal deficits. This finding demonstrates that alcohol-induced brain damage can occur in the absence of external dysmorphology in a primate species and strengthens the troubling notion that children with alcohol-induced brain damage may be far more common than those diagnosable with the full fetal alcohol syndrome.

ACKNOWLEDGMENTS This work was supported by a Fulbright grant from

the New Zealand-United States Educational Founda- tion to D.J.B., by a Resident Research Grant from the American Academy of Pediatrics to D.J.B. and by grants AA0523 to J.R.W. and AA05616 to S.K.C. from the National Institute on Alcohol Abuse and Alcohol- ism. We thank Dr. George Spears for help with the statistical analyses.

LITERATURE CITED Abel, E.L., and R.J. Sokol (1991) A revised conservative estimate of

the incidence of FAS and its economic impact. Alcoholism Clin. Exp. Res., 15:514-524.

Altman, J., and S.A. Bayer (1978) Prental development of the cerebellar system in the rat Cytogenesis and histogenesis of the deep nuclei and the cortex of the cerebellum. J. Comp Neurol., 179:23- 48.

Barnes, D.E., and D.W. Walker (1980) Prenatal ethanol exposure permanently reduces the number of pyramidal neurons in rat hippocampus. Dev. Brain Res., 1:333-340.

Bonthius, D.J., and J.R. West (1988) Blood alcohol concentration and microencephaly: A dose-response study in the neonatal rat. Tera- tology, 37:223-231.

Bonthius, D.J., and J.R. West (1990) Alcohol-induced neuronal loss in developing rats Increased brain damage with binge exposure. Al- coholism Clin. Exp. Res., 14:107-118.

Bonthius, D.J., and J.R. West (1991) Permanent neuronal deficits in rats exposed to alcohol during the brain growth spurt. Teratology,

Bonthius, DJ., N.E. Bonthius, R.M.A. Napper, and J.R. West (1992) Early postnatal alcohol exposure acutely and permanently reduces the number of granule cells and mitral cells in the rat olfactory bulb: A stereological study. J . Comp. Neurol., 324.557-566.

Borges, S., and P.D. Lewis (1983) Effects of ethanol on postnatal cell acquisition in the rat cerebellum. Brain Res., 271:388-391.

Bourne, G.H. (1975) Collected anatomical and physiological data from the rhesus monkey. In: The Rhesus Monkey. Vol 1. G.H. Bourne, ed. Academic Press, New York, pp. 1-63.

Clarren, S.K. (1986) Neuropathology in fetal alcohol syndrome. In: Alcohol and Brain Development. J.R. West, ed. Oxford University Press, New York, pp. 158-166.

Clarren, S.K., E.C. Alvord, S.M. Sumi, A.P. Streissguth, and D.W. Smith (1978) Brain malformations related to prenatal exposure to ethanol. J. Pediatr., 92:64-67.

Clarren, S.K., S.J. Astley, and D.M. Bowden (1987) Pregnancy out- comes after weekly oral administration of ethanol during gestation in pig-tailed macaques (Macaca nemestrima). Teratology, 35:345- 354.

Clarren, S.K., S.J. Astley, and D.M. Bowden (1988) Physical anomalies and developmental delays in non-human primate infants exposed to weekly doses of ethanol during gestation. Teratology, 37: 561-569.

Clarren, S.K., S.J. Astley, D.M. Bowden, H. Lai, A.H. Milam, P.K.

44~147-163.

Rudeen and W.J. Shoemaker (1990) Neuroanatomic and neuro- chemical abnormalities in nonhuman primate infanta exposed to weekly doses of ethanol during gestation. Alcoholism Clin. Exp. Res., 14:674-683.

Dobbing, J., and J. Sands (1979) Comparative aspects of the brain growth spurt. Early Hum. Dev., 3:79-83.

Golub, M.S. (1979) A primate model for detecting behavioral impair- ment in offspring after chronic parenteral drug exposure. Pharma- col. Biochem. Behav., 11:47.

Goodlett, C.R., B.L. Marcussen, and J.R. West (1992) A single day of alcohol exposure during the brain growth spurt induces brain weight restriction and cerebellar Purkinje cell loss. Alcohol 7:107- 114.

Hamre, K.M., and J.R. West (1993) The effects of the timing of ethanol exposure during the brain growth spurt on the number of cerebellar Purkinje and granule cell nuclear profiles. Alcoholism Clin. Exp.

Harvey, R.J., and R.M.A. Napper (1988) Quantitative study of granule and Purkinje cells in the cerebellar cortex of the rat. J . Comp. Neurol., 274:151-157.

Hendrickx, A.G., and R.H. Sawyer (1975) Embryology of the rhesus monkey. In: The Rhesus Monkey, Vol. 1. G.H. Bourne, ed. Academic Press, New York, pp. 142-169.

Jones, K.D., and D.W. Smith (1973) Recognition of fetal alcohol syndrome in early infancy. Lancet, 2~999-1001.

Jones, K.D., D.W. Smith, C.N. Ulleland, and A.P. Streissguth (1973) Pattern of malformation in offspring of chronic alcoholic mothers. Lancet, 1~1267-1271.

Little, R.E. and A.P. Streissguth (1978) Drinking during pregnancy in alcoholic women. Alcoholism. Clin. Exp. Res., 2~179-183.

Marcussen, B.L., C.R. Goodlett, J.C. Mahoney, and J.R. West (1994) Developing rat Purkinje cells are more vulnerable to alcohol-induced depletion during differentiation than during neurogenesis.

Miller, M.W. (1986) Effects of alcohol on the generation and migration of cerebral cortical neurons. Science, 233:1308-1311.

Miller, M.W., and G. Potempa (1990) Numbers of neurons and glia in mature rat somatosensory cortex: Effects of prenatal exposure to ethanol. J . Comp. Neurol., 293:92-102.

Riley, E.P., and L.S. Meyer (1984) Considerations for the design, im- plementation and interpretation of animal models of fetal alcohol effects. Neurobehav. Toxicol. Teratol., 6:97-101.

Sheller, B., S.K. Clarren, S.J. Astley, and P.D. Sampson (1988) Mor- phometric analysis of Macaca nemestrina exposed to ethanol during gestation. Teratology, 38:411-417.

Stephens, C.J. (1985) Alcohol consumption during pregnancy among southern city women. Drug Alcohol Depend., 16:19-29.

Streissguth, A.P., S. Landesman-Dwyer, J.C. Martin, and D.W. Smith (1980) Teratogenic effects of alcohol in humans and laboratory animals. Science, 209~353-361.

Verbitskaya, L.B. (1969) Some aspects of the ontophylogenesis of the cerebellum. In: Neurobiology of Cerebellar Evolution and Develop- ment. R. Llinas, ed. American Medical Association, New York, pp. 859-874.

Webster, W.S., D.A. Walsh, S.E. McEwen, and A.H. L i p n (1983) Some teratogenic properties of ethanol and acetaldehyde in C57BL/6J mice: Implications for the study of fetal alcohol syndrome. Teratology, 27:231-243.

Weibel, W.R. (1979) Stereological Methods. Vol. 1. Practical Methods for Biological Morphometry. Academic Press, London.

West, J.R., W.4.A. Chen, and N.J. Pantazis (1994) Fetal alcohol syndrome: The vulnerability of the developing brain and possible mechanisms of damage. Metab. Brain Dis., 9:291-322.

Wilsnack, S.C., A.D. Klassen, and R.W. Wilsnack (1984) Drinking and reproductive dysfunction among women in a 1981 national sur- vey. Alcoholism Clin. Exp. Res., 8~451-458.

Wonnacott, T.H., and R.J. Wonnacott (1977) Nonparametric statistics. In: Introductory Statistics. John Wiley & Sons, New York, p. 471.

Res., 17:610-622.

Alcohol, 11:147-156.

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Purkinje cell deficits in nonhuman primates following weekly exposure to ethanol during gestation