Developmental Brain Research, 1 (1981) 333-340 333

© Elsevier/North-Holland Biomedical Press

PRENATAL E T H A N O L EXPOSURE P E R M A N E N T L Y REDUCES THE

N U M B E R OF P Y R A M I D A L NEURONS IN RAT HIPPOCAMPUS

DAVID E. BARNES* and DON W. WALKER

Veterans Administration Medical Center and Department of Neuroscience, University of Florida,
College of Medicine, Gainesville, Fla. 32610 (U.S.A.)
(Accepted December 1st, 1980)

Key words: ethanol -- hippocampus -- prenatal ethanol exposure -- hippocampal development

fetal alcohol syndrome

SUMMARY

The effect of prenatal ethanol exposure in the rat on the development of dorsal
hippocampal pyramidal and dentate gyrus granule cells was examined. Ethanol was
administered in a nutritionally adequate liquid diet to pregnant rats during days 10-21
of gestation. Control groups were either pair-fed the same liquid diet, except for
equicaloric substitution of sucrose for ethanol, or received free access to pelleted
laboratory food. The brains of 60-day-old offspring exposed to ethanol during
gestation were found to have 20 ~o fewer dorsal hippocampal pyramidal cells than did
those of controls. Prenatal ethanol exposure, however, did not affect the number of
dentate gyrus granule cells. Prenatal exposure to ethanol permanently reduced the
number of prenatally formed hippocampal neurons without altering physical growth,
which suggested that the developing nervous system is particularly sensitive to the
toxic effects of ethanol.

INTRODUCTION

In the past few years a number of clinical reports have suggested that maternal
alcoholism is correlated with a pattern of abnormal development in children of such
mothers3,17, 22. This pattern of altered development and morphogenesis is commonly
called the 'fetal alcohol syndrome'16, is. Major features of the syndrome include pre-
and postnatal growth deficiency, unusual facial features, joint and skeletal abnor-
malities, cardiac and renal defects, microcephaly and mental deficiency. Not all of

* Address for correspondenceand reprints: Research Service(15l), Veterans Administration Medical

Center, Gainesvill¢, Fla. 32602, U.S.A.
334

these abnormalities are observed in each affected child. There are naturally gradations
of severity and patterns of defects that most likely depend on the amount and pattern
of alcohol consumption during pregnancy, as well as the specific time during the
gestational period in which the fetus is most heavily exposed. The correlative relation-
ship between alcohol consumption during pregnancy and subsequent physical and
mental abnormalities in the fetus seems well established. However, the possibility that
malnutrition, exposure to other drugs or other pre- or postnatal environmental factors
during pregnancy are responsible for the physical and intellectual abnormalities
associated with the fetal alcohol syndrome cannot be dismissed, since adequate control
over these co-existing variables is difficult or impossible in clinical studies of humans.
It is clear that proper experimental control over nutritional, environmental and
genetic variables can be obtained in animal experiments. A number of investigations of
the effect of prenatal exposure to ethanol in animals were made in the past, but these
early studies failed to control for nutrition or were methodologically unsound; thus
their results were unconvincing and contradictory z~. More recently, several investi-
gators have used nutritionally controlled animal models to demonstrate convincingly
the teratogenic effect of ethanol in mice4,~,s,24, 25 and dogs a3. Rats are evidently more
resistent to the teratogenic effect of ethanol because gross malformation of organ
systems has not been observedV,14,a~,z6. There have been a number of reports of
behavioral deficits on a variety of tasks in rats exposed to ethanol during gestation 6,7,
14,26-28,32. Some studies have noted that ethanol consumption during pregnancy
resulted in reduced physical growth of the offspring11,14,15,2°,27. However, the
majority of such reports have used a calorically dilute (1.0 kcal per ml) liquid diet or
continued exposure of the dams after birth, or both, without using a cross-fostering
procedure 11,14,15,2°,27. Ethanol is known to inhibit lactation 1°, and thus postnatal
ethanol consumption by the mother may lead to undernutrition of the developing
pups. The use of a calorically dilute diet could lead to a deficit of calories or protein or
both, resulting in altered physical development. Those experiments using a more
calorically concentrated diet and only prenatal exposure have not found deficits in
physical growth of ethanol-exposed offspringV,26,zs.
The suggestion that anomalies of brain development can occur in the absence of
detectable external anomalies is supported by a recently reported postmortem
neuropathological case study of human neonates that were exposed to ethanol during
gestation 9,28. That the developing nervous system is particularly sensitive to the effects
of prenatal ethanol exposure is also supported by the observations of behavioral
deficits 6,7,14,28-28,32 and delayed20, 33 cerebellar development in exposed offspring in
the absence of other observable teratology.
To date there have been no reports of permanent structural alterations of the
brain in animals exposed to ethanol during gestation. A permanent reduction in the
number of Purkinje and granule cells in the cerebellar cortex after early postnatal
exposure has been reported, howeverL The objective of the present experiment was to
determine if prenatal ethanol exposure results in a permanent alteration in the
complement of hippocampal neurons. The hippocampus was chosen as one model
system by which to assess the effects of prenatal ethanol exposure on neural
 335

development because of the availability of detailed knowledge of its normal develop-

ment and because its uniquely laminated structure is readily amenable to quantitative
measurement. We report here that prenatal ethanol exposure during gestational days
10-21 results in a permanent reduction of the prenatally formed dorsal hippocampal
pyramidal neurons, but does not affect the final complement of the postnatally formed
granule cells of the dentate gyrus.

MATERIALSAND METHODS

Animals and treatment groups

Parent animals were Long-Evans hooded rats purchased from Charles River.
After acclimatization to the laboratory, nulliparous rats approximately 3 months of
age and ranging in weight between 225 and 275 g were placed overnight with adult
male rats. Insemination was established the following morning (gestation day 1) by
vaginal smear. The pregnant rats were housed in standard individual maternity cages
under controlled temperature and humidity conditions and with a 07.00-19.00 h light
cycle. Lab Chow (Ralston Purina, St. Louis, Mo.) and water were freely available
except as noted below.
On gestation days 10-21, pregnant dams were given 1 of 3 treatments. One group
(n ----- 6) was given free access to an ethanol-containing liquid diet, in which ethanol
comprised 35 ~o of the available calories (8.1 ~o ethanol v/v). Control groups received
either a sucrose-containing liquid diet (n ----- 6), in which sucrose was substituted
equicalorically for ethanol and pair-fed to the ethanol group, or continued access to
Lab Chow throughout gestation (n = 13).

Liquid diets
The liquid diets were prepared by mixing the appropriate volumes of isocaloric
stock solutions of ethanol or sucrose with Sustacal (Mead Johnson) to result in diets
with 35 ~o ethanol or sucrose-derived calories and a total caloric concentration of 1.3
kcal/ml. Liquid diets were additionally supplemented with Vitamin Diet Fortification
mixture, 3.0 g/l, and Salt mixture X1V, 5.0 g/l (both from ICN Nutritional Bio-
chemicals, Cleveland, Ohio).

General procedures
Diets were prepared fresh daily and administered in calibrated bottles through
stainless-steel drinking tubes with openings enlarged to 4.0 mm. Pregnant dams were
weighed daily throughout gestation, and ethanol consumption was calculated as g/kg
body weight. At birth all pups were weighed and reduced to pooled litters of 8 males.
All litters were fostered to additional Lab Chow control dams. Pups were weighed at 5,
10, 15 and 21 days of age. At postnatal day 21, pups were either sacrificed by cardiac
puncture for fresh brain weights or were housed in groups of 4 in standard plastic
cages until they were 60 days old.
336

Histological procedures
At 60 days of age the offspring were transcardially perfused under Nembutal
anesthesia with 0.9 ~ saline followed by 10 ~ neutral buffered formalin. After removal
each brain was hemisected by a midsagittal cut, and one hemisphere was embedded in
Paraplast (Sherwood Medical, St. Louis), sectioned sagittally at 4.0 #m and a 2 in 10
series saved. Alternate slides were stained with cresyl violet or hematoxylin and eosin.
Anatomically matched sets of sections from each brain, approximately 1500 ,urn from
midline 19, were coded and used for quantitative histological analysis of the dorsal
hippocampus and dentate gyrus. Based on the section thickness (4.0 #m), the sections
calculated to be 1500 #m from midline were identified for each brain. These selected
coded sections were then compared in regard to intra- and extrahippocampal
landmarks and the matching thus further refined. Three sections were counted from
each brain; the matched section and the sections 40 #m medial and 40 #m lateral to the
matched section. Since there were no significant within-group differences in counts as a
function of these mediolateral locations, the values determined for each of the 3
sections per brain were averaged. Determination of the mean number of CAI and
CA2-4 hippocampal pyramidal cells per section was made by counting (magnification,
675 x ) throughout each section the total number of soma containing a clearly defined
nucleolus. Cytoarchitectonic regions of the hippocampus were defined according to
the criteria and nomenclature of Lorente de N621. The mean number of dentate gyrus
granule cells per section was determined by counting the number of cells in unit areas
and multiplying by the total cross-sectional area of the granule cell layer. The number
of granule cells per sq.mm was determined by calculating the average number of
granule cells contained within a grid area of 0.0025 sq.mm at a magnification of
1500 ×. Six samples of granule cell density were taken in each section, 3 samples from
each of the dorsal and ventral blades of the dentate gyrus. The total cross-sectional
area of the granule cell layer was determined by projecting each section onto paper by
use of a projecting microscope, tracing the boundary of the cell layer and measuring
the area with a Numonics digitizer (model 1224) programmed for area determination.

RESULTS

Consumption and physical growth

There were no significant differences in body weight gain among the 3 groups of
pregnant dams. The ethanol and pair-fed sucrose groups consumed an average of 9 l
± 6 kcal/day. The ethanol-treated groups consumed an average of 15.5 ~: 0.9 g/kg of
ethanol per day during the 12 days of ethanol treatment. A summary of the litter size
and physical growth data is presented in Table I. Prenatal exposure to ethanol
significantly reduced body weight of the offspring at birth, relative to control groups,
but had no effect on body weight at 5, I 0, 15 or 60 days of age or on fresh brain weight
at 21 days of age. Thus physical growth was not significantly altered by prenatal
ethanol exposure under the nutritionally adequate conditions of this experiment.

Histological measurements
The effect of prenatal ethanol exposure during gestational days 10-21 on the
 337

TABLE I
Litter size and body and brain weights of rat pups exposed to ethanol or control diets during days 10-21
of gestation
Results expressed as means 4- S.E. of the average values for each litter. Sucrose group was maintained
on sucrose-containing liquid diets individually pair fed with ethanol group. The liquid diets provided
35 % of total calories as ethanol or sucrose and contained 1.3 kcal/ml. The ethanol diet contained 8.1%
ethanol (v/v).

Variable Measured Treatment group (maternal diet)

Lab chow control Sucrose control Ethanol
(13 litters) (6 litters) (6 litters)

Litter size 14.2 :l: 0.5 13.5 4- 0.6 12.3 4- 1.2

Birth weight (g) 5.8 + 0.1 5.7 4- 0.3 5.2 + 0.2*
Weight at 5 days 12.0 4- 0.5 12.0 4- 0.6 11.8 + 0.6
Weight at 10 days 22.2 4- 1.3 23.2 4- 1.1 22.7 4- 0.6
Weight at 15 days 33.3 4- 1.2 34.2 4- 1.2 32.7 q- 0.8
Weight at 21 days 49.3 4- 1.4 48.6 + 1.9 47.4 -4- 1.4
Weight at 60 days 296.3 4- 5.6 296.6 4- 5.2 295.7 4- 5.6
Brain weight at 21 days (g) 1.390 + 0.012 1.390 4- 0.015 1.383 4- 0.015

* Significantly different from control groups, P < 0.05.

n u m b e r o f d o r s a l h i p p o c a m p a l p y r a m i d a l cells at 60 days o f age is s u m m a r i z e d in

T a b l e II. T h e n u m b e r o f C A I p y r a m i d a l cells was r e d u c e d b y 20 ~o in the e t h a n o l -
e x p o s e d group, relative to the sucrose a n d L a b C h o w controls ( P < 0.001). T h e
n u m b e r o f C A 2 - 4 p y r a m i d a l cells, however, was n o t altered b y p r e n a t a l e t h a n o l
exposure. T h e significant ( P < 0.001) r e d u c t i o n in the t o t a l n u m b e r o f p y r a m i d a l cells
p e r section was a l m o s t c o m p l e t e l y a c c o u n t e d f o r b y the specific r e d u c t i o n o f the CA1
p o p u l a t i o n o f cells. I n contrast, neither the cross-sectional a r e a o f the d e n t a t e gyrus
g r a n u l e cell layer, the density o f g r a n u l e cells n o r the t o t a l n u m b e r o f granule cells p e r
section was significantly altered b y p r e n a t a l e t h a n o l e x p o s u r e (Table III).

TABLE II
The effect of maternal ethanol consumption during gestational days 10-21 on the number of dorsal
hippocampalpyramidal cells at 60 days of age in male offspring
Values are the mean 4- S.E. of the number of pyramidal cells per 4.0/~m section.

Treatment group Hippocampal subdivision

C,41 CA2-4 Total (CA1-4)

Ethanol 255.2 4- 8.5* 155.4 4- 5.4 410.6 4- 11.5"

Sucrose 320.8 + 7.8 166.6 + 8.4 487.4 i 14.3
Lab Chow 324.1 4- 7.6 166.3 4- 7.8 490.4 4- 11.5

* Significantly different from control groups, P < 0.001.

338

T A B L E 111

The lack of effect of maternal ethanol consumption during gestational days 10-21 on the morphol~Jgy
of the granule cell layer of the dorsal dentate g.vrus at 60 days of age in male off:~pring
See text for m e t h o d s of m e a s u r e m e n t . Values are the m e a n i S.E. for each p a r a m e t e r measured.

Treatment group Area of granule cell Granule cells Granule cells

layer ( sq.mm ) per sq.mm per section

Ethanol 0.339 ~: 0.020 5883 :k 217 1987 _~ 124

Sucrose 0.310 j: 0.016 6448 _~ 240 1998 L: 80
Lab Chow 0.338 :a 0.024 6086 ± 270 2021 :~ 125

DISCUSSION

The present work demonstrates that prenatal ethanol exposure can permanently
alter the number of dorsal hippocampal pyramidal cells in the absence of observable
physical anomalies, and without significant alterations in physical growth. DelaYed
postnatal development of the cerebellum has been reported in rats prenatally exposed
to ethanol 2°,~3. These studies reported, however, that the cerebellar development was
only slowed, not permanently altered; no histological difference could be detected
between 30-day-old controls and animals prenatally exposed to ethanol z3.
In the rat, 85 ~o of the dentate gyrus granule cells originate postnatally, whereas
the neurogenesis of hippocampal pyramidal cells is completed before birth 1,~°,31.
Interestingly, in the present experiment the number of postnatally developing granule
cells was not permanently altered by prenatal ethanol exposure, while the number of
prenatally developing hippocampal pyramidal cells was decreased by 20 ~. Since the
dentate granule cells continue to proliferate after birth and the cessation of ethanol
exposure, it is impossible to determine whether the pyramidal cells are selectively
affected or whether the granule cell population recovers as had been shown after X-
irradiation 1. Studies in progress will examine whether ethanol exposure affects cell
proliferation or whether macroneurons, such as pyramidal cells, are susceptible to the
toxic effects of ethanol exposure at all stages of differentiation. Such an idea receives
support from experiments demonstrating that early postnatal (days 3-20) ethanol
exposure (by inhalation) permanently reduced the number of both cerebellar Purkinje
cells and cerebellar granule cellsL These investigators suggested that the initial targets
of ethanol were the immature Purkinje cells that were reduced in number before the
onset of peak granule cell neurogenesis 2. It is of interest that prenatal phenobarbital
exposure in mice has been reported to induce almost identical effects on hippocampal
development as we report here, i.e. the prenatally forming hippocampal pyramidal
cells, but not the postnatatly forming granule cells, were reduced in treated offspring 36.
Although many other brain regions need to be histologically examined after
prenatal ethanol exposure, the present results in combination with other reports of
delayed cerebellar developmentZ0,z 3 and permanent structural alterations of the
cerebellum (ref. 2 and Barnes and Walker, in preparation) suggest that the developing
hippocampus and cerebellum are particularly sensitive to the effects of ethanol. Recent
 339

studies suggest that the hippocampus in the adult rat is also especially sensitive to the
neurotoxic effects of chronic ethanol exposure 29,a4.
The permanent structural alterations of the rat hippocampus by prenatal ethanol
exposure may be relevant to the many reports of the behavioral deficits induced by
such exposureO,7,14,26-2a, 32. The fact that many of these deficits after prenatal ethanol
exposure can be characterized as lack of response inhibition 26-28 also suggests a
functional relationship between altered hippocampal structure and the occurrence of
behavioral deficits after prenatal ethanol exposure because the hippocampus has been
long thought to be involved in response inhibition 12.
The nutritionally controlled model used in this experiment can be used to
address a number of important issues concerning the effect of prenatal ethanol
exposure on brain development. Future experiments in which the hippocampus and
cerebellum are used as model neural systems to delineate the effect of concentration,
duration and timing of prenatal ethanol exposure on rat neurogenesis will facilitate
our knowledge of the neurological and behavioral disturbances associated with the
fetal alcohol syndromeg,16, is.

ACKNOWLEDGEMENTS

This work was supported by the Medical Research Service of the Veterans
Administration and N I A A A Grants AA03965 and AA00200.
We thank Pat Burnett, Larry Ezell, Douglas Goldberg and Dorothy Robinson
for excellent technical assistance.

REFERENCES
1 Altman, J. and Bayer S., Postnatal development of the hippocampal dentate gyrus under normal
and experimental conditions. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocampus, Vol.
1, Plenum Press, New York, 1975, pp. 95-122.
2 Bauer-Moffett, C. and Altman, J., The effect of ethanol chronically administered to preweanling
rats on cerebellar development: a morphological study, Brain Res., 119 (1977) 249-268.
3 B/erich, J. R., Majewski, R., Michaelis, R. and Tilner, 1., On the embryo-fetal alcohol syndrome,
Europ. J. Pediat., 121 (1976) 155-179.
4 Boggan, W. O., Randall, C. M., deBcukelaer, M. and Smith, R., Renal anomalies in mice prena-
tally exposed to ethanol, Res. Commun. Chem. Pathol. Pharmacol., 23 (1979) 12%142.
5 Boggan, W. O., Randall, C. L. and Dodds, H. M., Delayed sexual maturation in female C57BL/6J
mice prenatally exposed to alcohol, Res. Commun. Chem. Pathol. Pharmacol., 123 (1979) 117-125.
6 Bond, N. W. and DiGiusto, E. L., Effects of prenatal alcohol consumption on open-field behavior
and alcohol preference in rats, Psychopharmacology, 46 (1976) 163-168.
7 Bond, N. W. and DiGiusto, E. L., Avoidance conditioning and Hebb-Williams maze performance
in rats treated prenatally with alcohol, Psychopharmacology, 58 (1978) 69-71.
8 Chernoff, G. F., The fetal alcohol syndrome in mice: an animal model, Tertology, 15 (1977)
223-230.
9 Clarren, S. K., Alvord, A. C., Sumi, S. M., Streissguth, A. P. and Smith, D. W., Brain malforma-
tions related to prenatal exposure to ethanol, J. Pediat., 92 (1978) 64-67.
10 Cobo, E., Effect of different doses of ethanol in the milk-ejectingresponse in lactating women,
Amer. J. Obstet. Gynec., 115 (1973) 817-821.
11 Detering, N., Reed, W. D., Ozand, P. T. and Karahasan, A., The effects of maternal ethanol con-
sumption in the rat on the development of their offspring, J. Nutr., 109 (1979) 999-1009.
12 Douglas, R. J., The hippocampus and behavior, Psychol. Bull., 67 (1967) 416-442.
340

13 Ellis, F. W., Pick, R. and Sawyer, M., Effects of ethanol administration during pregnancy on fetal
development in beagle dogs, Fed. Proc., 36 (1977) 285.
14 Harris, R. A. and Case, J., Effects of maternal consumption of ethanol, barbital, or chlorodiaze-
proxide on the behavior of the offspring, Behav. Neural Biol., 26 (1979) 234-247.
15 Henderson, G. 1., Hoyumpa, A. M., MacClain, C. and Schenker, S., The effects of chronic and
alcohol administration on fetal development in the rat, Alcoholism: Clin exp. Res., 3 (1979)
99-106.
16 Jones, K. L. and Smith, D. W., Recognition of the fetal alcohol syndrome in early infancy, Lancet,
2 (1973) 999-1001.
17 Jones, K. L., Smith, D. W., Utleland, C. N. and Streissguth, A. P., Pattern of malformation in
offspring of chronic alcoholic mothers, Lancet, 1 (1973) 1267-1271.
18 Jones, K. L. and Smith, D. W., The fetal alcohol syndrome, Teratology, 12 (1975) 1-10.
19 K~nig, J. F. R. and Klippel, R. A., The Rat Brahl, A stereotaxic Atlas, Williams and Wilkins,
Baltimore, 1963.
20 Kornguth, S. E., Rutledge, J. J., Sunderland, E., Siegel, F., Carlson, I., Snollens, J., Juhl, U. and
Young, B., Impeded cerebellar development and reduced serum thyroxine levels associated with
fetal alcohol intoxication, Brain Res., 177 (1979) 347-360.
21 Lorente de N6, Studies on the structure of the cerebral cortex. II. Continuation of the study of the
anamonic system, J. Psychol. Neurol. (Lpz.), 46 (1934) 133-177.
22 Mulvihill, J. J. and Yeager, A. M., Fetal alcohol syndrome, Teratology, 13 (1976) 345-348.
23 Peiffer, J., Majewski, F., Fishback, H., Bierich, J. R. and Volk, B., Alcohol embryo and fetopathy,
J. neurol. Sci., 41 (1979) 125-137.
24 Randall, C. L., Taylor, W. J. and Walker, D. W., Ethanol-induced malformations in mice, Alco-
holism: Clin. exp. Res., 1 (1977) 219-224.
25 Randall, C. L. and Taylor, W. J., Prenatal ethanol exposure in mice: teratogenic effects,
Teratology, 19 (l 979) 305-312.
26 Riley, E. P., Lochry, E. A. and Shapiro, N. R., Lack of response inhibition in rats prenatally
exposed to alcohol, Psychopharrnacology, 62 (1979) 47-52.
27 Riley, E. P., Shapiro, N. R. and Lochry, E. A., Nose-poking and head-dipping behaviors in rats
prenatally exposed to alcohol, Pharmacol. Biochem. Behav., 11 (1979) 513-519
28 Riley, E. P., Lochry, E. A., Shapiro, N. I. and Baldwin, J., Response perseveration in rats exposed
to alcohol prenatally, Pharmacol. Biochem. Behav., 10 (1979) 255-259.
29 Riley, J. N. and Walker, D. W., Morphological alterations in hippocampus after long-term
alcohol consumption in mice, Science, 201 (1978) 646-648.
30 Rodier, P. M., Correlations between prenatally-induced alterations in CNS cell populations and
postnatal function, Teratology, 16 (t977) 235-246.
31 Schlessinger, A. R., Cowan, W. M. and Gottlieb, D. I., An autoradiographic study of the time of
orgin and the pattern of granule cell migration in the dentate gyrus of the rat, J. comp. NeuroL,
159 (1975) 149-176.
32 Shaywitz, B. A., Klopper, J. H. and Gordon, J. W., A syndrome resembling minimal brain dys-
function (MBD) in rat pups born to alcoholic mothers, Pediat. Res., 10 (1976) 451.
33 Volk, B., Delayed cerebellar histogenesis in the 'embryofetal alcohol syndrome', Acta Neuropath.
(Ber)., 39 (1977) 157-163.
34 Walker, D. W., Barnes, D. E., Zornetzer, S. F., Hunter, B. E. and Kubanis, P., Neuronal loss in
hippocampus induced by prolonged ethanol consumption in rats, Science, 209 (1980) 711-713.
35 Warner, R. H. and Rosett, H. L., The effects of drinking on offspring: a historical survey of the
American and British literature, J. Stud. Alcohol, 36 (1975) 1395-1420.
36 Yanai, J., Rosselli-Austin, L. and Tabakoff, B., Neuronal deficits in mice following prenatal
exposure to phenobarbital, Exp. Neurol, 64 (1979) 237-244.