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Effects of prenatal alcohol exposure on the
hippocampus: Spatial behavior,
electrophysiology, and neuroanatomy
Impact Factor: 4.3 · DOI: 10.1002/(SICI)1098-1063(2000)10:1<94::AID-HIPO11>3.0.CO;2-T · Source: PubMed







Robert F Berman

John H Hannigan

University of California, Davis

Wayne State University





Available from: John H Hannigan
Retrieved on: 12 July 2015

HIPPOCAMPUS 10:94–110 (2000)

Effects of Prenatal Alcohol Exposure on the
Hippocampus: Spatial Behavior, Electrophysiology,
and Neuroanatomy
Robert F. Berman1* and John H. Hannigan2

of Neurological Surgery, Center for
Neuroscience, University of California at Davis,
Davis, California
2Departments of Obstetrics and Gynecology,
and Psychology, C.S. Mott Center for Human Growth
and Development, Wayne State University School of
Medicine, Detroit, Michigan

Prenatal exposure to alcohol can result in fetal alcohol
syndrome (FAS), characterized by growth retardation, facial dysmorphologies, and a host of neurobehavioral impairments. Neurobehavioral effects
in FAS, and in alcohol-related neurodevelopmental disorder, include poor
learning and memory, attentional deficits, and motor dysfunction. Many of
these behavioral deficits can be modeled in rodents. This paper reviews
the literature suggesting that many fetal alcohol effects result, at least in
part, from teratogenic effects of alcohol on the hippocampus. Neurobehavioral studies show that animals exposed prenatally to alcohol are impaired
in many of the same spatial learning and memory tasks sensitive to
hippocampal damage, including T-mazes, the Morris water maze, and the
radial arm maze. Direct evidence for hippocampal involvement is provided by neuroanatomical studies of the hippocampus documenting
reduced numbers of neurons, lower dendritic spine density on pyramidal
neurons, and decreased morphological plasticity after environmental
enrichment in rats exposed prenatally to alcohol. Electrophysiological
studies also demonstrate changes in synaptic activity in in vitro hippocampal brain slices isolated from prenatal alcohol-exposed animals. Considered together, these observations demonstrate that prenatal exposure to
alcohol can result in abnormal hippocampal development and function.
Such studies provide a better understanding of neurological deficits
associated with FAS in humans, and may also contribute to the development of strategies to ameliorate the effects of prenatal alcohol exposure on
behavior. Hippocampus 2000;10:94–110. r 2000 Wiley-Liss, Inc.
fetal alcohol syndrome; radial maze; Morris maze;
environmental enrichment; neural plasticity

Fetal alcohol syndrome (FAS) was first defined in the early 1970s as a
pattern of growth retardation, facial anomalies, and mental retardation in
Grant sponsor: National Institute on Alcohol Abuse and Alcoholism; Grant
number: P50-AA-07606.
*Correspondence to: Robert F. Berman, Ph.D., Department of Neurological Surgery, University of California at Davis, Medical Neurosciences
Building, Room 502B, 1515 Newton Court, Davis, CA 95616. E-mail:
Accepted for publication 19 October 1999


infants born to alcoholic women (Jones and Smith,
1973). Similar patterns of development had been reported earlier but were not widely recognized (Lemoine
et al., 1968). Children with FAS show distinctive,
persistent, and subtle patterns of cognitive dysfunction
that become increasingly evident with more sophisticated psychological assessments (e.g., Mattson and Riley,
1998; Jacobson, 1998; Roebuck et al., 1999). Prenatal
alcohol-exposed children with alcohol-related neurodevelopmental disorders (ARNDs) show similar cognitive
effects (Stratton et al., 1996; Tanaka, 1998), but without
the characteristic facial features of FAS (Aase, 1994;
Mattson et al., 1998). Although the diagnostic criteria
for FAS and other fetal alcohol effects continue to evolve
(Sokol and Clarren, 1989; Stratton et al., 1996), early on
it was proposed that changes in central nervous system
(CNS) function may be the most sensitive signs of
prenatal alcohol exposure (Clarren, 1982). Considerable
research continues to investigate the CNS abnormalities
and dysfunction responsible for the cognitive and behavioral fetal alcohol effects, in both human and animal
Even prior to the definition of FAS, it was known that
placentally transferred alcohol accumulates in the fetal
hippocampus of rodents and primates (Ho et al., 1972).
It is now evident that hippocampal anatomy and
electrophysiology are altered by prenatal or early postnatal alcohol exposure. One recent review of clinical and
experimental outcomes concluded that damage to the
vulnerable hippocampus may be responsible for many of
the behavioral sequelae (Tanaka, 1998). Riley et al.
(1986) argued that the results of many behavioral studies
up to that time, mostly in rats, supported the hypothesis
of enduring postnatal hippocampal dysfunction. This
was based upon the similarity of effects on behavior after
prenatal alcohol exposure and direct hippocampal lesions. That analysis focused on response inhibition



deficits in various avoidance paradigms (Riley et al., 1986). More
recent studies have characterized the involvement of the hippocampus in neurobehavioral dysfunction after prenatal alcohol exposure by using tasks assessing spatial learning and memory (e.g.,
Blanchard et al., 1987; Hall and Berman, 1995). In this paper we
review evidence that the hippocampus is profoundly susceptible to
the teratogenic effects of prenatal alcohol exposure. Findings from
behavioral, neuroanatomical, and neurophysiological experiments from
our laboratories and others, including new research, are presented.


revised to allow the pups to remain with either biological or foster
dams throughout the period of early neonatal alcohol administration (Light et al., 1989; Goodlett and Johnson, 1997; Goodlett et
al., 1998). The great majority of all published studies of fetal
alcohol effects in animals have relied on these procedures,
although direct intraperitoneal injections of alcohol solutions and
inhalation of vaporized ethanol in special exposure chambers have
also been used (e.g., Pal and Alkana, 1997; Bellinger et al., 1999).
Each procedure has advantages and disadvantages in the experimental study of prenatal alcohol exposure (Hannigan and Abel, 1996).
The interpretation of results among studies can be influenced by
procedural differences.

Animal models, particularly those using rodents, are powerful
tools for determining the mechanisms and outcomes of early
alcohol exposure because the physiological responses to alcohol in
development are similar to those in humans (Hannigan and Abel,
1996). The neurobehavioral sequelae of prenatal alcohol exposure
in animals can also be remarkably consonant with the clinical and
behavioral outcomes in humans (Driscoll et al., 1990; Hannigan,
1996). However, rats do not normally voluntarily consume
enough alcohol to maintain chronically high blood alcohol
concentrations (BACs) during pregnancy. As a result, several
procedures have been developed for alcohol administration to
pregnant dams (Hannigan and Abel, 1996). One of the most
widely used procedures limits food and water consumption to an
alcohol-containing liquid diet (Sherwin et al., 1979), often made
from commercially available nutritional formulations (e.g., Liquidiett, Bio-Serv, Frenchtown, NJ or Sustacalt, Mead Johnson and
Co., Evansville, IN). Sufficient alcohol is added to this diet (about
12 g/kg/day, and up to ,18 g/kg/day in some studies) so that
,18% (low dose) or ,35% (high dose) of total daily calories are
provided by the alcohol (e.g., 35% ethanol-derived calories (35%
EDC)). Control diets can be made isocaloric to the alcoholcontaining diets by the substitution of another carbohydrate for
alcohol. A second procedure involves direct intragastric gavage or
intubation of dams with alcohol solutions, producing doses
typically between 2–6 g/kg/day. A third technique involves
artificial rearing, the so-called ‘‘pup-in-the-cup’’ technique, designed to expose neonatal rat pups to alcohol during a defined
period within the first 10 postnatal days (e.g., Kelly et al., 1988).
This early neonatal period of development in rodents shares a
number of functional and maturational similarities with thirdtrimester gestational development in humans (Dobbing and
Sands, 1979), and neonatal alcohol exposure in the rat is used to
model third-trimester alcohol exposure in human FAS. In artificial rearing, rat pups are surgically implanted on postnatal day 4
(PN4) with a chronically indwelling intragastric tube through
which an alcohol-containing liquid diet is delivered periodically
over 3–7 days. This protocol requires controls for the effects of
separating the pups from their dams and litters. Finally, a method
for direct intragastric administration of alcohol to neonates,
neonatal gavage (Sonderegger et al., 1982), has been revived and

The general findings for most of the studies reviewed here are
that early alcohol exposure impairs spatial response-dependent
learning, that deficits are greater upon reversal training, and are
most evident when tested in younger animals.

Deficits in acquisition and retention of spontaneous and
conditional alternation in T-mazes, and in spatial discrimination
reversal learning in animals exposed prenatally to alcohol, are
consistent with the behavioral effects of hippocampal damage
(O’Keefe and Nadel, 1978). Lochry and Riley (1980) first
reported that rats exposed to alcohol via maternal liquid diets
(17.5% EDC and 35% EDC) had dose-dependent increases in
errors on a T-maze during both acquisition and retention testing,
with or without reversal training. Abel (1982) and Zimmerberg et
al. (1989) later found deficits in spontaneous alternation in a
T-maze in rats after prenatal exposure to liquid diets providing
35% EDC. Wainwright et al. (1990) reported that prenatal
alcohol-exposed B6D2F2 mice that had learned to escape a simple
water T-maze by always turning in one direction (right or left)
showed deficits in learning the reverse. Deficits in acquisition and
reversal of a spatial alternation were also reported after either
prenatal alcohol exposure (Zimmerberg et al., 1991) or neonatal,
binge-like alcohol exposure (Thomas et al., 1996, 1997). In
Zimmerberg et al. (1991), rats were tested in a T-maze with two
choice points. The first choice point tested spatial reference
memory because the animal always had to go to the right. The
correct choice (left or right) at the second choice point alternated
with previous choice at that point, providing a test of spatial
working memory. Offspring exposed prenatally to alcohol were
impaired on both the spatial reference memory (males and
females) and the spatial working memory components (males

Morris Maze
In the basic Morris maze task (Morris, 1981), rodents are
required to swim to a hidden escape platform submerged in a large



tank of opaque water. The task appears to require spatial
‘‘mapping’’ based upon localization of extra-maze cues (Morris,
1981). Certain ‘‘probe’’ measures taken with the platform removed (e.g., percent time spent near the platform location, angle
of first approach) help discriminate spatial learning and memory
performance (i.e., knowing where the platform is) from some
motor responses (e.g., learning a swim pattern that leads to
escape). Hippocampal damage substantially impairs rodents’
ability to learn platform location (Morris et al., 1982). Studies
identifying spatial learning deficits in a Morris maze by young rats
(,PN20–PN30) after perinatal (i.e., prenatal or neonatal) alcohol
exposure, and thereby suggesting hippocampal dysfunction, were
first published in 1987 (Blanchard et al., 1987; Goodlett et al.,
1987). In one study, young Long-Evans rats born to dams fed
35% EDC liquid diets took longer paths to reach the escape
platform in the Morris maze than controls. The prenatal alcoholexposed females tended to perform more poorly than the males
over the 4 days of acquisition training. Probe trials revealed
specific spatial rather than general response deficits, and here
males tended to be more affected on spatial learning than females
(Blanchard et al., 1987).
Goodlett et al. (1987) tested juvenile Sprague-Dawley rats after
early neonatal alcohol exposure on postnatal days 4–10 (PN4–
10). The ‘‘pup-in-the-cup’’ artificial rearing procedure was used to
generate high peak BACs (i.e., binge exposures) during the period
of postnatal brain growth spurt. In a detailed analysis of spatial
learning and memory over 12 days of testing, both male and
female alcohol-exposed pups had significantly longer escape
latencies and traveled more circuitous paths in the Morris maze
than controls. Analyses of probe trials without a platform
confirmed specific deficits in spatial learning (Goodlett et al.,
1987). Similar spatial deficits in juvenile rats were reported
following direct intubations of alcohol to neonates from PN7–9,
without the complications of the artificial rearing method (Goodlett
and Johnson, 1997). Matthews and Simon (1998) modified the
Morris task by increasing the delay between training and testing
from 1 to 3 days. They found that the offspring of Long-Evans rats
intubated with 3 g/kg/day ethanol during gestation showed
significantly increased escape latencies as adults. Kim et al. (1997)
compared the effects of prenatal alcohol exposure via a 36% EDC
diet on nonspatial and spatial learning in male Sprague-Dawley
rats. There were no deficits on a nonspatial delayed nonmatchingto-sample object recognition task. However, the same rats showed
increased latencies in Morris maze acquisition, demonstrating the
specificity of spatial deficits.

Radial Arm Maze
Spatial learning deficits can also be demonstrated in Olton-type
radial arm mazes. Reyes et al. (1989) were the first to report
deficits in eight-arm radial maze acquisition in surrogate-fostered,
food-restricted, prenatal alcohol-exposed, male and female rats
(17% EDC or 35% EDC) tested on ,PN60. Only half of the
alcohol-exposed offspring were able to reach criterion, and those
that did required significantly more training trials relative to
pair-fed controls. Pick et al. (1993) reported similar effects in mice

injected with ethanol (3 g/kg or 5 g/kg; sc) on PN2–14. At PN50,
these mice took twice as many trials as controls to reach criterion,
and continued to require significantly more trials than controls to
enter all eight arms (Pick et al., 1993). Hall et al. (1994) showed
that midgestational alcohol exposure (i.e., gestational days (GD)
7–13), via 35% EDC liquid diets, was sufficient to impair radial
maze acquisition when rats were tested as either juveniles
(,PN26) or adults (,PN80). In contrast, Opitz et al. (1997)
showed no differences in an unrewarded compact version of a
radial maze testing offspring of mice intubated with 3.19 g/kg/day
of alcohol from GD14–18, even though litter mates of these mice
had significant deficits in acquiring a conditioned taste aversion.
Stone et al. (1996) also reported deficits in radial arm maze
performance in prenatal alcohol-exposed rats. The same rats were
not impaired in passive avoidance, suggesting that spatial performance may be a more sensitive behavioral measure of alcohol
teratogenicity in the hippocampus than response inhibition (Riley
et al., 1986; Greene et al., 1992).
To elaborate the role of learning and attention in spatial
behavior after prenatal alcohol, we adapted a radial arm maze to
assess attentional processes by requiring animals to use visual or
spatial cues in a food-search task. In the visual cue task, rats were
trained to find food in three black arms in an eight-arm maze with
white, black, or gray arms. The position of the colored arms varied
from day to day so that spatial position was irrelevant. Animals
had to attend to the color of the arms to find food in this ‘‘cued’’
condition. After 3 weeks, the food was switched from the black to
the white arms for 3 more weeks of training. Next, a ‘‘spatial’’
contingency was used where the location of three goal arms was
reinforced and the color cues became irrelevant. As shown in
Figure 1, under the spatial cue condition, rats exposed prenatally
to either 4 g/kg/day or 6 g/kg/day showed significantly longer
latencies to locate food in the three reinforced arms compared to
alcohol-naive controls. Prenatal alcohol-exposed rats also made
more errors than controls when the contingency was shifted from
the ‘‘cued’’ to the ‘‘spatial’’ condition (data not shown), again
suggesting a spatial learning deficit in prenatal alcohol-exposed

Spatial Deficits and Age of Testing
Growth retardation and neurobehavioral deficits after prenatal
alcohol exposure may reflect delayed maturation of the CNS
(Sherwin et al., 1979; Abel, 1982; Pettigrew, 1986; Rintelmann et
al., 1994). This argument is based in part on experimental studies
that show a ‘‘catching up’’ of alcohol-exposed animals to alcoholnaive animals in some tasks (Meyer et al., 1990), as well as in
humans where the most severe deficits are often observed in the
adolescent (Sampson et al., 1994). The spatial deficits revealed
above in T-mazes, Morris mazes, and radial arm mazes also
illustrate the variable age-dependency of fetal alcohol effects.
While the effects of fetal alcohol exposure in children and rodents
can be long-lasting (Riley, 1990; West et al., 1990), there are also
reports that fetal alcohol effects are sometimes transient or
‘‘outgrown’’ (e.g., Meyer and Riley, 1986). Such reports support




Mean total latency (sec 6 SEM) to locate food
reinforcement in 3 of 8 arms of a radial arm maze by either visual
‘‘cues’’ or by ‘‘spatial’’ location. In the cued condition, food was found
in three arms of the same color (e.g., black), while under the spatial
condition, food was associated with three specific arm locations

regardless of arm color. Animals exposed prenatally to alcohol
performed as well as controls under the visual cue condition, but
were impaired in learning spatial locations of food reward as
indicated by increased latencies to complete the maze. *P F .05 vs.
none and zero.

the possibility that some fetal alcohol effects may modifiable with
age, and indeed may respond to treatment.
Spatial and temporal serial pattern learning and memory in rats
are disrupted by prenatal alcohol (e.g., Riley et al., 1993; LaFiette
et al., 1994). Working memory and spatial learning deficits were
reported in both younger and older rats, but older rats with prior
experience in the maze apparently remembered, showing no
deficits in relearning (Hall et al., 1994). The effects of prenatal
alcohol exposure on these learning tasks may also indicate
attention problems, although such attention deficits have not
been identified reliably in rats (e.g., Hayne et al., 1992). However,
even a single in utero episode of high peak BACs in mice was able
to produce a profound deficit in memory retrieval in 2-year-old
mice that was not evident in 3-month-old mice (Dumas and
Rabe, 1994).

We have examined the role of age at testing, using a radial arm
maze task that appears to be solved by young (PN21) and adult
(PN90) rats using fundamentally different arm selection ‘‘strategies.’’ At least two general strategies can be used by rats to solve the
eight-arm radial maze. Animals can select a highly variable
patterns of arms each day, as shown in Figure 2A, or they can
select adjacent arms, as indicated in Figure 2B. The adoption of
highly variable, and seemingly random, arm selections by adult
rats has been argued to indicate the use of a spatial mapping
strategy that is highly dependent on hippocampal function
(O’Keefe and Nadel, 1978). Adult animals typically use the
‘‘random’’ choice of arms when solving the radial maze, and
younger rats (e.g., tested beginning on PN21) tend to adopt the
adjacent arm choice strategy. Data from Hall and Berman (1995)
supporting this conclusion are shown in Figure 2C. These results



Examples of two general search strategies for locating food reward in the radial arm maze. A: Highly variable daily arm
selection pattern typical of adult rats. B: An adjacent arm selection
strategy observed in juvenile rats. C: Juvenile rats (26 days of age) use

an adjacent arm selection strategy, while adult rats (90 days of age) do
not (adapted from Hall and Berman, 1995). D: Animals exposed
prenatally to 35% EDC tend to use an adjacent arm strategy
compared to alcohol-naive controls (P F 0.05).

are similar to those reported earlier by Einon (1980). Therefore,
we hypothesized that if prenatal alcohol delayed development of
the CNS, then adult rats exposed prenatally to alcohol might use
the adjacent arm strategy.
This hypothesis was tested in offspring of Sprague-Dawley rats
given 35% EDC liquid diet from GD8 through parturition. Rats
were cross-fostered to alcohol-naive dams and weaned on PN21.

On PN90, radial maze training began. Briefly, animals were
placed on a restricted feeding schedule and habituated to the
eight-arm maze for 5 min per day for 5 days without food reward.
The arms of the maze were then baited, and adult rats (PN90)
were trained to enter each arm a single time within 5 min to
retrieve a food reward. The pattern of arm choices was analyzed
over 12 days of training.



The results are shown in Figure 2D. Rats exposed prenatally to
alcohol showed a pattern of arm choices that indicated adoption
of an adjacent arm strategy, similar to the maze strategy observed
in younger animals. Although the use of the adjacent arms choice
strategy was not as robust as in juvenile animals, the difference in
average adjacent arms selected between animals exposed prenatally
to alcohol and alcohol-naive controls was statistically significant
(P , 0.05). As suggested by Hall and Berman (1995), one
possible explanation for the shift in strategies from young to adult
rats could be maturation of a hippocampus capable of supporting
a spatial strategy for solving the maze. This explanation would be
consistent with the hypothesis that prenatal alcohol exposure
delays development of the hippocampus.

Persistence of Fetal Alcohol Effects
Spatial learning deficits can be long-lasting. Nagahara and
Handa (1997) assessed delayed spatial alternation in a T-maze for
food reward in male offspring of Long-Evans dams fed 35% EDC
liquid diets during pregnancy. At three separate ages tested,
,PN45, ,PN85, or ,PN175, there were significant decreases in
percent correct alternations at delays greater than 30 sec. The
youngest animals were also impaired at the 10-sec delay, and no
animals were impaired in the no-delay condition (Nagahara and
Handa, 1997). Stone et al. (1996) reported similar prenatal
alcohol-induced deficits in spontaneous alternation in a T-maze in
rats tested as old as 18 months of age (i.e., ,PN540).
Gianoulakis (1990) reported spatial navigation deficits in the
Morris water maze after prenatal alcohol exposure (35% EDC
liquid diet). Alcohol-exposed Sprague-Dawley rats showed increased escape latencies at 40, 60, and 90 days of age, as well as
evidence of poor spatial memory during probe trials without the
escape platform. Gianoulakis (1990) also controlled for maternal
influences by cross-fostering and found that being raised by a dam
that had been exposed to alcohol did not contribute to the spatial
performance deficits. Westergren et al. (1996) tested SpragueDawley rats at 6 months of age in the Morris water maze and
reported marginally significant impairments in acquisition. Sutherland et al. (2000) reported small but significant increases in escape
latencies in alcohol-exposed adult (5–8 months of age) males, in a
standard Morris maze with a fixed platform position. In contrast,
when the platform was moved from day to day, prenatal
alcohol-exposed rats had shorter latencies than controls on the
first trial. This was interpreted as evidence for poor memory of the
previous days’ position compared to alcohol-naive controls (e.g.,
less interference, or for the use of a nonspatial strategy; Sutherland
et al., 2000).

Dose, Gender Effects, and Critical Periods
of Exposure
Goodlett and Johnson (1999) recently reviewed the literature
demonstrating the importance of dose, gender, age of testing, and
especially period of alcohol exposure, to behavioral effects in
general. Each of these factors can influence how prenatal alcohol
exposure affects spatial learning. Dose effects were evident in
several of the studies reviewed so far. Tomlinson et al. (1998)


reported that Morris maze escape latency was increased in
,62-day-old Wistar rat offspring exposed to 6 g/kg of alcohol on
PN5, but not to lower doses (2 g/kg or 4 g/kg). However, the
magnitude of prenatal alcohol effects is not always related to dose
in any simple way. For example, Clausing et al. (1995) assessed
spatial learning in a seven choice-point complex maze that thirsty
rats completed for a water reward. Offspring of rats exposed to
18% EDC or 36% EDC in liquid diets were tested between
PN69–80. Among the multiple measures of performance, three
reflected spatial learning. In each of three measures of spatial
learning, the 18% EDC group, but not the higher-dose 36%
EDC group, was significantly impaired relative to controls.
Clausing et al. (1995) noted that the high alcohol dose group, but
not the low-dose group, evidenced behavioral signs of increased
stress/anxiety (i.e., freezing) that could have masked spatial
learning deficits.
Prenatal alcohol effects can also be influenced significantly by
gender, although the critical factors influencing gender differences
in response to alcohol are unclear at this time. For example,
Blanchard et al. (1997) found that male rats exposed prenatally to
alcohol (i.e., 35% EDC throughout gestation) were more impaired then females in spatial memory tested during probe trials in
the Morris water maze. Both were impaired in water maze
acquisition. In contrast, Minetti et al. (1996), using a single
prenatal alcohol exposure (i.e., 5.8 mg/kg, ip) on GD8, found
greater deficits in females than males during probe trials in the
Morris water maze. Initial acquisition did not differ between sexes.
Goodlett et al. (1987) reported that both male and female rats
showed Morris maze acquisition deficits following alcohol exposure on postnatal days 4–10. However, females were reported to
show greater deficits when high, repeated, binge-like, peak BACs
were generated during this same postnatal period (Kelly et al.,
1988). The timing and duration of exposure may also affect
gender differences in the developmental effects of alcohol exposure. Goodlett and Peterson (1995) reported spatial learning
deficits in the Morris water maze in both male and female rats
exposed to ethanol on postnatal days 4–10. However, the duration
of exposure was important, in that males exposed to alcohol over 2
postnatal days (i.e., PN4–6 or PN7–9) also showed spatial
learning deficits, whereas the full exposure period (i.e., PN4–9)
was required to reveal deficits in females. Even a single neonatal
day of alcohol exposure on either PN5 or PN10 may be sufficient
to impair Morris maze performance in male rats tested between
41–54 days of age (Pauli et al., 1995).

Behavioral Summary
Considered together, the pattern of effects on spatial learning
and memory indicates consistent deficits in animals exposed
prenatally to ethanol. Such deficits have been observed in
spontaneous and conditional alternation, the Morris water maze,
and radial arm maze tasks. Despite the fact that the details can
differ among various experiments, significant deficits in spatial
learning and/or memory are consistently seen across alcohol
exposure procedures, alcohol dose, age of testing, and gender. In
general, deficits tend consistently to be greater in younger animals,



following longer delays between training and testing, and after
higher doses of alcohol (i.e., higher peak BACs). These results also
indicate that the pattern of spatial learning and memory deficits is
similar to that observed after direct hippocampal damage, and is
consistent with the hypothesis that prenatal alcohol exposure
interferes with the normal development and function of the
hippocampus (Riley et al., 1986). Of course, the effects of prenatal
alcohol exposure are not limited to the hippocampus, and growth
retardation throughout the CNS is a hallmark of FAS.

Cell Loss
Barnes and Walker (1981) reported a significant 20% reduction
in the numbers of dorsal hippocampal CA1 region pyramidal
neurons, whereas there was only a nonsignificant 3% decrease in
granule cells in the dentate gyrus in young adult (PN60) rats after
prenatal alcohol exposure via liquid diet. Perez et al. (1991)
reported significant ,10% reductions in cell numbers in all
hippocampal CA fields on PN36 in Wistar rats, using a simple
cell-counting procedure in Luxol fast-blue-stained tissue. On
PN65 these reductions ranged from 31% in CA3 to 46% in CA1
(Perez et al., 1991). These relatively large effects were remarkable
in that the 12 g/kg alcohol dose was administered in four
intraperitoneal injections over a single day, GD7, a date which
anticipates the period of initial hippocampal pyramidal cell
generation by about 4–5 days (Perez et al., 1991). Significant
32–40% reductions in the numbers of CA3 pyramidal neurons
were also reported by Ba et al. (1996) following alcohol exposure
during development. However, the attribution of these effects to a
particular period of perinatal exposure is impossible because
alcohol (i.e., 12% solution) was administered as the sole drinking
fluid to dams for 2 months before pregnancy, throughout
pregnancy and lactation, and directly to offspring until PN45 (Ba
et al., 1996).
Similar to the effects of high BAC binge exposures on behavior,
BAC influences neuroanatomical effects in the hippocampus.
Greene et al. (1992) reported significant reductions in the CA1
area and pyramidal cell counts on both PN21 (,10% decrease)
and PN60 (,20% decrease) after neonatal binge alcohol exposure
concentrated into four of 24 daily administrations. However,
when the same total daily dose was distributed across all 24
administrations each day, there were no significant effects in the
hippocampus (Greene et al., 1992). Also, using an uncorrected
sampling procedure to count cells, Wigal and Amsel (1990)
reported that prenatal alcohol exposure via 35% EDC liquid diet,
but not neonatal exposure via artificial rearing methods, significantly reduced the estimated numbers of CA1 pyramidal cells and
mature granule cells on PN21. Combining prenatal plus neonatal
exposures in the same animals did not further reduce the numbers
of either CA1 pyramidal cells or granule cells (Wigal and Amsel,
1990). In a later study from this same laboratory, the opposite
temporal pattern was found, with higher peak BACs than those

used in Wigal and Amsel (1990). Specifically, early neonatal but
not prenatal alcohol exposure reduced CA1 pyramidal cell numbers and the CA1 area in weanling Sprague-Dawley rats (DiazGranados et al., 1993). In addition, the density of mature granule
cells was significantly reduced relative to untreated offspring after
combined prenatal plus neonatal alcohol exposure. Prenatal or
neonatal exposure alone also reduced granule cell density, but did
not do so significantly (Diaz-Granados et al., 1993).
Changes in cell counts can vary as the animals age. Lobaugh et
al. (1991) tested offspring of Sprague-Dawley dams weaned onto
progressively greater alcohol concentrations in liquid diet procedure beginning on GD1, up to 35% EDC. At PN21 or PN180,
following nonspatial behavioral testing, the number of pyramidal
cells and cell density were measured in the midtemporal hippocampal CA1. In contrast to the previous studies, there were no
significant differences in cell counts among prenatal treatment
groups at either age, although there was a significant decrease in
cell density in all groups with age.
Recent morphological studies using unbiased stereological
procedures indicate that reduced neuronal populations in the
hippocampus following prenatal alcohol exposure are regionally
selective and are due to decreases in neuronal generation or
proliferation, rather than to cell death or changes in density due to
changes in area or volume. Miller (1995) assessed hippocampal
development in Long-Evans rats exposed prenatally to alcohol
from GD6–21, or postnatally from PN4–12. Injections of
[3H]-thymidine were used to assess the ‘‘birthdays’’ of neuronal
populations in the hippocampus. Stereological cell-counting
procedures were used to correct for potential overestimation
caused by cell fragments, or by underestimating mean cell
diameter. A significant 17% reduction was found in the number
of pyramidal neurons in the hippocampal CA1 region after
prenatal exposure. Similar reductions were seen in CA1 after
neonatal alcohol exposure to high BACs (339 6 17 mg/dl).
Lower BACs (i.e., 132 6 14 mg/dl to 222 6 24 mg/dl) did not
affect pyramidal cell number. In contrast, prenatal alcohol exposure did not affect the numbers of granule cells in the dentate
gyrus, while postnatal alcohol exposure affected granule-cell
numbers as a biphasic function of postnatal BAC. At ‘‘moderate’’
BACs, dentate gyrus-cell numbers were significantly higher; at
‘‘moderately high’’ BACs, there was no effect; and only at the
highest BACs were cell numbers significantly reduced. Based on
an analysis of [3H]-thymidine uptake, Miller (1995) concluded
that alcohol exposure during hippocampal development delayed
the generation of neurons in both the CA1 and dentate gyrus.

Neuronal Branching and Spines
West et al. (1981) first reported work with a Timm’s stain
showing that prenatal alcohol exposure via 35% EDC in a liquid
diet caused abnormal branching of mossy fibers in the ventral
hippocampus. Mossy fibers invaded the CA3 infrahippocampal
region, where they are not normally present (West and HodgesSavola, 1983). This was confirmed with a horseradish peroxidase
(HRP) stain (West and Pierce, 1984). Wigal and Amsel (1990)
later found that neonatal alcohol exposure, whether or not



combined with prenatal exposure, but not prenatal exposure
alone, increased the area of CA4 region. Hoff et al. (1984) found
no evidence of significant change in the dimensions of the
hippocampus or dentate gyrus on PN10 or PN20 in offspring of
Sprague-Dawley rats fed a 35% EDC liquid diet. Davies and
Smith (1981) studied dendritic arbors in hippocampal pyramidal
neurons in 14-day-old mice and reported a 20% ‘‘stunting’’ in
total basilar dendritic length. Dendritic arbors were ‘‘simpler in
configuration’’ following maternal feeding with a 25% EDC diet
(,5.5 g/kg/day) during gestation and early lactation, although
complexity was not quantified.
Abel et al. (1983) counted dendritic spines on ,PN90 and
found significant 27% and 31% decreases in numbers of spines on
apical and basilar tertiary dendritic branches, respectively, in the
CA1 hippocampal region after prenatal alcohol exposure via
intragastric intubation (6 g/kg/day) throughout gestation. In a
brief report by Perez et al. (1991), using a single-day dosing
regime in Wistar rats, there were significant 27–47% decreases in
apical and basilar dendritic spine densities, which appeared to be
summed across pyramidal cells in all of hippocampus proper
(CA1, CA2, CA3, and CA4). Ferrer et al. (1988) reported ,50%
reduction in apical and basilar secondary, rather than tertiary,
dendritic spine density in CA1 on PN15, following up to 25%
ethanol in drinking water for 4 weeks before, and a 35% EDC diet
during gestation. However, in contrast to Abel et al. (1983) and
Perez et al. (1991), there were no significant differences in
dendritic spine densities on PN90 in Wistar rats (Ferrer et al.,
1988). Ferrer et al. (1988) interpreted their findings to suggest
that there was recovery from the teratogenic effects of alcohol on
the hippocampus during postnatal maturation between PN15–
90, although other reports suggest that effects on spine density in
CA1 are persistent.

Prenatal alcohol-induced changes in the hippocampus are also
evident at the ultrastructural level. Smith and Davies (1990)
assessed the ultrastructure of hippocampal CA1 pyramidal cells in
C57B1/6J mice after alcohol exposure extending from GD12 to
PN9. Pyramidal neurons were more densely packed in adult
alcohol-exposed rats with less elaborate dendritic arbors in a
manner typical of younger animals, suggesting developmental
delay. There were also fewer dendritic spines, with no changes in
spine length or numbers of microtubules. Morphological changes
were also found in mitochondria and in the distribution of
endoplasmic reticulum in CA1 (Smith and Davies, 1990). Tanaka
et al. (1983) had previously reported dilated, rough endoplasmic
reticulum in the hippocampus of offspring of Wistar dams given
10% ethanol in drinking water. They also found decreased density
of synapses quantified under electron microscopy in the hippocampal CA3 field (Tanaka et al., 1991).

Anatomical Plasticity
Changes in numbers of dendritic spines, synapses, or complexity of dendritic fields with development or environmental stimulation reflect neuronal plasticity within the hippocampus. Ferrer et


al. (1988) suggested that the absence of the ,50% reduction in
secondary dendritic spine density in CA1 on PN90 meant that
there was recovery between PN15–90, perhaps mediated by some
kind of residual neuronal plasticity. On the other hand, in an
electron microscopy study, Hoff (1988) argued that a significant
decrease in synapse turnover, measured as changes in the proportion of synapses of different shapes, indicated that synaptic
plasticity in the hippocampus was impaired after prenatal alcohol
exposure. Dewey and West (1985b,c) demonstrated that although
prenatal alcohol exposure itself did not influence the development
of hippocampal commisural or perforant path projections to the
dentate gyrus, subsequent neuronal plasticity within these projections was impaired. This was evident as a decrease in collateral
sprouting of hippocampal fibers in response to unilateral entorhinal lesions and a decrease in neurite outgrowth in prenatal
alcohol-exposed rats (Dewey and West, 1984, 1985a; West et al.,

The effects of prenatal alcohol exposure on hippocampal
electrophysiology have been studied at several levels, ranging from
EEG measurements to studies of in vitro hippocampal slices.
Evidence exists at each level for abnormal neural electrical activity
consistent with the behavioral and neuroanatomical studies
demonstrating the sensitivity of the hippocampus to the teratogenic effects of alcohol.

In Vitro Hippocampal Slice Electrophysiology
The in vitro rat brain slice preparation has been used extensively to characterize the electrophysiology of the hippocampus
isolated from rats exposed prenatally to alcohol (Berman and
Krahl, 1996). Stimulation-evoked extracellular field potentials
have been characterized in brain slices from these animals,
including measures of synaptic strength (i.e., input/output responses), synaptic inhibitory circuitry (i.e., paired-pulse inhibition), and synaptic plasticity (i.e., long-term potentiation or LTP).
The initial report of abnormal hippocampal electrophysiology
after prenatal alcohol exposure was by Hablitz (1986). In this
study, hippocampal subregion CA1 was examined at PN40–60 in
rats that had been exposed prenatally to a 35% EDC liquid diet
from GD3–21. Paired-pulse response inhibition typically observed at short interpulse intervals (,50 msec) in slices from
controls was absent in slices from the prenatal alcohol-exposed
animals. In addition, paired-pulse potentiation seen at pairedpulse intervals longer than 100 msec was greatly enhanced. There
were no significant effects of prenatal alcohol on input/output
curves, although hippocampal slices isolated from prenatal alcoholexposed offspring were described as less responsive than those of



These results were essentially replicated by Tan et al. (1990). In
this study, field potentials evoked in CA1 by stimulation of
Schaffer collateral axons were examined in hippocampal slices
isolated from rats exposed prenatally to alcohol, using a liquid diet
containing either 17.5% EDC or 35% EDC. Controls received
either an isocaloric alcohol-free liquid diet or were fed normal rat
chow ad libitum. A dose-related reduction in birth weight and
retarded growth on PN21 were observed in prenatal alcoholexposed animals (Tan et al., 1990). As in Hablitz (1986), there
was a loss of paired-pulse response inhibition at short intervals and
a marked enhancement of paired-pulse potentiation at longer
inter-pulse intervals (Tan et al., 1990).
LTP, a neurophysiological correlate of synaptic plasticity, has
also been evaluated in animals exposed prenatally to alcohol. In
Tan et al. (1990), the magnitude of LTP in hippocampal
subregion CA1 was lowest in the 35% EDC group, but did not
differ significantly among groups. Swartzwelder et al. (1988)
examined hippocampal LTP in animals exposed prenatally to a
diet containing 18.8% EDC. LTP was elicited in CA1 using a
10-sec, 60-Hz stimulus train to the Schaffer collaterals at 20% of
the maximum evoked population spike response. The magnitude
of LTP in the prenatal alcohol-exposed animals showed only a
75% increase over baseline measured 30 min after tetanus. This
was significantly less than the 140% increase elicited in CA1 of
alcohol-naive animals fed rat chow, and less than the 260%
increase in animals maintained on an alcohol-free liquid diet. The
apparent difference in LTP magnitude between control groups
was not significant. Savage et al. (1998) demonstrated that the rate
of decay in LTP was accelerated in hippocampal slices from
prenatal alcohol-exposed rats.
In a recent study from our laboratory (Krahl et al., 1999),
input/output responses, paired-pulse inhibition, and LTP were
examined in region CA1 of in vitro hippocampal slices. Slices were
isolated from adolescent (PN25–32) and young adult animals
(PN63–77) exposed prenatally to 0, 4, or 6 g/kg/day ethanol
administered to the dams via intragastric gavage from GD8–21.
Slices from the higher-dose, 6 g/kg/day prenatal alcohol-exposed
animals on ,PN30 showed significantly reduced maximal evoked
population spike amplitudes compared to animals receiving lower
alcohol doses (i.e., 4 g/kg/day), or to age-matched alcohol-naive
controls. In contrast, there were no differences in maximal
population spike amplitude in the hippocampus of prenatal
alcohol-exposed animals examined as young adults (,PN70).
There were also no differences at any age among groups in
input/output profiles or paired-pulse responses at either short or
long interpulse intervals. The Kral et al. (1999) study supports the
conclusion that prenatal alcohol exposure results in abnormal
hippocampal electrophysiological responses, and demonstrates
that the effects of prenatal alcohol exposure are both dose-related
and age-dependent.
The route and pattern of prenatal alcohol exposure may be
important variables contributing to the electrophysiological outcomes of prenatal alcohol exposure. The liquid diet procedures
used by Hablitz (1986), Tan et al. (1990), and Swartzwelder et al.
(1993) all produced abnormal paired-pulse responses and interfered with the generation of LTP in CA1. These evoked responses

were intact in CA1 in the study by Krahl et al. (1999) that used an
intragastric gavage exposure technique. This may be important,
because liquid diet exposure procedures typically result in lower
mean peak and longer sustained BACs than those achieved by
intragastric intubation. Similarly, Sutherland et al. (1997) assessed
field excitatory postsynaptic potentials (EPSPs) in the dentate
gyrus of rats exposed prenatally to a liquid diet containing 5%
ethanol. This regime produced ‘‘moderate’’ BACs in the dams
(,83 mg/dl). At PN120–150, these rats had no differences in
input-output curves but had smaller LTPs than controls. Recently,
Bellinger et al. (1999) reported that hippocampal slices prepared
at PN45–60 from rats that had been exposed to ethanol vapors
from PN4–9, yielding BACs of 350 mg/dl, had similar reductions
in input/output characteristics without changes in LTP or pairpulse potentiation. Liquid diet procedures may be more representative of steady alcohol consumption in humans, while gavage
procedures and their concomitant higher BACs may be more
similar to binge-drinking patterns. Each pattern of alcohol
consumption, therefore, could result in different levels and peaks
of prenatal alcohol exposure that lead to different neurobehavioral, anatomical, and electrophysiological effects. Understanding
the influence of patterns of drinking and alcohol exposure to the
fetus is clearly important, and studies addressing this question
need to be carried out in this area.

EEG Studies
Two EEG studies in animals demonstrated fetal alcohol
exposure effects on the hippocampus. Kaneko et al. (1993)
studied auditory event-related potentials (ERPS) in offspring of
rats fed an alcohol-containing liquid diet during gestation.
Electrophysiological data revealed that the prenatal alcoholexposed animals had significantly longer latencies of the P1 and
N1 ERP components recorded in the hippocampus, again
suggesting that the hippocampus is altered by prenatal alcohol
exposure. Cortese et al. (1997) reported that prenatal alcohol
exposure altered hippocampal theta activity, a slow rhythmic EEG
activity in the 4–12-Hz frequency range that is characteristic of
the hippocampus (Bland, 1986). Rats were either untreated or
exposed prenatally to either 4 or 6 g/kg/day ethanol via an
intragastric gavage procedure. Theta activity was measured while
animals were moving or still, and the ratio of the relative power of
theta during these two periods (i.e., ratio of ‘‘movement’’ theta to
‘‘still’’ theta) was analyzed. A significantly higher ratio of movingto-still theta was observed in the 6 g/kg/day male animals
compared to controls, again demonstrating changes in a hippocampal electrophysiological response that has been associated with
processing spatial information (O’Keefe and Nadel, 1978) after
prenatal alcohol exposure. However, this study could not determine whether the change in ratio was the result of an increase in
type I (i.e., ‘‘movement-related’’) or a decrease in type II
(‘‘still-related’’) theta, or both.
In summary, in vivo and in vitro findings from a variety of
animal studies clearly demonstrate abnormal hippocampal electrophysiological activity resulting from prenatal alcohol exposure.
These electrophysiological changes are consistent with neuroana-



tomical studies demonstrating loss of principal neurons in the
hippocampus as well as aberrant mossy fiber projections, as
described above. These changes are also consistent with behavioral
studies demonstrating deficits in spatial learning tasks, tasks
known to be impaired after direct hippocampal damage (e.g.,
lesions). Considered together, the weight of the evidence leads to
the conclusion that the hippocampus is a prime target for fetal
alcohol effects, and further supports the possibility that at least
some of the cognitive deficits seen with FAS may result from
hippocampal damage. Additional evidence for compromised
plasticity and growth in the hippocampus after prenatal alcohol
exposure comes from our recent studies on the effects of
environmental enrichment on hippocampal spine development
(Hannigan et al., 1993; Berman et al., 1996).

Rearing animals in enriched or complex environments can
reliably stimulate CNS development, facilitate recovery of function, and enhance behavioral performance (Weiler et al., 1995;
Rosenzweig, 1996; Kolb et al., 1998). Therefore, we predicted
that environmental enrichment might ameliorate some fetal
alcohol effects in our rodent models. We have been systemically
examining the effects of postnatal environment enrichment on
both brain development and behavior in rats exposed prenatally to
alcohol. These studies are designed to assess the influence of the
postnatal environment on the expression of fetal alcohol effects on
hippocampal function, and to evaluate the potential of environmental enrichment to ameliorate some of the damaging effects of
alcohol exposure on brain growth and behavioral development
(Hannigan et al., 1993; Berman et al., 1996).

Morris Maze Learning After Environmental
We and others have documented that rearing rats in an
enriched environment after prenatal alcohol exposure can significantly improve behavioral performance, including learning,
memory, and motor performance (Hannigan et al., 1993; Wainwright et al., 1993; Berman et al., 1996; Klintsova et al., 1998;
but see Opitz et al., 1997). In our first studies, Long-Evans rats
were exposed prenatally to either 6 kg/day alcohol or sucrose
vehicle from GD8–19, using an intragastric gavage technique.
There were two separate equal intubations of 3 g/kg delivered 2 hr
apart to optimize both the concentration and volume of the
alcohol solution. This method of alcohol administration results in
BACs between 155–220 mg/dl measured 30 min after the second
daily intubation (e.g., Church et al., 1990). Animals in the
untreated control group were not intubated.
At weaning on PN21, male and female pups from each prenatal
treatment group were assigned to one of two postnatal environmental rearing conditions: Isolated or Enriched. In the Isolated control


condition, pups were housed individually in small hanging
steel-wire cages and not disturbed. Rats reared in the Enriched
condition were housed in same-sex groups of 10–12, with rats
from each of the three prenatal treatment groups housed together.
The enriched environment arenas were large Nalgenet (Nalge
Nunc International, Rochester, NY) tubs (75 3 75 3 60 cm
high) with hardwood chip bedding, feed, and water sources, and
were supplied with ‘‘toys’’ (e.g., dowels, plastic pipe, ladders)
which the animals could manipulate, chew, and climb on. The
toys were changed every 3–4 days.
After the enrichment period, at about PN63–72, rats were
trained in the Morris water maze. Animals were given four trials
per day over 4 consecutive days. The platform was moved to a new
location on the fifth training day, and learning of this new
position was evaluated (Fig. 3). Although there was no effect of
prenatal alcohol exposure on Morris water maze acquisition in this
study, there was an effect in learning the new platform position on
test day 5. Environmental enrichment improved water maze
performance in all groups, regardless of prenatal treatment, and
the behavioral impairment in prenatal alcohol-exposed animals on
day 5 was reduced (Hannigan et al., 1993). Although these effects
were subtle, we recently replicated these findings using a modified
Morris maze protocol with a longer intertrial interval (i.e.,
Hannigan et al., 1999). Because Matthews and Simon (1998)
reported Morris maze impairments following longer delays between training and testing, we increased the intertrial interval
from ,5 min to 60 min and reduced the number of trials per day
from five to three. Animals were again required to learn a new
platform position on the fifth day. Despite these changes, the
magnitude of fetal alcohol effects was unchanged. When the goal
platform was moved to a new position within the maze after 4
training days (i.e., a fifth ‘‘reversal’’ day), prenatal alcohol-exposed
rats reared in the Isolated condition took significantly longer than
controls to find the platform in its new location. However, after
enrichment there were no significant differences due to prenatal
alcohol exposure. These results with Sprague-Dawley rats on
reversal training replicate and extend to our earlier findings of
amelioration of fetal alcohol effects in Long-Evans rats (Hannigan
et al., 1993).

Effects of Enrichment on Hippocampal Dendritic
Spine Density
As described above, 6 weeks of an enriched environment can
improve learning, memory, and motor performance in prenatal
alcohol-exposed rats (Hannigan et al., 1993). However, we have
also found that the ability of environmental enrichment to
increase dendritic spine density on CA1 and CA3 hippocampal
pyramidal cells is severely compromised by prenatal alcohol
exposure (Berman et al., 1996).
In these studies, spine densities on hippocampal pyramidal
neurons in CA1 and CA3 were quantified. Briefly, slices from the
dorsal hippocampal CA1 region were processed with a modified
version of the rapid Golgi method. Single apical shaft pyramidal
cells were selected that were well-impregnated, contained minimal
truncation, and were not obscured by surrounding cells, blood



Morris water-maze performance after environmental
enrichment. Rats reared in an enriched environment (dashed lines)
showed shorter escape latencies (mean sec) than animals reared under
isolated conditions (solid lines). This was true for both prenatal
alcohol-exposed rats and alcohol naive rats. During ‘‘reversal’’
training (inset bar graph), isolated rats exposed prenatally to alcohol

took significantly longer to find the new location of the escape
platform than isolated untreated controls (P F 0.05). In contrast,
environmentally enriched rats exposed prenatally to alcohol did not
differ significantly from enriched alcohol-naive rats (data adapted
from Hannigan et al., 1993).

vessels, or artifact. Tertiary apical or basilar terminal (nonbranching) dendrites (one per cell) were drawn. Ten cells per rat (five for
apical and five for basilar dendrites) were analyzed. The numbers
of spines were quantified using a digital morphometric analysis
system, and unbiased correction procedures were used to estimate
three-dimensional spine density distributed about a dendrite shaft
(Feldman and Peters, 1979). Data were expressed as the estimated
number of spines per 10 mm tertiary dendritic segment.
Mean spine densities in CA1 are depicted in Figure 4 for males
and females demonstrating the effects of postweaning environment and prenatal alcohol. There were no significant differences
in apical spine density among the prenatal treatment groups
reared in the Isolated condition. Postweaning environmental
enrichment significantly increased apical or basilar dendritic spine
density in the two control groups, but did not increase spine
densities in CA1 in animals exposed prenatally to alcohol (Berman
et al., 1996). Among Isolated groups, the fact that prenatal
exposure to alcohol did not affect dendritic spine density in CA1

may reflect in part the negative impact of the Isolated condition
itself on controls.
The refractory hippocampal response to neuroanatomical signs
of environmentally induced plasticity may reflect a developmental
delay. Waters et al. (1997) demonstrated that the maturation of a
spatial behavior (spontaneous alternation in a ‘‘Y’’-maze) was
associated with the developmental emergence of a noveltyinduced elevation in FOS-like immunoreactivity (FL-IR) in the
hippocampus. Normal rats younger than PN30 do not alternate
and do not increase FL-IR. Perhaps a delay in this or similar
neuroplastic responses to the environment can account for poor
spine growth after prenatal alcohol exposure, but a delay does not
account for the general improvement in spatial behavior in the
Morris maze after enrichment.
We recently extended our analyses of the ameliorative effects of
postnatal environmental enrichment on hippocampal neuroanatomy to CA3. Damage to this region has been implicated in the
memory impairments seen after prenatal alcohol exposure (Tanaka,




al., 1991). There may be enduring, possibly life-long fetal alcohol
effects on brain growth and neuronal plasticity in the hippocampus, even in the presence of postnatal stimulation that is able to
improve performance and eliminate deficits on learning and
motor tasks (Hannigan et al., 1993). Our results suggest that
prenatal alcohol exposure compromises the ability of the CNS to
increase the complexity of its neuropil in response to environmental stimulation. Because environmental enrichment can improve
spatial learning and memory and motor performance in prenatal
alcohol-exposed rats, these findings suggest that the beneficial
effects of environmental enrichment on behavior may not be
directly mediated by any hippocampal anatomical response to


Analysis of dendritic spine densities on CA1 and CA3
hippocampal pyramidal neurons after prenatal alcohol exposure and
6 weeks of differential rearing in either an isolated or enriched
environment. Data are presented separately for males and females. In
CA1, prenatal alcohol exposure did not significantly affect spine
density for males and females. However, only alcohol-naive rats (i.e.,
none, zero) showed significant increases in spine density after
environmental enrichment (data adapted from Berman et al., 1996).
In CA3, male and female rats exposed prenatally to alcohol showed
significantly reduced dendritic spine densities compared to controls,
regardless of rearing conditions. The effect of environmental enrichment on spine densities for control groups (i.e., none and zero) was
not statistically significant.

1998), and CA3 also appears to be responsive to environmental
manipulations. The same brains described above from animals
exposed prenatally to 6 g/kg/day of alcohol were assessed in this
new analysis. There were no main effects or interactions due to
gender. The results are depicted in Figure 4. Unlike in CA1, there
was a significant main effect of prenatal treatment (F(2, 40) 5 4.50,
P , 0.02), which planned comparisons indicated was due to
significantly reduced spine density in the prenatal alcohol group
relative to both the untreated (F(1, 40) 5 4.52, P , 0.04) and
intubated control groups (F(1, 40) 5 7.74, P , 0.01). The
control groups did not differ from each other (P . 0.65). There
were no significant effects or interactions due to postnatal
environmental enrichment in CA3 (P 5 0.11 and 0.70). In our
model, hippocampal pyramidal cell spine density in CA3 may be
more sensitive to prenatal alcohol exposure than in CA1, but not
to enrichment. Other studies have also reported teratogenic effects
in CA3 anatomy, as noted above (e.g., Perez et al., 1991; Tanaka et

There have been relatively few studies of spatial learning in
children or adolescents with FAS of ARNDs. A recent review of
neuropsychological findings in children with FAS and ARNDs
included reports of ‘‘visuospatial deficits’’ (Roebuck et al., 1999).
Sampson et al. (1987) proposed, on the basis of a partial least
squares multiple covariate analysis using several overlapping
measures of alcohol consumption, that there are ‘‘latent’’ indications of poor spatial organization. However, it is difficult to
discern the nature of such deficits with this type of analysis. Hunt
et al. (1995) measured 442 14-year-old children with known
levels of prenatal alcohol exposure on a computerized test of
spatial visualization involving recall of shapes. Performance correlated with two measures of maternal self-report of drinking,
including an index of binge drinking (number of drinks per
occasion in the month before the pregnancy). The more the
mothers drank, the faster and less accurately the adolescents
responded. Children exposed prenatally to ‘‘high’’ levels of
alcohol, with or without diagnoses of FAS, were impaired on tests
of visuomotor integration (Mattson and Riley, 1998) or ‘‘globallocal’’ visuospatial abilities (Mattson et al., 1998). Steinhausen et
al. (1982) reported significant deficits in spatial relationships in
severe FAS compared to mild FAS. Olson et al. (1998) also
reported deficits in visual-spatial skills in 9 nonretarded adolescents with FAS. In another study, 4 boys diagnosed with ‘‘fetal
alcohol effects’’ built only nonstacking asymmetrical designs with
blocks. After the boys were shown a video model of stacked,
symmetric designs, none imitated, suggesting not only an inability
to model, but also a deficit in spatial perceptual tasks (Meyer,
It is unclear what the relationships are between these kinds of
tests and the spatial deficits seen in animal models. Uecker and
Nadel (1996, 1998) argued that deficits in certain spatial tasks
could be indicative of hippocampal damage after prenatal alcohol
exposure, and that hippocampal-associated deficits identified in
rats should be explored in children. They also argued that many
‘‘spatial’’ tasks ‘‘have no clear association with the hippocampus’’



(Uecker and Nadel, 1996). Deficits on some tasks (e.g., Milner’s
‘‘stepping-stone’’ maze) are apparently not indicative of exclusive
hippocampal involvement (Milner, 1965). Uecker and Nadel
(1996) administered the ‘‘memory for 16 objects’’ task to 15
children with FAS and 15 matched controls (4 girls/11 boys; 10
years old). There were no deficits in immediate recall, although
upon delayed (24 hr) recall, there were significant deficits in
memory for what the 16 objects were (e.g., screwdriver, whistle,
toy car). FAS children recalled 17% fewer objects than did
controls after a 24-hr delay. However, the larger effect was in
remembering where the objects were located in the 60-cm2 field.
Spatial recall was measured as the mean difference in distance (cm)
from the original positions of the objects and where the objects
were placed upon recall. Regardless of delay, FAS children had
distance differences that were ,42% larger that controls. An
analysis of the object displacement patterns, relative to the original
positions in a fairly uniform dispersal of objects across the field,
indicated that most FAS children tended to line up objects left to
right across the field. In contrast, the few control children who
had poorer spatial recall tended to displace objects around the
Uecker and Nadel (1998) recently elaborated their analyses of
hippocampal damage in FAS children using an Ellis-type spatial
memory task (cf. Ellis et al., 1989). Performance on the Ellis
spatial memory task can dissociate object memory from spatial
memory. Children with mental retardation and Down’s syndrome
perform poorly on both object and spatial memory in this task
(Ellis et al., 1989). Although FAS children took longer to recall
objects seen in a ‘‘picture book,’’ there were no significant
differences in either immediate or delayed recall of what those
objects were. In contrast, the FAS children recalled significantly
fewer correct locations than did controls at both immediate recall
(49% vs. 73%) and delayed recall (38% vs. 49%) (Uecker and
Nadel, 1998). Uecker and Nadel (1998) argued that the pattern of
spatial deficits in this task for FAS is consistent with the
hypothesis that these children have hippocampal damage. The
results on both the ‘‘memory for 16 objects’’ and the Ellis-type
spatial memory task in these FAS children are very similar to the
dissociation between object memory and spatial memory deficits
reported in rats (e.g., Kim et al., 1997). Overall, the authors
concluded that these results indicate ‘‘at least hippocampal
damage’’ (Uecker and Nadel, 1996).
We recognize two important limitations to interpretations of
behavior, in children and animals, as an index of localized alcohol
teratogenic effects within the CNS. Similarity of outcome does
not imply causality: spatial deficits in themselves do not prove
hippocampal involvement in teratogenic outcome. In fact, Uecker
and Nadel (1996) also identified spatial deficits in FAS children
on other tests that were indicative of involvement of the prefrontal
cortex. Indeed, prenatal alcohol exposure has profound neuroanatomical and neurophysiological effects on many other brain areas
in FAS children, including the neocortex and cerebellum (Berman
and Krahl, 1996; Roebuck et al., 1998, 1999). Damage to these
other areas certainly also contributes to behavioral dysfunction,
including spatial learning and memory deficits.

A few autopsy studies of fetal or infant brain list damage to the
hippocampus as part of the gross alterations (cited in Mattson and
Riley, 1996), although studies assessing dendritic spines in the
human brain are restricted to the neocortex (Ferrer et al., 1988).
To date, there have been no reports from imaging studies (e.g.,
MRI or CAT) identifying hippocampal damage (Mattson and
Riley, 1996; Roebuck et al., 1998; Moore et al., 1998). Information about abnormal hippocampal electrophysiology after prenatal alcohol exposure has all been derived from research in animal
models of fetal alcohol effects (Berman and Krahl, 1996). No
human studies to date have directly examined hippocampal
electrophysiology in FAS. However, cortical EEG and evoked
potential studies in humans clearly document abnormal brain
electrical activity associated with prenatal alcohol exposure (see
Berman and Krahl, 1996).

Cognitive deficits, including impaired learning, memory, and
attentional problems, are hallmarks of fetal alcohol effects on the
CNS. The studies summarized in this review strongly implicate
damage to the hippocampus as an important mediator of some of
the behavioral and cognitive deficits associated with FAS. This
conclusion is based on the weight of evidence demonstrating
spatial learning and memory deficits in animals exposed prenatally
and/or neonatally to alcohol in the same behavioral tasks that are
impaired, sometimes selectively, by direct hippocampal damage
(e.g., lesions, pharmacological manipulations). This behavioral
evidence is supported by neuroanatomical studies demonstrating
abnormal axonal projections in the hippocampus of prenatal
alcohol-exposed rodents, as well as direct loss of hippocampal
pyramidal neurons. Electrophysiological studies also show abnormal electrical activity in the hippocampus, both in vivo (EEG,
ERPs) and in vitro using hippocampal brain slices. The variability
in many of these outcomes in hippocampal function illustrates the
importance of the details of alcohol administration (e.g., dose,
route, diet), patterns of exposure (e.g., bingeing), critical periods
(e.g., prenatal vs. neonatal), and individual differences (e.g.,
gender, genetics (strain)). Finally, the ability of postnatal environmental enrichment to stimulate increased dendritic spine density
in the hippocampus is blunted by prenatal alcohol exposure.
Reduced neuronal plasticity long after removal of alcohol demonstrates that the effects of prenatal alcohol exposure extend well
beyond the prenatal or early neonatal periods, when the CNS is
undergoing rapid development (i.e., neuronal proliferation, neurite extension, and synaptogenesis) in the presence of alcohol.
Several issues remain concerning the effects of prenatal alcohol
on brain growth and development, and development and function
of the hippocampus in particular. First, it is now abundantly clear
that the hippocampus is not unique in its sensitivity to the
teratogenic effects of prenatal alcohol. Other brain regions,
including the cerebellum (Klintsova et al., 1997; Goodlett and
Johnson, 1999) and frontal cortex (Miller, 1986; Moore et al.,



1998; Hannigan et al., 1999), are also adversely affected by
prenatal alcohol exposure and contribute to the motor and
cognitive deficits in FAS. Understanding how different regions of
the brain are affected by perinatal alcohol and by enrichment
should improve diagnosis of FAS and provide insight into the
nature of neurological deficits. The development of more effective
treatments for fetal alcohol effects might then be possible.
Second, just as critical periods are being described for effects of
neonatal alcohol exposure (West and Goodlett, 1990; Goodlett
and Johnson, 1999), there are likely to be critical periods for the
effects of prenatal alcohol exposure on brain development. If so,
‘‘critical periods’’ may exist for the postnatal ameliorative effects of
environmental enrichment, and these should be identified.
Third, more research is needed to provide insight into the
molecular and biochemical mechanisms by which prenatal alcohol
damages the hippocampus (e.g., second messenger systems, gene
expression, trophic factors). Finally, more human studies of
hippocampal structure and function after prenatal alcohol exposure are needed. Relatively few human studies have attempted to
examine the effects of prenatal alcohol exposure in FAS, but the
findings from animal studies clearly indicate the potential for
hippocampal damage in humans (e.g., Uecker and Nadel, 1996,
1998). The technology for carrying out such studies may be
available (i.e., MRI, fMRI).
In conclusion, the behavioral signs of FAS or ARNDs,
including spatial learning and memory deficits, can have understandable neurological bases. The effects of perinatal alcohol
exposure can extend in time far beyond the period of actual
exposure and influence neural plasticity throughout life. Finally,
and potentially of the most importance, basic research on animal
models indicates that prenatal alcohol-damaged animals can
benefit from being reared in an appropriately structured postnatal
environment. Such findings proffer hope that useful behavioral
strategies for amelioration of fetal alcohol can be developed to take
advantage of this remaining responsiveness of the brain. Such
remediation would improve the lives of the many children born
each year damaged by prenatal alcohol.

We appreciate the substantive and editorial comments on an
earlier version of this paper by B. Cortese, and the secretarial
assistance of A. English. The technical assistance of M. Sperry, T.
Smith, C. Siebert, and L. DiCerbo is also appreciated.

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