behavioral and neurochemical effects on rat offspring after prenatal exposure to ethanol
TRANSCRIPT
www.elsevier.com/locate/neutera
Neurotoxicology and Teratol
Behavioral and neurochemical effects on rat offspring after prenatal
exposure to ethanol
Lyvia M.V. Carneiro, Joao Paulo L. Diogenes, Silvania M.M. Vasconcelos, Gislei F. Aragao,
Emmanuelle C. Noronha, Patrıcia B. Gomes, Glauce S.B. Viana *
Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara, Rua Cel. Nunes de Melo, 1127, Fortaleza 60430-270, Brazil
Received 13 February 2005; accepted 22 March 2005
Abstract
The work studied behavioral and neurochemical alterations in 21-day-old pups, from both sexes (26 g on average) born from female
Wistar rats administered daily with ethanol (0.5 or 4.0 g/kg, p.o.), for 30 days before mating, and throughout their gestational period. Ethanol
administration continued from delivery up to weaning. The open field, elevated plus maze and forced swimming tests were used to evaluate
effects of ethanol on locomotion, anxiety and depression, respectively. Binding assays were used to identify dopaminergic (D1- and D2-like)
and muscarinic (M1 plus M2) receptors. Results of the plus maze test indicated significant and dose-dependent increases in the number of
entrances in the open arms and in the time of permanence in the open arms, in the prenatally ethanol-exposed offspring, as compared to
controls, indicating an anxiolytic effect. In the open field test, this group presented decreases in spontaneous locomotor activity as well as in
the occurrences of rearing and grooming. Offspring also showed dose-dependent increases in their immobility time in the forced swimming
test, characterizing despair behavior. Decreases in the hippocampal (D2: 32%; D1: 25%) and striatal (D2: 30%; D1: 52%) dopaminergic
binding were detected in ethanol-exposed offspring. On the other hand, significant increases were observed in muscarinic binding in the
hippocampus (40%) as well as in the striatum (42%). This study shows evidence that in utero ethanol exposure produces a long-lasting effect
on development and pharmacological characteristics of brain systems that may have important implications in behavioral and neurochemical
responsiveness occurring in adulthood.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Rat; Prenatal ethanol; Behavior; Dopaminergic receptors; Muscarinic receptors
1. Introduction
The central nervous system is markedly affected by
alcohol consumption that causes sedation and relief of
anxiety and, at higher concentrations, slurred speech, ataxia,
impaired judgment and uninhibited behavior. Like other
sedative–hypnotic drugs, ethanol is a central nervous system
depressant and, at high blood concentrations, induces coma,
respiratory depression and death. Ethanol has been shown to
affect a large number of membrane proteins that participate
in signaling pathways, including neurotransmitter receptors
for amines, aminoacids and opioids, enzymes such as Na+/
K+ ATPase, adenylate cyclase, phospholipase C and ion
0892-0362/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ntt.2005.06.006
* Corresponding author. Tel.: +55 85 288 8337; fax: +55 85 288 8333.
E-mail address: [email protected] (G.S.B. Viana).
channels [10]. Various studies suggest that alcohol acts
not only by enhancing inhibitory neurotransmission at
GABAA receptors, but also by reducing the excitatory
neurotransmission at N-methyl-d-aspartate (NMDA) recep-
tors [28].
Chronic maternal ethanol abuse during pregnancy is
associated with important teratogenic effects on the off-
spring [1,14], and alcohol appears as a leading cause of
mental retardation and congenital malformation in humans.
However, the extent and severity of a child’s condition
depend on several factors, such as how much alcohol the
pregnant mother consumed, how often and in what period of
pregnancy [29]. The abnormalities that have been charac-
terized as Fetal Alcohol Syndrome (FAS) include retarded
body growth, microcephaly, poor coordination, underdevel-
opment of the mid-facial region and minor joint anomalies
ogy 27 (2005) 585 – 592
L.M.V. Carneiro et al. / Neurotoxicology and Teratology 27 (2005) 585–592586
[3]. More severe cases may include congenital heart defects
and mental retardation. The consequences of heavy drinking
in the 2nd and 3rd trimesters of pregnancy are not well
defined, but animal studies suggest that the brain is vulne-
rable to ethanol throughout its development [29]. A recent
study in children with FAS [21] reported that the pattern of
impaired place learning and spared cued-navigation, as
measured by a computerized (virtual) Morris water task, is
similar to that observed in rats exposed to ethanol during
periods of prenatal or early postnatal brain growth, as well as
in animals with hippocampal damage.
Ethanol rapidly crosses the placenta and reaches con-
centrations in the fetus that are similar to those in the
maternal blood. Children prenatally exposed to alcohol can
suffer from serious cognitive deficits and behavioral
problems. Brain imaging studies have identified structural
changes in various brain regions of these children including
the basal ganglia, corpus callosum, cerebellum and hippo-
campus that may account for the cognitive deficits [29]. In
addition, brain growth continues to be adversely affected
long after the prenatal insult and brain regions most affected
may be consistent with the neurochemical deficits character-
istic of children prenatally exposed to alcohol [36].
Furthermore, clinical as well as experimental evidence
have demonstrated that ethanol is a teratogenic drug, and its
consumption during pregnancy induces harmful effects on
the developing fetus, leading to the Fetal Alcohol Effects.
Experimental evidence demonstrates that alcohol interferes
with many molecular, neurochemical and cellular events
occurring during the normal development of the brain. The
impairment of several neurotransmitter systems and their
receptors as well as changes in the endocrine environment
are also important factors involved in the neurodevelop-
mental liabilities observed after in utero alcohol exposure
[18].
Thus, the objectives of the present work were to study
behavioral (anxiolytic, sedative, muscle relaxant and anti-
depressant) as well as neurochemical (dopaminergic and
muscarinic binding assays) effects presented by rat offspring
from dams exposed during mating and during their gesta-
tional periods to daily oral administration of ethanol.
2. Materials and Methods
2.1. Drugs
Diazepam was purchased from Cristalia (Sao Paulo,
Brazil), and Imipramine was from Novartis (Sao Paulo,
Brazil). Mianserin, butaclamol and atropine were from
Sigma, USA. The ligands l-[N-methyl-3H] scopolamine
methyl chloride (3H-NMS, 60-85 Ci/mmol), [N-methyl-3H]
SCH 23390 (3H-SCH 23390, 60–90 Ci/mmol), and [3H]
spiroperidol (3H-spiroperidol, 60–100 Ci/mmol) were from
New England Nuclear, USA. All other drugs were of
analytical grade.
2.2. Animals and treatment
A total of 80 21-day-old Wistar male and female rats (26
g on average) from the Animal House of the Federal
University of Ceara were used. These pups were born from
dams administered daily for 30 days, before mating and
throughout their gestational period, with either 0.5 or 4 g/kg
ethanol, p.o., from 10 and 20% v/v ethanol solutions in
distilled water. The ethanol administration continued from
delivery up to weaning, when 21-day-old pups were
submitted to behavioral testing and sacrificed afterwards
for neurochemical measurements. Control dams were also
administered by gavage an equivalent volume of distilled
water (10 ml/kg body weight), for the same period of time.
Female rats had free access to a commercial diet (Purina,
Brazil) and water and were mated (5 female to 1 male) and
housed in plastic cages, in a 25 -C room with a 12-h on-and-
off lighting schedule. Experiments were performed accord-
ing to the Guide for the care and use of laboratory animals,
from the US Department of Health and Human Services,
Institute of Laboratory Animal Resources, Washington DC,
1985. Except for diazepam- and imipramine-treated pups
(which were not prenatally exposed to ethanol, but orally
administered with diazepam or imipramine 1 h before the
test), no other drug challenge was administered during the
behavioral testing. These two drugs were used as standards
in the plus maze and forced swimming tests, respectively.
All 21-day-old pups from the four groups (control, ethanol
0.5 and 4 g/kg, and drug treated) were submitted initially to
the open field test, followed by the elevated plus maze test
and by a 5-min trial in the forced swimming test. The next
day, animals were submitted again to the forced swimming
test.
2.3. Behavioral testing
2.3.1. Open field test
The animal was placed in the open field arena, made of
acrylic (transparent walls and black floor, 30�30�15 cm)
divided into nine squares of equal areas. The open field test
[2] was used to evaluate the exploratory activity of the
animal, for 5 min. The observed parameters were number of
squares crossed (locomotor activity) and occurrences of
grooming (number of times the rat scratched its face with its
forepaws) and rearing (number of times the rat stood
completely erect on its hind legs).
2.3.2. Elevated plus maze test
The plus maze was that used typically for mice [24],
because the study was performed with 21-day-old rats. The
apparatus consisted of two perpendicular open arms (30�5
cm) and two closed arms (30�5�25 cm) also in perpen-
dicular position. The open and closed arms were connected
by a central platform (5�5 cm). The platform and the lateral
walls of the closed arms were made of transparent acrylic.
The floor was made of black acrylic. The maze was 45 cm
Table 1
Effects of ethanol on the open field test in 21-day-old rats
Group/dose
(mg/kg, p.o.)
Number of squares
crossed
Rearing Grooming
Control 47.93T1.59 (15) 16.36T0.86 (14) 2.33T0.24 (18)
Ethanol 0.5 34.00T4.23 (10)* 8.36T0.99 (11)* 2.30T0.32 (13)
Ethanol 4 32.40T3.07 (10)* 8.92T1.22 (13)* 1.41T0.17 (17)*
Values are meansTSEM of the number of animals, in parenthesis.
Experiments were performed with litters from dams exposed to ethanol
(EtOH 0.5 or 4 g/kg, p.o.) during their gestational period up to weaning.
* p <0.05 as compared to control (ANOVA and Dunnett, as the post hoc
test).
L.M.V. Carneiro et al. / Neurotoxicology and Teratology 27 (2005) 585–592 587
above the floor. The rat was placed at the center of the plus
maze with its nose in the direction of one of the closed arms,
and observed for 5 min for the measurement of the following
parameters: number of entries in the open (NEOA) and
closed (NECA) arms, and times of permanence in the open
(TPOA) and closed (TPCA) arms. The time of permanence
measures the time spent by the rat in the open and closed
arms. Anxiolytic compounds such as diazepam (2 mg/kg,
p.o., 1 h before the test), used as standard, reduce the natural
aversion of the animal to the open arms. The plus maze test
was performed immediately after animals were submitted to
the open field test.
2.3.3. Forced swimming test
In this test, animals were placed two times (trial and test
condition), on a despair behavior situation by forcing them
to swim in a restricted area. Animals were observed for 5
min [34]. After a short period of activity, they adopt a
characteristic and reproducible floating behavior. This
behavior is reduced by antidepressant drugs, and by CNS
stimulant substances. Imipramine (30 mg/kg, p.o.) was used
as standard and administered 1 h before the test. The animal
was submitted to the trial immediately after the plus maze
test, and again 24 h later.
2.4. Neurochemical measurements
2.4.1. Determination of muscarinic receptor numbers
Brains from 21-day-old pups were dissected on ice and
their cerebral areas (hippocampus and striatum) were
immediately frozen at �20 -C until use. Receptor numbers
were measured through binding assays with a 10%
homogenate prepared (w/v) in 150 mM sodium phosphate
buffer, pH 7.4, at 4 -C, using [3H]-N-methylscopolamine
([3H]-NMS, 85 Ci/mmol, New England Nuclear, Boston,
MA), according to the method described by Dombrowski et
al. (1983) [11]. Total homogenates (80–160 Ag protein)
were incubated in a buffer containing 2.35 nM of [3H]-NMS
in a final volume of 0.2 ml. After incubation at 37 -C, for 30min, the reaction was terminated by filtration through
Whatman GF/B filters which were then washed five times
with 4 ml of ice-cold saline, dried at 60 -C and placed in
vials containing 3 ml of a toluene-based scintillation fluid.
The radioactivity was measured with a Beckman scintilla-
tion counter, at a counting efficiency of 48%. Specific
binding was calculated as total minus nonspecific binding in
the presence of atropine (12.5 AM), and results were
expressed as femtomoles per milligram of protein. Protein
was determined according to Lowry et al. (1951) [26], using
bovine serum albumin as standard.
2.4.2. Determination of dopaminergic receptor numbers
The method described by Meltzer et al. (1989), and
Kessler et al. (1991) [30,23], was used for determination of
D1 and D2 receptor numbers. In the case of D1 receptors, the
specific ligand [3H]-SCH 23390 (87 Ci/mmol, from New
England Nuclear, USA) was used. Total homogenates were
incubated in 50 mM Tris–HCl buffer (pH 7.4) with the
following composition (mM): NaCl (120), CaCl2 (2), MgCl2(1), NaEDTA (1) and ascorbic acid (1). A concentration of
5.75 nM of [3H]-SCH 23390 in a final volume of 0.2 ml was
used. For the determination of D2 receptor numbers, the
specific ligand [3H]-spiroperidol (114 Ci/mmol, from New
England Nuclear, USA) was utilized. Total homogenates
were incubated in a 50 mM Tris–HCl buffer (pH 7.4),
containing 5 AM mianserin for blocking serotonergic
receptors and 17.3 nM of [3H]-spiroperidol in a final volume
of 0.2 ml. In both cases (D1 and D2 receptor assays), specific
binding was defined as total minus nonspecific binding
carried out in the presence of 10 AM butaclamol. After
incubation at 37 -C for 60 min, experiments proceeded as
described above for the muscarinic binding. Protein was
determined according to Lowry et al. (1951), using bovine
serum albumin as standard.
2.5. Statistical analysis
Data are presented as meanTSEM. Behavioral tests were
analyzed by one-way ANOVA and Dunnett as the post hoc
test, while the results from binding assays were analyzed by
the Student’s t test. Results were considered significant at
p <0.05.
3. Results
In the open field test (Table 1), used to measure the
animals’ exploratory behavior and stereotypies, ethanol at
both doses significantly reduced the number of crossings,
indicating a decreased locomotor spontaneous activity (29
and 32% for the groups exposed to 0.5 and 4 g/kg ethanol
respectively). In this parameter, a dose-related effect was not
observed. Although no alteration in grooming was demon-
strated in offspring from dams treated with 0.5 g/kg ethanol,
a significant 40% decrease in grooming was detected in
offspring from dams treated with the higher dose of ethanol,
as compared to controls. These results indicate that ethanol
at a higher dose is able to decrease stereotyped behavior.
The number of rearings also decreased significantly by 49
Table 2
Effects of ethanol in the elevated plus maze test in 21-day-old rats
Group/dose (mg/kg, p.o.) NEOA TPOA
Control 2.15T0.29 (13) 35.50T5.31 (14)
Ethanol 0.5 4.5T0.59 (14)* 75.43T5.52 (07)*
Ethanol 4 5.61T0.59 (13)* 169.9T21.13 (12)*
Diazepam 2 5.37T0.73 (08)* 224.9T27.79 (08)*
Values are meansTSEM of the number of animals, in parenthesis.
Experiments were performed with litters from dams exposed to ethanol
(EtOH 0.5 or 4 g/kg, p.o.) during their gestational period up to weaning.
Diazepam (2 mg/kg, p.o.) was used as standard. NEOA—numbers of
entries in the open arms. TPOA—time of permanence in the open arms
(time the animal stays in the open arms).
* p <0.05 as compared to control (ANOVA and Dunnett, as the post hoc
test).
control 0,5g/kg 4g/kg IMI 30mg/kg0
50
100
150
****
*
Ethanol
Imm
ob
ility
Tim
e (s
)
Fig. 1. Immobility time measurements in 21-day-old rats prenatally exposed
to ethanol, as determined by the forced swimming test. Experiments were
performed with litters from dams exposed to ethanol (0.5 or 4g/kg, p.o)
during their gestational period. *p <0.05 and ***p <0.01 respectively as
compared to control (ANOVA and Dunnett as the post hoc test).
200
Control
4g/kg EtOH
Bin
din
gte
in)
L.M.V. Carneiro et al. / Neurotoxicology and Teratology 27 (2005) 585–592588
and 46% respectively, in the two groups prenatally treated
with ethanol (0.5 and 4 g/kg) as compared to controls.
Tables 2 and 3 show the effects of ethanol (0.5 and 4 g/
kg) on the plus maze test in 21-day-old rats. Results indicate
that offspring from dams exposed to ethanol, at both doses,
showed significant and dose-dependent increases in NEOA
values (105 and 161% for the doses of 0.5 and 4 g/kg
ethanol, respectively) as compared to controls. Effects were
similar to those observed with diazepam (150% increase)
used as standard. These results point to the anxiolytic effect
of ethanol that is still manifested in exposed offspring.
Furthermore, a dramatic increase was also detected in the
time of permanence in the open arms, TPOA (2 and 4.8
times increases in the groups whose mothers were treated
with 0.5 or 4 g/kg ethanol), as compared to controls. The
diazepam-treated group showed a 534% increase, corre-
sponding to values 6 times higher than controls.
Offspring prenatally exposed to 0.5 and 4 g/kg ethanol
showed dose-dependent increases of 89 and 111% in their
immobility time, as compared to controls, in the forced
swimming test. These results indicate a depressant effect,
since CNS depressors generally enhance this typical floating
behavior. On the contrary, this behavior was significantly
decreased by imipramine, a tricyclic antidepressant drug
used as standard (Fig. 1).
A 25% decrease in D2 receptor concentration was
detected in the hippocampus from prenatally ethanol (4 g/
Table 3
Effects of ethanol in the elevated plus maze test in 21-day-old rats
Group/dose (mg/kg, p.o.) NECA TPCA
Control 4.71T0.43 (14) 249.7T6.48 (14)
Ethanol 0.5 3.42T0.20 (07)* 175.7T8.62 (07)**
Ethanol 4 2.91T0.22 (12)** 153.9T16.82 (12)**
Diazepam 2 2.12T0.22 (08)** 91.00T14.83 (08)**
Values are meansTSEM of the number of animals, in parenthesis.
Experiments were performed with litters from dams exposed to ethanol
(EtOH 0.5 or 4 g/kg, p.o.) during their gestational period up to weaning.
Diazepam (2 mg/kg, p.o.) was used as standard. NECA—number of entries
in the closed arms. TPCA—time of permanence in the closed arms.
* p <0.05 and ** p <0.01 as compared to control (ANOVA and
Dunnett, as the post hoc test).
kg) exposed offspring, as compared to controls. A similar
percentage of decrease was also observed in the striatum
from the same ethanol-treated group (Fig. 2). Ethanol (4 g/
kg) also caused 30% decrease in hippocampal D1 receptor
concentration and a dramatic decrease (52%) in the striatal
D1 receptor concentration, in litters from ethanol-exposed
mothers (Fig. 3). On the contrary, a significant increase was
observed in muscarinic receptors in the hippocampus from
rats prenatally treated with ethanol (4 g/kg). A similar in-
crease in muscarinic receptors (42%) was demonstrated in
the striatum in the 4 g/kg group, as compared to controls
(Fig. 4).
Except for non-significant 10% increase and 10%
decrease in body weights of 21-day-old pups from the 4
and 0.5 g/kg ethanol groups respectively, as related to
controls, no other change was observed (control: 25.6T0.87
Control 4 g/kg EtOH Control 4 g/kg EtOH0
100
**
***
Hippocampus Striatum
3H-S
pir
op
erid
ol
(fm
ol/m
g p
ro
Fig. 2. Dopaminergic D2 receptor measurements in the hippocampus and
striatum from 21-day-old rats. Experiments were performed with litters
from dams exposed to ethanol (EtOH, 4 g/kg, p.o.) during their gestational
period up to weaning. **p <0.01 and ***p <0.001 as compared to control
(Student’s t test).
0
100
200
300
400
500
control
4g/kg EtOH
****
Hippocampus Striatum
SC
H 2
3390
Bin
din
g(f
mo
l/mg
pro
tein
)
Fig. 3. Dopaminergic D1 receptor measurements in the hippocampus and
striatum from 21-day-old rats. Experiments were performed with litters
from dams exposed to ethanol (EtOH, 4 g/kg, p.o.) during their gestational
period up to weaning *p <0.05 and ***p <0.001 as compared to control
(Student’s t test).
L.M.V. Carneiro et al. / Neurotoxicology and Teratology 27 (2005) 585–592 589
g; ethanol 0.5 g: 23.7T0.72 g; ethanol 4 g: 28.3T1.85 g).
However, ethanol-exposed groups had smaller number of
litters compared to controls (control group: 9.8T0.60 litters;
ethanol 0.5 g: 6.3T1.03 litters; ethanol 4 g: 5.5T0.91 litters)
(data not shown).
0
100
200
300
400
500
600
700
***
***
control
4g/kg EtOH
N-m
eth
yl-
3H S
cop
ola
min
e B
ind
ing
(fm
ol/m
g pr
otei
n)
Hippocampus Striatum
Fig. 4. Muscarinic (M1+M2) receptor measurements in the hippocampus
and striatum from 21-day-old rats. Experiments were performed with litters
from dams exposed to ethanol (EtOH, 4 g/kg, p.o.) during their gestational
period up to weaning. ***p <0.001 as compared to control (Student’s t
test).
4. Discussion
Distinguishing features of prenatal alcohol exposure in
humans as well as in rodents are impaired cognitive and
behavioral functions, resulting from damage to the central
nervous system [8,36,21]. The consumption of significant
quantities of ethanol during pregnancy is responsible for the
Fetal Alcohol Syndrome (FAS), which is characterized by
low birth weight, microcephaly, facial abnormalities (flat-
tening), mental retardation, heart defects and other abnor-
malities [28]. Central nervous system dysfunctions are the
most severe and permanent consequences of maternal
alcohol intake. The neocortex, hippocampus and cerebellum
are especially susceptible to alcohol and have been
associated with behavioral deficits [18]. Both human and
animal research provide evidence that the CNS is vulnerable
to the damaging effects of ethanol during development, and
one particular form of damage is neuronal loss. A recent
report [39] indicates that the hippocampal CA1 area is
highly susceptible to prenatal ethanol exposure.
In our work, we showed that rat offspring prenatally
exposed to a low or a high doses of ethanol presents a
significant increase in the number of entrances and time
of permanence in the open arms, in the elevated plus
maze test. Our results are indicative of an anxiolytic effect
of ethanol. It is widely known that, in rodents, ethanol at
moderate doses typically causes motor incoordination,
hyperactivity and hypothermia, besides acting as an
anxiolytic drug. Each of these responses can be analyzed
by a specific test. The level of anxiety is measured, in the
elevated plus maze test, as the relative amount of time the
animal spends in the open arms, compared to that in the
closed arms, and thus this test is designed for detecting
the anxiolytic effect.
There is evidence [15] indicating a role of nitric oxide-
dependent pathways in ethanol-induced anxiolytic effects,
as measured by the elevated plus maze test. These results
showed that the inhibition of NO-dependent pathways
enhances, whereas the stimulation of these pathways
decreases, the efficacy of ethanol to produce anxiolytic
effects in rats. The authors postulate that NO-dependent
increases in the guanylate cyclase activity and cGMP levels
oppose the anxiolytic effects produced by the acute ethanol
administration.
In addition, we showed that rat offspring presented
significant decreases not only in the number of crossings but
also in the occurrences of rearing and grooming, as
determined by the open field test. Substances like alcohol,
classified as a central nervous system depressant, are
expected to cause a decrease in the spontaneous locomotor
activity. A significant role in alcohol consumption behavior
and its reinforcement is played by the neurotransmitter
dopamine.
Our results also demonstrated that the in utero
exposure of offspring to ethanol caused a significant
increase in their immobility times, indicating a depressant
effect, as determined by the forced swimming test. It is
widely accepted that antidepressants act on monoamine
neurotransmitter systems, such as NE, DA and mainly 5-
L.M.V. Carneiro et al. / Neurotoxicology and Teratology 27 (2005) 585–592590
HT. The serotonin system is then thought to play a role in
the pharmacology of alcohol, probably by modulating DA
release. In addition, 5-HT neurotransmission has been
shown to play an important role in anxiety [13,17], and
the cellular actions of various neurotransmitters, such as
5-HT, NE, DA and ACh, in the brain, are mediated
through the activation of adenylate cyclase. This causes
the formation of the intracellular second messenger cAMP,
and subsequently leads to the activation of the cAMP-
dependent protein kinase A [16].
The present work showed that while prenatal exposure to
4 g/kg ethanol significantly decreased dopaminergic D1 as
well as D2 receptors, it increased muscarinic cholinergic
receptors in rat hippocampus and striatum. Earlier bio-
chemical and behavioral studies showed that in utero
ethanol exposure produces a long-lasting effect in the
development of electrophysiological and pharmacological
characteristics of midbrain DA systems, in adulthood
[37,38]. These results suggest that in utero ethanol exposure
may produce a downregulation in the function of DA
receptors distinct from the somatodendritic impulse-regulat-
ing D2 autoreceptors.
Our results agree with others [35] showing that daily
prenatal exposure to 3 g/kg ethanol causes a significant
decrease in the number of DA D2 binding sites within the
dorsal and ventral striatum, but no alteration after exposure
to a higher (5 g/kg) dose of ethanol. Earlier findings [7]
indicated that prenatal ethanol exposure may predomi-
nantly produce diminished reactivity of the D2 but not D1
subtypes of DA receptors or an opposite outcome [12].
Others [6] reported that prenatal ethanol exposure did not
alter DA concentration or turnover and produced a
transient increase in D1 but not D2 receptor binding, in
mice. Furthermore, we have recently shown [40] that
ethanol administered for 1 week to rats produces decreases
in D1 and D2 receptor densities and no changes in
dissociation constants. Ethanol is a reinforcing substance
and, as such, manifests its effects through activation of
brain mesolimbic dopaminergic reward pathways. Reduced
dopamine levels and D2 receptor numbers have been
shown in brains of alcohol-preferring animals, in genetic
models of alcoholism [32]. Furthermore, dopamine ago-
nists reduce alcohol consumption, whereas antagonists
show the opposite effect.
Effects on other receptor systems were also observed
after alcohol consumption. Earlier reports [41] showed an
ontogenic increment in muscarinic binding, while others
[20] found a decreased affinity of muscarinic receptors, in
the hippocampus of rat pups prenatally exposed to
ethanol. However, Black et al. (1995) [5], testing the
hypothesis that prenatal ethanol exposure alters hippo-
campal muscarinic receptors, showed a significant
decrease in the number of muscarinic receptors in rat
hippocampus, leading to long-lasting alterations in mus-
carinic cholinergic receptors. Alterations in the cholinergic
system in alcohol-exposed rat fetuses may underlie some
of the cognitive deficits observed after prenatal alcohol
exposure [31].
Research [18,39,33] has shown that in utero exposure to
ethanol is deleterious to fetal brain development. An
interaction of ethanol with glial cells, particularly astrocytes,
has been suggested to contribute to developmental alcohol
neurotoxicity. Recent works [9,19] demonstrated that, at low
concentrations, ethanol inhibits the proliferation of astroglial
cells in vitro, particularly when stimulated by acetylcholine.
These data suggest that intracellular signal transduction
pathways activated by muscarinic receptors may represent a
relevant target for the development of ethanol neurotoxicity
in humans.
In addition to the cognitive deficits associated with FAS,
clinical and animal studies indicate that alcohol exposure
might also have detrimental effects on social behavior [27].
A recent work [25] showed that CA1 and CA3 volumes,
pyramidal cell density and number were reduced in the 4–9
postnatal days (equivalent to the third human trimester), in
the hippocampus from rat pups exposed to alcohol in utero.
The resulting damage to the hippocampus may contribute to
the behavioral deficits, related to learning and memory,
noted after prenatal ethanol exposure in rodents and humans
as well [4]. There is evidence showing that many of the
effects of ethanol on learning and memory stem from altered
cellular activity in the hippocampus and related structures
[42].
We showed that litters prenatally exposed to ethanol
present behavioral as well as neurochemical alterations
which were acquired during prenatal development. The
predominant profile as far as the CNS effects are
concerned is, as expected, a central benzodiazepine-like
depressant effect, with sedative as well as anxiolytic
actions. However, a dramatic increase in immobility time,
in the forced swimming test, also revealed an enhanced
despair behavior, characteristic of depression. This effect
might be a consequence of ethanol withdrawal. It has been
shown that [22] while acute ethanol administration to mice
(2 or 2.5 g/kg) exhibited an antidepressant-like effect, its
prolonged consumption produced tolerance to this effect,
and its withdrawal (similar to our experimental conditions)
elicited a depression.
As far as the neurochemical alterations are concerned,
we also showed that ethanol caused opposite effects on
dopaminergic and muscarinic receptors, decreasing D1
and D2 receptors (downregulating), while increasing
(upregulating) muscarinic receptors. The effects on the
dopaminergic system could explain some of the behav-
ioral alterations, such as those seen in rearing and
grooming. On the other hand, the significant increase in
muscarinic receptors could indicate a disturbance in the
balance state normally existing between the central
dopaminergic and muscarinic systems. These findings
may have important implications for understanding neuro-
chemical and behavioral responsiveness occurring in
ethanol-exposed animals.
L.M.V. Carneiro et al. / Neurotoxicology and Teratology 27 (2005) 585–592 591
Acknowledgments
The authors thank the financial support of the Brazilian
National Research Council (CNPq) and the technical
assistance of Ms. Vilani Rodrigues Bastos and Ms. Jacque-
line Viana.
References
[1] E.L. Abel, Fetal Alcohol Syndrome, CRC Press, New York, US,
1981.
[2] J. Archer, Tests for emotionality in rats and mice: a review, Anim.
Behav. 21 (1973) 205–235.
[3] S.L. Archilbald, C. Fennena-Notestine, A. Gamst, E.P. Riley, S.N.
Mattson, T.L. Jernigan, Brain dysmorphology in individuals with
severe prenatal alcohol exposure, Dev. Med. Child Neurol. 43 (2001)
148–154.
[4] R.F. Berman, J.H. Hannigan, Effects of prenatal alcohol exposure on
the hippocampus: spatial behavior, electrophysiology and neuro-
anatomy, Hippocampus 10 (2000) 94–110.
[5] A.C. Black, L.W. Goolsby, G.A. Cohen, H.E. Young, Effects of
prenatal ethanol exposure on the hippocampal neurochemistry of
albino rats at 90 days of postnatal age, Am. J. Obstet. Gynecol. 173
(1995) 514–519.
[6] W.O. Boggan, W. Xu, C.L. Shepherd, L.D. Middaugh, Effects of
prenatal ethanol exposure on dopamine systems in C57BL/6J mice,
Neurotoxicol. Teratol. 18 (1996) 41–48.
[7] R. Brus, W. Felinska, M. Rykaczewska, R.M. Kostrzewa, R.
Szkilnik, A. Plech, Prenatal ethanol diminishes reactivity of pre-
sumed dopamine D3 receptors in rats, Pol. J. Pharmacol. 47 (1995)
109–114.
[8] W.J. Chen, S.E. Maier, S.E. Parnell, J.R. West, Alcohol and the
developing brain: neuroanatomical studies, Alcohol Res. Health 27
(2003) 174–180.
[9] L.G. Costa, M. Guizetti, Inhibition of muscarinic receptor-induced
proliferation of astroglial cells by ethanol: mechanisms and implica-
tions for the Fetal Alcohol Syndrome, Neurotoxicology 23 (2002)
685–691.
[10] I. Diamond, A.S. Gordon, Cellular and molecular neuroscience of
alcoholism, Physiol. Rev. 77 (1997) 1–20.
[11] A.M. Dombrowski, A.A. Jerkins, F.C. Kauffman, Muscarinic receptor
binding and oxidative activities in the adult rat superior cervical
ganglion: effects of 6-hydroxydopamine on nerve growth factor,
J. Neurosci. 3 (1983) 1963–1970.
[12] M.J. Druse, N. Tajuddin, A. Kuo, M. Connertz, Effects of in utero
exposure on the developing dopaminergic system in rats, J. Neurosci.
Res. 27 (1990) 223–240.
[13] A.S. Eison, M.S. Eison, Serotonergic mechanisms in anxiety, Prog.
Neuro-psychopharmacol. Biol. Psychiatry 18 (1994) 47–62.
[14] C.B. Ernhart, R.J. Sokol, S. Martier, P. Moron, D. Nadler, J.W. Ager,
A. Wolf, Alcohol teratogenicity in the human: a detailed assessment of
specificity, critical period, and threshold, Am. J. Obstet. Gynecol. 156
(1987) 33–39.
[15] V.M. Ferreira, C.F. Valenzuela, G.S. Morato, Role of nitric oxide-
dependent pathways in ethanol-induced anxiolytic effects in rats,
Alcohol Clin. Exp. Res. 23 (1999) 1898–1904.
[16] A.G. Gilman, Transmembrane signaling, G proteins, and adenylyl
cyclase, Harvey Lect. 85 (1989) 153–172.
[17] F.G. Graeff, F.S. Guimaraes, T.G. Andrade, J.F. Deakin, Role of 5-HT
in stress, anxiety and depression, Pharmacol. Biochem. Behav. 54
(1996) 129–141.
[18] C. Guerri, Mechanisms involved in central nervous system dysfunc-
tions induced by prenatal ethanol exposure, Neurotox. Res. 4 (2002)
327–335.
[19] M. Guizetti, T. Moller, L.G. Costa, Ethanol inhibits muscarinic
receptor-mediated DNA synthesis and signal transduction in human
fetal astrocytes, Neurosci. Lett. 344 (2003) 68–70.
[20] K. Hadjiivanova, V.D. Petkov, L. Alova, V.V. Petkov, Y. Vuglenova,
Changes in brain muscarinic and beta-adrenoreceptors of the offspring
of ethanol-consuming mothers (experiments on rats), Acta Physiol.
Pharmacol. Bulg. 17 (1991) 91–97.
[21] D.A. Hamilton, P. Kodituwakku, R.J. Sutherland, D.D. Savage,
Children with Fetal Alcohol Syndrome are impaired at place learning
but not cued-navigation in a virtual Morris water task, Behav. Brain
Res. 143 (2003) 85–94.
[22] K. Hirani, R.T. Khisti, C.T. Chopde, Behavioral action of ethanol in
Porsolts forced swim test: modulation by 3 alpha-hydroxy-5 alpha-
pregnan-20-one, Neuropharmacology 43 (2002) 1339–1350.
[23] R.M. Kessler, M.S. Ansari, D.E. Schmidt, T. Paulis, J.A. Clanton,
R. Innis, M. Ai-Tikriti, R.G. Manning, D. Gillespie, High affinity
dopamine D2 receptor radioligants: 2. [125I] M Epidepride, a
potent and specific radioligand for the characterization of striatal
and extra-striatal dopamine D2 receptors, Life Sci. 49 (1991)
617–618.
[24] R.G. Lister, The use of a plus-maze to measure anxiety in the mouse,
Psychopharmacology 92 (1997) 180–185.
[25] D.J. Livy, E.K. Miller, S.E. Maier, J.R. West, Fetal alcohol exposure
and temporal vulnerability: effects of binge-like alcohol exposure on
the developing rat hippocampus, Neurotoxicol. Teratol. 25 (2003)
447–458.
[26] O.H. Lowry, N.J. Rosebrough, A.C. Farr, R.J. Randall, Protein
measurement with Folin phenol reagent, J. Biol. Chem. 193 (1951)
265–275.
[27] J.N. Lugo Jr., M.D. Marino, K. Cronise, S.J. Kelly, Effects of alcohol
exposure during development on social behavior in rats, Physiol.
Behav. 78 (2003) 185–194.
[28] S.B. Masters, The alcohols, in: B.G. Katzung (Ed.), Basic and Clinical
Pharmacology, 8th edition, Lange Medical Books, New York, USA,
2001, pp. 382–392.
[29] S.N. Mattson, A.M. Schoenfeld, E.P. Riley, Teratogenic effects of
alcohol on brain and behavior, Alcohol Res. Health 25 (2001)
185–191.
[30] H.Y. Meltzer, S. Matsubara, J.C. Lee, Classification of typical and
atypical antipsychotic drugs on the basis of dopamine D1- and D2-
and serotonin pKi values, J. Pharmacol. Exp. Ther. 251 (1989)
238–246.
[31] A.H. Nagahara, R.J. Handa, Fetal alcohol-exposed rats exhibit
differential responses to cholinergic drugs on a delay-dependent
memory task, Neurobiol. Learn. Mem. 72 (1999) 230–243.
[32] E.P. Noble, Alcoholism and the dopaminergic system: a review,
Addict. Biol. 1 (1996) 333–348.
[33] T. Othman, D. Legare, P. Sadri, W.W. Lautt, F.E. Parkinson, A
preliminary investigation of the effects of maternal ethanol intake
during gestation and lactation on brain adenosine A1 receptor
expression in rat offspring, Neurotoxicol. Teratol. 24 (2002)
275–279.
[34] R.D. Porsolt, M. LePichnon, M. Jalfre, Depression: a new animal
model sensitive to antidepressant treatment, Nature 277 (1977)
730–732.
[35] S. Randall, J.H. Hannigan, In utero alcohol and postnatal methyl-
phenidate: locomotion and dopamine receptors, Neurotoxicol. Teratol.
21 (1999) 587–593.
[36] E.P. Riley, C.L. McGee, E.R. Sowell, Teratogenic effects of
alcohol: a decade of brain imaging, Am. J. Med. Genet. 127C
(2004) 35–41.
[37] R.Y. Shen, L.A. Chiodo, The effects of in utero ethanol administration
on the electrophysiological activity of rat nigrostriatal dopaminergic
neurons, Brain Res. 624 (1993) 216–222.
[38] R.Y. Shen, J.H. Hannigan, G. Kapatos, Prenatal alcohol reduces the
activity of adult mid brain dopamine neurons, Alcohol Clin. Exp. Res.
23 (1999) 1801–1807.
L.M.V. Carneiro et al. / Neurotoxicology and Teratology 27 (2005) 585–592592
[39] T.D. Tran, S.J. Kelly, Critical periods for ethanol-induced cell loss
in the hippocampal formation, Neurotoxicol. Teratol. 25 (2003)
519–528.
[40] S.M. Vasconcelos, D.S. Macedo, L.O. Lima, F.C. Sousa, M.M.
Fonteles, G.S. Viana, Effect of one-week ethanol treatment on
monoamine levels and dopaminergic receptors in rat striatum, Braz.
J. Med. Biol. Res. 36 (2003) 503–509.
[41] S.B. Wigal, A. Amsel, R.E. Wilcox, Fetal ethanol exposure diminishes
hippocampal beta-adrenergic receptor density while sparing muscar-
inic receptors during development, Brain Res. Dev. Brain Res. 55
(1990) 161–169.
[42] A.M. White, D.B. Matthews, P.J. Best, Ethanol, memory, and hippo-
campal function: a review of recent findings, Hippocampus 10 (2000)
88–93.