behavioral and neurochemical effects on rat offspring after prenatal exposure to ethanol

8
Behavioral and neurochemical effects on rat offspring after prenatal exposure to ethanol Lyvia M.V. Carneiro, Joa ˜o Paulo L. Dio ´genes, Silvania M.M. Vasconcelos, Gislei F. Araga ˜o, Emmanuelle C. Noronha, Patrı ´cia 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 channels [10]. Various studies suggest that alcohol acts not only by enhancing inhibitory neurotransmission at GABA A 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 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). Neurotoxicology and Teratology 27 (2005) 585 – 592 www.elsevier.com/locate/neutera

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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.

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