Acute ethanol intoxication during pregnancy: Postnatal effects on the behavioral response to serotonin agents

Alcohol, Vol. 9, pp. 523-527, 1992 Printed in the U.S.A. All rightsreserved.

0741-8329/92 $5.00 + .00 Copyright©1992PergamonPress Ltd.

Acute Ethanol Intoxication During Pregnancy: Postnatal Effects on the Behavioral Response to Serotonin Agents S U S A N A F U L G I N I T I l, N O R A S. V I G L I E C C A

AND SILVIA A. MINETTI

Departamento de Farmacologia, Facultad de Ciencias Quimicas, Universidad Nacional de C6rdoba, C6rdoba, Argentina Received 11 J u l y 1991; A c c e p t e d 14 M a y 1992 FULGINITI, S., N. S. VIGLIECCA AND S. A. MINETTI. Acute ethanol intoxication duringpregnancy in rats: Postnatal effects on the behavioral response to serotonin agents. ALCOHOL 9(6) 523-527, 1992.-Pregnant wistar rats were treated on the eighth day of gestation (GD 8) with two IP injections, spaced by 4 h, of either ethanol (2.9 g/kg in 24e70 v/v saline solution, EG) or saline (SG). Other pregnant females did not received any type of IP injections (absolute control group, ACG). Offspring were tested at 45 or 90 days of age. At 45 days of age, EG showed an increased behavioral response (forepaw treading and hindlimb abduction) to the 5-HT~ agonist, 5-methoxy-N,N-dymethyltryptamine. In addition, an enhanced "wet-dog" shakes behavioral response to 5-HT 2 agonist, 5-hydroxy-L-tryptophan, was also observed in EG as compared to ACG and SG. On the contrary, at 90 days, EG exhibited a diminished behavioral reactivity to 5-HTI and 5-HT2 agonists as compared to SG. These results demonstrated that acute administration of ethanol on GD 8 induced long-lasting changes in the functioning of central serotonergic systems. Prenatal alcohol

5-HT syndrome

5-HT receptors

Behavioral reactivity

A L C O H O L exposure during pregnancy is a risk factor for long-lasting abnormalities in the fetus. It is well known that offspring of alcoholic women often present morphological, physiological, and behavioral abnormalities (6,34), which have been called Fetal Alcohol Syndrome (FAS) (17). Studies with different animal models o f FAS have demonstrated that rats exposed to alcohol in utero show comparable behavioral alterations to those observed in humans (20,34). In addition, biochemical changes at the synaptic level of central monoaminergic neurons (8,10,21,32-34), as well as an altered reactivity to several pharmacological agents, have also been reported in offspring of rats prenatally exposed to alcohol (1,3,23, 24,36). We have previously described that 45-day-old offspring of rats, exposed to ethanol on gestational day 8 (GD 8), exhibited a shorter sleeping time in response to an ethanol challenge dose, and higher blood ethanol levels upon awakening (15). On the contrary, we have also observed that offspring of 90day-old animals had an increased sensitivity to the hypnotic effect of ethanol (unpublished results). Monoamines, particularly serotonin (5-HT) and catecholamines (CA), have both been suggested to be involved in the CNS depressant effects induced by ethanol (13,14,25). Taking into account these data and, in order to elucidate potential causes of the altered sensi-

5-HT agonists

tivity to ethanol, in the present report, we studied the functioning of central serotonergic systems in 45- and 90-day-old offspring of rats which have been intoxicated with ethanol on GD 8. This purpose was undertaken by means of the analysis of the behavioral reactivity of 5-HT sites induced by the injection of 5-HT receptor agonists. The reactivity of central 5-HT, sites was measured by means of 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), which evokes various motor syndromes, mainly forepaw treading (FT) and hindlimb abduction (HA) (22,37,38). Behavioral response to 5-HT2 receptors was measured by the "wet-dog" shakes (WDS) elicited by injection of the 5-HT precursor, 5-hydroxytryptophan (5-HTP) in the presence of a peripheral decarboxylase inhibitor (22,39). METHOD

Subjects The procedure used to expose rats to ethanol during GD 8 has been previously described (15). Parent animals were male and nulliparous female Wistar rats (90-120 days old), born and bred in our laboratory. They were housed under a 12-h dark-fight cycle (light onset: 07:00 h), maintained with food and water ad fib and under constant temperature conditions (22 + 1 °C). In the evening of the proestrus day, they were

Requests for reprints should be addressed to Susana Fulginiti, Departmento de Farmacologia, Facultad de Ciencias Quimicas, Universidad Nacional de C6rdoba, Sucursal 16, C.C. 61, 5016 C6rdoba, Argentina. 523

524 housed overnight with male rats in a 3 : 1 relation. The presence of spermatozoa in the vaginal smears during the early hours of the following morning was taken as an index of pregnancy and was referred to as gestationai day 9 (GD 1). Dams were weighed and housed with other pregnant females until GD 8, when they were randomly divided into three groups: absolute control (ACG), saline (SG), and ethanol (EG). While the EG received two IP injections of ethanol (2.9 g/kg in 24% v/v saline solution), spaced by an interval of 4 h (10:00 and 14:00 h), the SG received an identical volume of saline. A C G did not receive any IP injection on GD 8. Injections were made with considerable care to avoid uterine trauma. A C G and SG rats remained without food and water during approximately 12 h since alcohol-treated rats exhibited an inhibition of feeding habits during the same period of time. Maternal blood ethanol levels have been previously reported (15). Briefly, they were determined by gas chromatography (19) at different times after alcohol injections; the highest alcohol levels were observed 60 rain after the second dose (446 + 10 mg/dl, mean pm SEM). Females were then individually housed in maternity cages and weighed again on GD 18. Throughout gestation, pregnant females were fed with a lab-chow maternity diet (Nutrimentos S.A. pregnancy lab chow). From GD 20 to birth, breeding cages were checked three times daily for birth. Within 24 h after delivery [postnatal day 1 (PD 1)] all pups were weighed and the number of newborns counted in order to determine litter size. Litters were then randomly culled to five males and five females whenever possible. Offspring were weaned at 25 days of age and housed in groups of 8-12 rats according to sex and treatment. AT 45 or 90 days of age, ACG, SG, and EG offspring (three males and one female) from each of eight ECG, 11 SG, and 11 EG litters were kept in the experimental room for at least 2 days prior to the behavioral studies.

Drugs 5-MeODMT (Sigma Chemical Co.) was dissolved in saline using a few drops of glacial acetic acid and injected IP in a volume of 0.1 mi/100 g b.w. Carbidopa (supplied by CibaGeigy, Buenos Aires) and 5-HTP (Sigma) were suspended in deionized water with Tween-80 and injected subcutaneously in a volume of 0.2 ml/100 g b.w. All drug solutions were prepared immediately before use.

Behavioral Procedures Rats were singly placed in plastic cages (50 x 35 cm) 5 min before the experiments. The observer of the behavior was always blind to the animal's experimental condition. Following the injection of 5-MeODMT, rats were observed for continuous scoring periods of 2 rain, separated by l-min nonscoring intervals for a total observation period of 13 min, beginning 2 min after 5-MeODMT administration. The ranked intensity scale was: 0 = absent, 1 = present, 2 = moderate, and 3 = intense. The values for each behavior from the four 2-min observation periods were summed over the total period (13 min) mad expressed as the total behavioral score. For WDS, only male animals were used. They were injected with a peripheral aromatic acid decarboxylase inhibitor, carbidopa (25 mg/kg), followed 30 rain later by the 5-HT precursor, 5-HTP (150 mg/kg), as described by Lucki et al. (22). Thirty minutes after 5-HTP injection, rats were observed for head and body shakes during six 5-min periods, which were spaced 30 min apart during a total of lg0 min. Scores

FULGINITI, VIGLIECCA AND MINETTI from the six 5-min periods were summed over the total observation time (180 min) and divided by the number of observation periods (six). RESULTS In this experiment, acute ethanol treatment of pregnant rats did not significantly affect body weight gain during pregnancy, gestational length, litter size, or body weight of pups at birth. Body weight gain (g) between GD 1 and 8 were (mean + SE): ACG = 19.4_+ 2.0 (n = 8), SG = 20.2 + 1.4(n = l l ) , a n d E G = 21.7 + 1.8 (n = 11), while weight gain between GD 8 and 18 were: ACG = 36.2 + 3.3, SG = 40.7 + 3.1, and EG = 34.8 +_ 3.0; length of gestational period (days): A C G = 23.0 _+ 0.0, SG = 22.8 + 0.1, and EG = 23.0 +_ 0.0; total litter size: A C G = 9.6 + 0.8, SG = 10.7 + 0.6, and EG = 10.4 + 0.4; and body weight of pups at birth (g): ACG = 6.1 + 0.3, SG = 6.0 + 0.1, and EG = 6.1 + 0.1. Similarly, there were no significant weight differences between groups at 45 or 90 days of age. The effects of prenatal alcohol on the 5-HT syndrome, produced by the injection of 2.5 mg/kg 5-MeODMT, in 45and 90-day-old offspring, are illustrated in Fig. 1. A three-way ANOVA (treatment x sex x age) was performed in order to analyze these data. There were significant interactions between prenatal treatment x age in FT, F(2, 66) = 19.31, p < 0.01, and HA, F(2, 66) = 20.04, p < 0.01. Post hoc comparisons (Mann-Whitney U test) further demonstrated that, at 45 days, the behavioral responses to 5-MeODMT (FT and HA) were significantly enhanced in alcohol pre-exposed subjects as compared to A C G and SG; whereas, at 90 days, these behaviors were significantly decreased compared only with SG. Post hoc comparisons also revealed that SG showed an enhanced HA behavior at 90 days, as compared with ACG and SG of 45-day-old rats. Besides, ethanol-treated rats had FT and H A scores significantly lower at 90 days of age as compared with younger rats of the same group, and did not differ from A C G and SG subjects of 45 days of age. There were no significant sex effects or treatment x sex interactions. Fig. 2 represents WDS behavior of male offspring following 5-HTP administration. A two-way ANOVA (treatment x age) showed a significant interaction between prenatal treatment x age, F(2, 35) = 10.07, p < 0.01. While 45-dayold ethanol-treated rats showed an enhanced behavioral response compared with both control groups (Newman-Keuls test, p < 0.05), 90-day-old SG rats presented an increased number of WDS compared with ACG and EG rats (p < 0.01). Comparisons between 45- and 90-day-old saiine-treated controls showed enhanced WDS behavior in older subjects 6o < o.oI). DISCUSSION The existence of multiple 5-HT receptors in the brain has been consistently reported. These sites were classified into two major subclasses, denominated 5-HT~ and 5-HT2 receptors (16,26). Moreover, the 5-HT~ site is composed of at least two or probably three different subtypes, 5-HT~A, 5-HTm, and 5-HTtc (4). Several reports have proposed that some components of the serotonerglc behavioral syndrome (forepaw treading and hindlimb abduction), after 5-HT agonists are mediated by the 5-HT~ site (22,37,38), while "wet-dog" shaking behavior induced by 5-HTP is probably mediated by 5-HT2 receptors (22,39). The present study demonstrates that prenatal acute ethanol

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(10,31). A decreased number of central 5-HTrbinding sites and a decreased presynaptic 5-HT uptake in the motor cortex have been reported in 19- and 35-day-old offspring of rats that consumed ethanol chronically prior to parturition (I 1,35). It is possible that acute ethanol exposure on GD 8 evokes increased behavioral reactivity of 5-HT receptors at 45 days of age as a consequence of developmental alterations in central 5-HT neurotransmission. Although the above-mentioned neurochemical evidence (35) appears to be contradictory with this suggestion, it might be that chronic exposure to ethanol during pregnancy does affect serotonergic systems in a different way than acute intoxication. The present study also showed that saline-treated subjects have an enhanced score in hindlimb abduction and "wet-dog" shake behavior at 90 days as compared to younger rats. Regarding this effect, it must be pointed out that relatively mild stressful conditions during pregnancy, such as crowding or one daily saline injection, modify the development of central 5-HT systems (27-30). Peters (29) described that 60-day-old offspring from mothers, exposed to a combined stress regimen of crowding and once-daily saline injection throughout pregnancy, showed an increased behavioral response to both 5MeODMT and 5-HTP. The data of 90-day-old ACG rats revealed that hindlimb abduction and "wet-dog" shake scores did not differ from younger offspring of the same group. Thus, it appears that two IP saline injections on GD 8 may by itself alter 5-HT~ and 5-HT 2 receptors' behavioral reactivity in the adult offspring. Since our long-lasting behavioral changes, probably produced by saline administrations during GD 8, are no longer observed in alcohol pretreated rats, it seems possible that mild maternal stress-induced changes could be prevented

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FIG. I. Behavioral responses (FT abnd HA) to 2.5 mg/kg of 5MeODMT (IP) in 45- and 90-day-old offspring. The intensity of the behavioral responses was scored every 2 min for 13 min postinjection of 5-MeODMT. Results are given as mean ± SEM total score for each parameter. Number of animals are given in parentheses. *p < 0.05, **p < 0.02 Compared to ACG and SG rats of 45 clays of age (FT and HA). **p < 0.02 Compared to SG rats of 90 days of age (FT). *p < 0.05, **p < 0.02 Compared to ACG and EG rats of 90 days of age (HA). +/7 < 0.05, ++/7 < 0.02 Compared to 45-day-old offspring of the same group.

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intoxication increases the behavioral responses to both 5MeODMT and 5-HTP at 45 days of age. These findings reflect an enhanced response of serotonergic systems mediated principally by either 5-HT~ or 5-HT2 receptors. These changes are short-lasting, being absent at 90 days of age, indicating a nonpermanent effect of alcohol prenatal treatment. Neurochemical evidence has shown that the development of central serotonergic systems is affected by prenatal ethanol exposure. Hence, offspring of mothers exposed to ethanol through all or much of gestation exhibited deficits of brain 5-HT and/or 5-hydroxyindolacetic acid (5-HIAA), which persists for weeks to months after the last exposure to ethanol

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administrated 30 min following pretreatment with carbidopa (25 mg/kg SC) in 45- and 90-day-old male offspring. Values are the means ± SEM of six 5-min periods spaced 30 min apart during a total time of 180 rain. *p < 0.05 Compared to ACG and SG rats of 45 days of age. **p < 0.01 Compared to ACG and EG rats of 90 days of age. ++p < 0.01 Compared to 45-day-old rats of the same group.

526

FULGINITI, VIGLIECCA AND MINETTI

by simultaneous ethanol injections. Whether this effect by prenatal ethanol is due to the well-known preventive effect of ethanol on stress-induced changes (5,9,18) remains to be elucidated. The selective involvement of 5-HT receptor subtypes in certain physiological processes has been frequently postulated (2,7,12). Therefore, it seems possible that the altered behavioral reactivity mediated by 5 - H T sites observed in offspring of dams exposed to a brief period o f ethanol intoxication, could be reflected in 5-HT-mediated functions at adult age.

Finally, behavioral disturbances as well as altered reactivity to pharmacological agents in ethanol prenatally treated offspring, could be in part due to modifications of 5-HT receptors. ACKNOWLEDGEMENTS This research was supported by grants from Consejo de Investigaciones Cienttficas y T6cnicas de C6rdoba (CONICOR) and from Consejo Nacional de Investigaciones Cientlficas y T~nicas (CONICET).

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