Neurotoxicologyand Teratology, Vol. 12, pp. 79-84. ©Pergamon Press plc. 1990. Printed in the U.S.A.
0892-0362/90 $3.00 + .00
Apomorphine-Induced Motor Behavior in Rats Exposed Prenatally to Alcohol I J O H N H. H A N N I G A N , 2 B E T T Y A. B L A N C H A R D , M E L I S S A P. H O R N E R , E D W A R D P. R I L E Y 3 A N D M I C H E L L E L. P I L A T I
Center for Behavioral Teratology and Department of Psychology, The University at Albany State University of New York, Albany, N Y R e c e i v e d 23 O c t o b e r 1988
HANNIGAN, J. H., B. A. BLANCHARD, M. P. HORNER, E. P. RILEY AND M. L. PILATI. Apomorphine-induced motor behavior in rats exposedprenatally to alcohol. NEUROTOXICOL TERATOL 12(2) 79-84, 1990.--We psychopharmacologically examined dopamine function in rats exposed to ethanol prenatally. Pregnant rats received liquid diets of 35% or 0% ethanol-derived calories (EDC), or ad lib lab chow (LC). Twenty-eight-day-old offspring received systemic doses of apomorphine chosen to stimulate predominantly presynaptic (0.02 or 0.1 mg/kg) or postsynaptic dopamine receptors (2.0 or 5.0 mg/kg). Behavior was scored automatically for 60 min in an "open field." For males, prenatal ethanol exposure resulted in a dose-response shift to the left for locomotor activity. Females exposed to the liquid diet, with or without ethanol, showed less of an increase in locomotor activity following the 5.0 mg/kg dose of apomorphine than did LC controls. There were no effects of prenatal treatment on repetitious motor behavior in the automated "open field" or on stereotypy scored by direct observation in separate groups of rats. The results are consistent with an hypothesis that prenatal ethanol exposure alters the sensitivity of postsynaptic (perhaps mesolimbic) dopamine systems important to locomotor activity in young male rats. Fetal Alcohol Syndrome
Dopamine
Sex differences
Undemutrition
FETAL Alcohol Syndrome (FAS) and fetal alcohol effects include behavioral dysfunctions such as hyperactivity, and learning and attentional deficits (8,26). Animals exposed prenatally to alcohol exhibit postnatal alcohol-related developmental effects that include similar behavioral dysfunction, including spontaneous overactivity and learning deficits (1, 2, 21). In rats, prenatal ethanol exposure can produce changes in various measures of CNS dopamine, norepinephine and acetylcholine function (10). Psychopharmacological assessment of prenatal ethanol-exposed animals can provide information about the relationship of such neurochemical alterations to behavior (15). Prenatal ethanol-exposed male rats are more sensitive to the locomotor stimulating effects of methylphenidate (20,27) and amphetamine (3). These effects suggest that dysfunctional catecholamine systems may be involved in spontaneous overactivity in these animals [cf. (4)]. The absence of differential effects of clonidine on wall-climbing and locomotor activity in rats exposed to ethanol in utero (14) suggests further that the effects of methylphenidate and amphetamine might not be due to their actions on noradrenergic systems. To clarify the role of putative dopamine dysfunction in the behavioral consequences of prenatal ethanol exposure, we assessed the sensitivity of fetal ethanol-exposed rats to the direct
dopamine agonist apomorphine in a test of locomotor activity at 28 days of age, an age at which we had observed differential responsiveness to amphetamine (3). Further, we chose to examine both low and high doses of apomorphine. Low doses of apomorphine, presumably by a preferential stimulation of presynaptic dopamine receptors, have been shown to decrease locomotor activity. High doses of apomorphine, which stimulate postsynaptic receptors and "override" the effects of simultaneous presynaptic stimulation, increase locomotor activity (9, 25, 29). Differential responses to low and/or high doses of apomorphine would allow us to further specify a site for the hypothesized dopaminergic dysfunction (12,17). METHOD The protocol for the production of alcohol-exposed rats has been detailed previously (3). Briefly, parent Long-Evans rats on a 12-hr light-dark cycle (lights on at 0700 hr) with free access to food and water were mated nightly. Upon detection of a vaginal plug, females were housed in individual breeding cages in a separate nursery and assigned to one of three treatment groups. The first group received a liquid diet providing 35% ethanolderived calories (35% EDC) on days 6 through 20 of pregnancy. A second group (0% EDC) was pair-fed the same liquid diet as the
IA preliminary report was made to the Annual Meeting of the Society for Neuroscience, New Orleans, LA, November, 1987. 2Requests for reprints should be addressed to J. H. Hannigan, Center for Behavioral Teratology, The University at Albany, SS-112, 1400 Washington Avenue, Albany, NY 12222. 3Current address: The Department of Psychology, San Diego State University, San Diego, CA 92182.
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35% EDC group, but with sucrose substituted isocalorically for ethanol. The third group (LC) was given ad lib lab chow and water throughout pregnancy. The liquid diets consisted of water, chocolate Sustacal (Mead Johnson, Inc.), Vitamin Diet Fortification Mixture and Salt Mixture XIV (ICN Nutritional Biochemicals), and either 95% ethanol or sucrose. The liquid diets provided approximately 1.3 kcal/ml, were the sole source of nutrition during the period of administration, and are nutritionally complete for rats. The day after parturition, litters were examined for obvious morphological anomalies, weighed, sexed, and culled to ten pups, providing an equal number of males and females when possible, Pups were weaned at 21 days of age and housed in pairs with same sex littermates until testing.
Behavioral Testing Subjects were male and female rats (5-8 per cell) from each of the three prenatal treatment groups, with no more than one animal per litter assigned to any cell of the design to randomize litter effects. At 27 days of age (Day 1 of testing), rats were transported from the colony to the testing room, handled and placed in an automated "open field" (Omnitech Digiscan Analyzer) for a 15-min "habituation" session of three 5-min blocks. The activity monitors were 39 x 39 x 30 cm-high chambers, crossed by infrared photobeams and housed in sound-attenuating, red-lit boxes with masking noise provided by ventilation fans. Considering the various measures of behavioral activation following stimulants, we specifically defined "locomotor activity" as the distance traveled in the automated "open field" as transduced by successive infrared photobeam breaks. Unless otherwise indicated, the terms "activity" or "locomotor activity" refer to this measure. Photobeam breaks were also decoded automatically by the Digiscan Analyzer into a measure of number of bouts of repetitious movement along any one side of the automated monitor, as an approximation of gross "stereotypy." On the following day (Day 2 of testing) each animal was weighed and injected subcutaneously with 0.0, 0.02, 0.1, 2.0 or 5.0 mg/kg/ml apomorphine hydrochloride (Sigma Chemical Co., No. A-4393) in 0.9% saline/0.1% ascorbate vehicle. These doses of apomorphine were chosen because they are thought to stimulate predominantly presynaptic (0.02 and 0.1 mg/kg) or postsynaptic (2.0 and 5.0 mg/kg) dopamine receptors (9,29). Behavior in the monitors was measured for 60 min in six 10-min blocks. All testing took place between 1300 and 1600 hr. To provide a more sensitive index of repetitious or stereotypic movements than is available with the automated activity monitor, we did a second study with separate groups of rats, which we observed directly following apomorphine injections. Rats were raised identically to those described above. Naive male and female rats from the three prenatal treatment groups ( n ' s = 6 - 1 4 ) were injected with 0, 2.0, 5.0 or 10.0 mg/kg/ml of apomorphine on postnatal day 28 and placed into an observation box (20 x 20 x 20 cm) for 40 min. Using a l-rain time-sampling technique, the following behaviors were scored by an experienced observer who was unaware of the prenatal history of the subject: Quiet (not moving while alert or sleeping), Exploratory Locomotor Activity (irregular movement and rearing), Pacing (repetitious movement about the chamber), and small-amplitude Stereotypic Movements without locomotion (e.g., apparent sniffing, prancing, or head bobbing). Several other scored behaviors (e.g., grooming, oral dyskinesia, gnawing) occurred rarely and were not analyzed further. RESULTS
Dam and Litter Characteristics Subjects were selected randomly from and are representative of
TABLE 1 BODY WEIGHTOF OFFSPRING Postnatal Day 1
Postnatal Day 28
Female
Male
Female
Male
6.74 (0.09)
7.04 (0.09)
74.09 (1.70)
78.47 (2.05)
0% EDC
6.38* (0.10)
6.70* (0.09)
70.42 (1.00)
77.17 (1.43)
35% EDC
5.67? (0.11)
5.905(0.10)
65.63+ (1.92)
69.71t (0.93)
Prenatal treatment LC
Body weights (in grams) on postnatal Day 1 represent means ( -+_S.E.M.) of litter means for each sex. Body weights on postnatal Day 28 are means (_+S.E.M.) of individual animals. Within each age, * indicates significantly different from the LC group of the same sex, and t indicates significantly different from both the LC and 0% EDC groups of the same sex. Males weighed more than females at both ages (p's
0892-0362/90 $3.00 + .00
Apomorphine-Induced Motor Behavior in Rats Exposed Prenatally to Alcohol I J O H N H. H A N N I G A N , 2 B E T T Y A. B L A N C H A R D , M E L I S S A P. H O R N E R , E D W A R D P. R I L E Y 3 A N D M I C H E L L E L. P I L A T I
Center for Behavioral Teratology and Department of Psychology, The University at Albany State University of New York, Albany, N Y R e c e i v e d 23 O c t o b e r 1988
HANNIGAN, J. H., B. A. BLANCHARD, M. P. HORNER, E. P. RILEY AND M. L. PILATI. Apomorphine-induced motor behavior in rats exposedprenatally to alcohol. NEUROTOXICOL TERATOL 12(2) 79-84, 1990.--We psychopharmacologically examined dopamine function in rats exposed to ethanol prenatally. Pregnant rats received liquid diets of 35% or 0% ethanol-derived calories (EDC), or ad lib lab chow (LC). Twenty-eight-day-old offspring received systemic doses of apomorphine chosen to stimulate predominantly presynaptic (0.02 or 0.1 mg/kg) or postsynaptic dopamine receptors (2.0 or 5.0 mg/kg). Behavior was scored automatically for 60 min in an "open field." For males, prenatal ethanol exposure resulted in a dose-response shift to the left for locomotor activity. Females exposed to the liquid diet, with or without ethanol, showed less of an increase in locomotor activity following the 5.0 mg/kg dose of apomorphine than did LC controls. There were no effects of prenatal treatment on repetitious motor behavior in the automated "open field" or on stereotypy scored by direct observation in separate groups of rats. The results are consistent with an hypothesis that prenatal ethanol exposure alters the sensitivity of postsynaptic (perhaps mesolimbic) dopamine systems important to locomotor activity in young male rats. Fetal Alcohol Syndrome
Dopamine
Sex differences
Undemutrition
FETAL Alcohol Syndrome (FAS) and fetal alcohol effects include behavioral dysfunctions such as hyperactivity, and learning and attentional deficits (8,26). Animals exposed prenatally to alcohol exhibit postnatal alcohol-related developmental effects that include similar behavioral dysfunction, including spontaneous overactivity and learning deficits (1, 2, 21). In rats, prenatal ethanol exposure can produce changes in various measures of CNS dopamine, norepinephine and acetylcholine function (10). Psychopharmacological assessment of prenatal ethanol-exposed animals can provide information about the relationship of such neurochemical alterations to behavior (15). Prenatal ethanol-exposed male rats are more sensitive to the locomotor stimulating effects of methylphenidate (20,27) and amphetamine (3). These effects suggest that dysfunctional catecholamine systems may be involved in spontaneous overactivity in these animals [cf. (4)]. The absence of differential effects of clonidine on wall-climbing and locomotor activity in rats exposed to ethanol in utero (14) suggests further that the effects of methylphenidate and amphetamine might not be due to their actions on noradrenergic systems. To clarify the role of putative dopamine dysfunction in the behavioral consequences of prenatal ethanol exposure, we assessed the sensitivity of fetal ethanol-exposed rats to the direct
dopamine agonist apomorphine in a test of locomotor activity at 28 days of age, an age at which we had observed differential responsiveness to amphetamine (3). Further, we chose to examine both low and high doses of apomorphine. Low doses of apomorphine, presumably by a preferential stimulation of presynaptic dopamine receptors, have been shown to decrease locomotor activity. High doses of apomorphine, which stimulate postsynaptic receptors and "override" the effects of simultaneous presynaptic stimulation, increase locomotor activity (9, 25, 29). Differential responses to low and/or high doses of apomorphine would allow us to further specify a site for the hypothesized dopaminergic dysfunction (12,17). METHOD The protocol for the production of alcohol-exposed rats has been detailed previously (3). Briefly, parent Long-Evans rats on a 12-hr light-dark cycle (lights on at 0700 hr) with free access to food and water were mated nightly. Upon detection of a vaginal plug, females were housed in individual breeding cages in a separate nursery and assigned to one of three treatment groups. The first group received a liquid diet providing 35% ethanolderived calories (35% EDC) on days 6 through 20 of pregnancy. A second group (0% EDC) was pair-fed the same liquid diet as the
IA preliminary report was made to the Annual Meeting of the Society for Neuroscience, New Orleans, LA, November, 1987. 2Requests for reprints should be addressed to J. H. Hannigan, Center for Behavioral Teratology, The University at Albany, SS-112, 1400 Washington Avenue, Albany, NY 12222. 3Current address: The Department of Psychology, San Diego State University, San Diego, CA 92182.
79
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35% EDC group, but with sucrose substituted isocalorically for ethanol. The third group (LC) was given ad lib lab chow and water throughout pregnancy. The liquid diets consisted of water, chocolate Sustacal (Mead Johnson, Inc.), Vitamin Diet Fortification Mixture and Salt Mixture XIV (ICN Nutritional Biochemicals), and either 95% ethanol or sucrose. The liquid diets provided approximately 1.3 kcal/ml, were the sole source of nutrition during the period of administration, and are nutritionally complete for rats. The day after parturition, litters were examined for obvious morphological anomalies, weighed, sexed, and culled to ten pups, providing an equal number of males and females when possible, Pups were weaned at 21 days of age and housed in pairs with same sex littermates until testing.
Behavioral Testing Subjects were male and female rats (5-8 per cell) from each of the three prenatal treatment groups, with no more than one animal per litter assigned to any cell of the design to randomize litter effects. At 27 days of age (Day 1 of testing), rats were transported from the colony to the testing room, handled and placed in an automated "open field" (Omnitech Digiscan Analyzer) for a 15-min "habituation" session of three 5-min blocks. The activity monitors were 39 x 39 x 30 cm-high chambers, crossed by infrared photobeams and housed in sound-attenuating, red-lit boxes with masking noise provided by ventilation fans. Considering the various measures of behavioral activation following stimulants, we specifically defined "locomotor activity" as the distance traveled in the automated "open field" as transduced by successive infrared photobeam breaks. Unless otherwise indicated, the terms "activity" or "locomotor activity" refer to this measure. Photobeam breaks were also decoded automatically by the Digiscan Analyzer into a measure of number of bouts of repetitious movement along any one side of the automated monitor, as an approximation of gross "stereotypy." On the following day (Day 2 of testing) each animal was weighed and injected subcutaneously with 0.0, 0.02, 0.1, 2.0 or 5.0 mg/kg/ml apomorphine hydrochloride (Sigma Chemical Co., No. A-4393) in 0.9% saline/0.1% ascorbate vehicle. These doses of apomorphine were chosen because they are thought to stimulate predominantly presynaptic (0.02 and 0.1 mg/kg) or postsynaptic (2.0 and 5.0 mg/kg) dopamine receptors (9,29). Behavior in the monitors was measured for 60 min in six 10-min blocks. All testing took place between 1300 and 1600 hr. To provide a more sensitive index of repetitious or stereotypic movements than is available with the automated activity monitor, we did a second study with separate groups of rats, which we observed directly following apomorphine injections. Rats were raised identically to those described above. Naive male and female rats from the three prenatal treatment groups ( n ' s = 6 - 1 4 ) were injected with 0, 2.0, 5.0 or 10.0 mg/kg/ml of apomorphine on postnatal day 28 and placed into an observation box (20 x 20 x 20 cm) for 40 min. Using a l-rain time-sampling technique, the following behaviors were scored by an experienced observer who was unaware of the prenatal history of the subject: Quiet (not moving while alert or sleeping), Exploratory Locomotor Activity (irregular movement and rearing), Pacing (repetitious movement about the chamber), and small-amplitude Stereotypic Movements without locomotion (e.g., apparent sniffing, prancing, or head bobbing). Several other scored behaviors (e.g., grooming, oral dyskinesia, gnawing) occurred rarely and were not analyzed further. RESULTS
Dam and Litter Characteristics Subjects were selected randomly from and are representative of
TABLE 1 BODY WEIGHTOF OFFSPRING Postnatal Day 1
Postnatal Day 28
Female
Male
Female
Male
6.74 (0.09)
7.04 (0.09)
74.09 (1.70)
78.47 (2.05)
0% EDC
6.38* (0.10)
6.70* (0.09)
70.42 (1.00)
77.17 (1.43)
35% EDC
5.67? (0.11)
5.905(0.10)
65.63+ (1.92)
69.71t (0.93)
Prenatal treatment LC
Body weights (in grams) on postnatal Day 1 represent means ( -+_S.E.M.) of litter means for each sex. Body weights on postnatal Day 28 are means (_+S.E.M.) of individual animals. Within each age, * indicates significantly different from the LC group of the same sex, and t indicates significantly different from both the LC and 0% EDC groups of the same sex. Males weighed more than females at both ages (p's
