Neurotoxicologyand Teratology, Vol. 13, pp. 335-339. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0892-0362/91 $3.00 + .00
Cocaine Treatment in Neonatal Rats Affects the Adult Behavioral Response to Amphetamine H A R R Y E. H U G H E S ,
GRAHAM
F. P R I N G L E , L O U I S A. S C R I B A N I A N D D I A N A L. D O W - E D W A R D S
~
Laboratory of Cerebral Metabolism, Department of Neurosurgery State University of New York Health Science Center, Brooklyn, NY 11203 R e c e i v e d 20 F e b r u a r y 1990
HUGHES, H. E., G. F. PRINGLE, L. A. SCRIBANI AND D. L. DOW-EDWARDS. Cocaine treatment in neonatal rats affects the adult behavioral response to amphetamine. NEUROTOXICOL TERATOL 13(3) 335-339, 1991.--This study investigated whether exposure to cocaine during critical periods of brain development alters the motor stimulating effects of amphetamine given in adulthood. Female rats received 50 mg/kg/day cocaine HC1 SC or vehicle during either postnatal days 1-10 or 11-20. At 60-65 days of age, activity counts were collected over a 15-min baseline period. Subjects then received one of 3 doses (0, 0.1, 0.25 mg/ kg) of d-amphetamine sulfate SC followed by a 90-min period of activity monitoring. Adult activity in 1-10-day cocaine-treated rats was different from vehicle-treated rats in response to 0.1 mg/kg amphetamine only. Adult activity in 11-20-day cocaine-treated rats was different from vehicle-treated rats in response to 0.25 mg/kg only. The observed differences represented an increase and decrease in activity, respectively. These alterations in amphetamine response may be related to the observed alterations in D-1 receptor concentrations as well as the altered rates of brain glucose metabolism we have observed in adult rats neonatally exposed to cocaine. Development
Locomotor activity
Cocaine
Amphetamine
COCAINE abuse by pregnant women can result in children born with neurobehavioral anomalies that include jitteriness, increased startle responses and deficient interactive behavior (1,4). The gap between simply describing these anomalies and understanding the mechanisms which underlie their development needs to be closed. Administration of cocaine to gravid rats during the last two weeks of gestation has been shown to result in heightened motor activity in offspring at around three weeks postnatal age (15). Other prenatal cocaine studies have found effects in the offspring that include startle reflex (10), male sexual behavior (20) and learning of an odor/milk association (27). While prenatal studies address the effects of drugs primarily on cell division and migration, the early postnatal period in rats more closely approximates the third trimester of human gestation in neural development, the period of synaptogenesis. Adult female rats neonatally exposed to cocaine exhibited significant increases in brain metabolic activity in limbic, motor and sensory structures (8). Some of these structures such as the caudate, hippocampus and ventral thalamic nuclei were also stimulated by acute cocaine administration in adult rats (22). It appears that neonatal cocaine treatment produces long-term changes in the function of these stimulant responsive brain systems. If, how and under what circumstances these neurochemical changes relate to changes in motor activity produced
by the same treatment represents an ongoing area of study. The current study investigated the extent to which early cocaine treatment alters the locomotor enhancing effects of amphetamine (17) given in adulthood. The ability of amphetamine to alter locomotor patterns across mammalian species (9) has been firmly established. Developmentally, amphetamine challenge has revealed different sensitivities to lead acetate in perinatally treated adult mice (26). Since both cocaine and amphetamine alter function in brain monoamine systems, long-term effects of cocaine can reasonably be assessed by studying the animal's motor response to different dosages of amphetamine. METHOD
Subjects Female offspring of Sprague-Dawley rats (Charles River, Wilmington, MA) reared in SUNY Health Science Center colony were used as subjects. Dams and their litters were housed in 44 × 24 × 20 cm plastic cages with wood chip bedding in a temperature- and humidity-controlled room. The light/dark regimen was 12:12 h. Food (Purina pellets 5012) and water were constantly available to the dams. Gravid rats were checked for newborn pups
1Requests for reprints should be addressed to Diana L. Dow-Edwards, Ph.D., Department of Pharmacology, Box 29, SUNY Health Science Center, 450 Clarkson Avenue, Brooklyn, NY 11203.
335
336
daily. Litters found during these checks were declared born on that date (postnatal day 1), and were culled to eight pups (six females and two males). The 122 pups used in this study were drawn from 21 litters. Although in our previous study female rats were more sensitive than males to perinatal cocaine treatment (8), gender differences were not addressed in this study. Only female rats were used because of the extended time requirements of the experimental design.
Dosage Procedure Female littermates received 50 mg/kg/day cocaine HC1 in 50 mg/ml solution or vehicle SC during either postnatal days 1-10 or 11-20. A Hamilton microliter syringe with a 30-gauge hypodermic needle inserted in a rostral direction at the thoracic level along the pup's dorsal midline surface was used. On days 1 and 2 only, the dose was divided into two 25 mg/kg injections separated by 8 h to prevent mortality. The four treatment groups were designated as follows: C ]-1° (cocaine treatment during postnatal days 1-10), V 1-1° (vehicle treatment during postnatal days 1-10), C j 1-2o (cocaine treatment during postnatal days 11-20), V 11-2° (vehicle treatment during postnatal days 11-20).
Apparatus A Digiscan Animal Activity Monitor, model RXYZCM(8) interfaced with an IBM computer and Onmitech software program. The inside of the activity chamber measured 60 × 60 × 37 cm and contained two 10-watt light bulbs for illumination, a fan, 24 infrared sensors spaced 2.5 cm apart (16 horizontal, 8 vertical) and a 42 × 42 × 31 cm Plexiglas, open box for the animal. The arrangement of sensors permitted activity monitoring along both X and Y axes. The row of sensors measuring vertical movements was located 16 cm from the floor of the box. A 20 × 20 cm glasscovered aperture on the lid of the chamber permitted direct observation into the box. Behaviors recorded included total distance traveled (cm), time spent in stereotypy, the number of vertical movements and the number of clockwise rotations.
Testing Beginning at 56 days of age, subjects from each of the four treatment groups were given daily vaginal smears to determine estrus cycle phase. Following one complete cycle, the rats were placed in the activity chamber on the first day of diestrus. Activity counts were collected over a 15-min baseline period. Subjects then received a single injection of one of three doses (0, 0.1, 0.25 mg/kg) at 1 ml/kg of d-amphetamine sulfate SC followed by a 90-min period of activity monitoring. On each test day, animals were monitored within a 6-h test window randomizing drug dosage and treatment groups.
Drugs Both cocaine HC1 and d-amphetamine sulfate were freshly prepared in sterile water and saline, respectively, and were purchased from Sigma Chemical Co., St. Louis, MO. All dosages are expressed as weight of the salt.
Statistics Because animals from the same litter were differentially treated, individual rats rather than litters served as experimental units (5). Data analysis was performed on a NEC computer using a SYSTAT software program. The 15-min baseline activity period and the 90-min postamphetamine injection period were ana-
HUGHES ET AL.
lyzed separately. A two-way factorial analysis of variance (ANOVA) compared the main effects of treatment schedule (1-10 vs. 11-20 day) and group (cocaine vs. vehicle) for baseline activity. A three-way ANOVA compared the main effects of treatment schedule, group and dosage for the 90-min postamphetamine injection period. Student t-tests were used as post hoc tests when applicable to determine the significance of interactions between groups and individual dosages of amphetamine. The minimum accepted level of significance was p = 0 . 0 5 . Student t-tests were used to compare body weights and age at testing of cocaine- and vehicletreated animals. RESULTS
Body Weights and Baseline Activity No significant differences were observed in body weights at either 60 days or on day of testing between cocaine- and vehicleexposed rats within each treatment schedule. Also, the estrus cycle as determined by vaginal smearing beginning on day 56 did not differ in cocaine- and vehicle-treated rats (data not shown). No significant differences in total distance traveled were noted between treatment groups for either early or late exposed animals during the 15-min, preinjection baseline period (Fig. i). Repeated measures ANOVAs indicate that activity levels for cocaine- and vehicle-treated rats diminished significantly over time for early, F(2,116)=8.88, p0.10. At 0.25 mg/kg amphetamine, V ~1-2° rats demonstrated a peak increase in activity at about 30 min followed by a gradual decline, just as seen in the early treated animals. However, C 11-z° subjects at this dosage traveled significantly less than V j 1-2o rats, F(1,20)= 11.48, p
0892-0362/91 $3.00 + .00
Cocaine Treatment in Neonatal Rats Affects the Adult Behavioral Response to Amphetamine H A R R Y E. H U G H E S ,
GRAHAM
F. P R I N G L E , L O U I S A. S C R I B A N I A N D D I A N A L. D O W - E D W A R D S
~
Laboratory of Cerebral Metabolism, Department of Neurosurgery State University of New York Health Science Center, Brooklyn, NY 11203 R e c e i v e d 20 F e b r u a r y 1990
HUGHES, H. E., G. F. PRINGLE, L. A. SCRIBANI AND D. L. DOW-EDWARDS. Cocaine treatment in neonatal rats affects the adult behavioral response to amphetamine. NEUROTOXICOL TERATOL 13(3) 335-339, 1991.--This study investigated whether exposure to cocaine during critical periods of brain development alters the motor stimulating effects of amphetamine given in adulthood. Female rats received 50 mg/kg/day cocaine HC1 SC or vehicle during either postnatal days 1-10 or 11-20. At 60-65 days of age, activity counts were collected over a 15-min baseline period. Subjects then received one of 3 doses (0, 0.1, 0.25 mg/ kg) of d-amphetamine sulfate SC followed by a 90-min period of activity monitoring. Adult activity in 1-10-day cocaine-treated rats was different from vehicle-treated rats in response to 0.1 mg/kg amphetamine only. Adult activity in 11-20-day cocaine-treated rats was different from vehicle-treated rats in response to 0.25 mg/kg only. The observed differences represented an increase and decrease in activity, respectively. These alterations in amphetamine response may be related to the observed alterations in D-1 receptor concentrations as well as the altered rates of brain glucose metabolism we have observed in adult rats neonatally exposed to cocaine. Development
Locomotor activity
Cocaine
Amphetamine
COCAINE abuse by pregnant women can result in children born with neurobehavioral anomalies that include jitteriness, increased startle responses and deficient interactive behavior (1,4). The gap between simply describing these anomalies and understanding the mechanisms which underlie their development needs to be closed. Administration of cocaine to gravid rats during the last two weeks of gestation has been shown to result in heightened motor activity in offspring at around three weeks postnatal age (15). Other prenatal cocaine studies have found effects in the offspring that include startle reflex (10), male sexual behavior (20) and learning of an odor/milk association (27). While prenatal studies address the effects of drugs primarily on cell division and migration, the early postnatal period in rats more closely approximates the third trimester of human gestation in neural development, the period of synaptogenesis. Adult female rats neonatally exposed to cocaine exhibited significant increases in brain metabolic activity in limbic, motor and sensory structures (8). Some of these structures such as the caudate, hippocampus and ventral thalamic nuclei were also stimulated by acute cocaine administration in adult rats (22). It appears that neonatal cocaine treatment produces long-term changes in the function of these stimulant responsive brain systems. If, how and under what circumstances these neurochemical changes relate to changes in motor activity produced
by the same treatment represents an ongoing area of study. The current study investigated the extent to which early cocaine treatment alters the locomotor enhancing effects of amphetamine (17) given in adulthood. The ability of amphetamine to alter locomotor patterns across mammalian species (9) has been firmly established. Developmentally, amphetamine challenge has revealed different sensitivities to lead acetate in perinatally treated adult mice (26). Since both cocaine and amphetamine alter function in brain monoamine systems, long-term effects of cocaine can reasonably be assessed by studying the animal's motor response to different dosages of amphetamine. METHOD
Subjects Female offspring of Sprague-Dawley rats (Charles River, Wilmington, MA) reared in SUNY Health Science Center colony were used as subjects. Dams and their litters were housed in 44 × 24 × 20 cm plastic cages with wood chip bedding in a temperature- and humidity-controlled room. The light/dark regimen was 12:12 h. Food (Purina pellets 5012) and water were constantly available to the dams. Gravid rats were checked for newborn pups
1Requests for reprints should be addressed to Diana L. Dow-Edwards, Ph.D., Department of Pharmacology, Box 29, SUNY Health Science Center, 450 Clarkson Avenue, Brooklyn, NY 11203.
335
336
daily. Litters found during these checks were declared born on that date (postnatal day 1), and were culled to eight pups (six females and two males). The 122 pups used in this study were drawn from 21 litters. Although in our previous study female rats were more sensitive than males to perinatal cocaine treatment (8), gender differences were not addressed in this study. Only female rats were used because of the extended time requirements of the experimental design.
Dosage Procedure Female littermates received 50 mg/kg/day cocaine HC1 in 50 mg/ml solution or vehicle SC during either postnatal days 1-10 or 11-20. A Hamilton microliter syringe with a 30-gauge hypodermic needle inserted in a rostral direction at the thoracic level along the pup's dorsal midline surface was used. On days 1 and 2 only, the dose was divided into two 25 mg/kg injections separated by 8 h to prevent mortality. The four treatment groups were designated as follows: C ]-1° (cocaine treatment during postnatal days 1-10), V 1-1° (vehicle treatment during postnatal days 1-10), C j 1-2o (cocaine treatment during postnatal days 11-20), V 11-2° (vehicle treatment during postnatal days 11-20).
Apparatus A Digiscan Animal Activity Monitor, model RXYZCM(8) interfaced with an IBM computer and Onmitech software program. The inside of the activity chamber measured 60 × 60 × 37 cm and contained two 10-watt light bulbs for illumination, a fan, 24 infrared sensors spaced 2.5 cm apart (16 horizontal, 8 vertical) and a 42 × 42 × 31 cm Plexiglas, open box for the animal. The arrangement of sensors permitted activity monitoring along both X and Y axes. The row of sensors measuring vertical movements was located 16 cm from the floor of the box. A 20 × 20 cm glasscovered aperture on the lid of the chamber permitted direct observation into the box. Behaviors recorded included total distance traveled (cm), time spent in stereotypy, the number of vertical movements and the number of clockwise rotations.
Testing Beginning at 56 days of age, subjects from each of the four treatment groups were given daily vaginal smears to determine estrus cycle phase. Following one complete cycle, the rats were placed in the activity chamber on the first day of diestrus. Activity counts were collected over a 15-min baseline period. Subjects then received a single injection of one of three doses (0, 0.1, 0.25 mg/kg) at 1 ml/kg of d-amphetamine sulfate SC followed by a 90-min period of activity monitoring. On each test day, animals were monitored within a 6-h test window randomizing drug dosage and treatment groups.
Drugs Both cocaine HC1 and d-amphetamine sulfate were freshly prepared in sterile water and saline, respectively, and were purchased from Sigma Chemical Co., St. Louis, MO. All dosages are expressed as weight of the salt.
Statistics Because animals from the same litter were differentially treated, individual rats rather than litters served as experimental units (5). Data analysis was performed on a NEC computer using a SYSTAT software program. The 15-min baseline activity period and the 90-min postamphetamine injection period were ana-
HUGHES ET AL.
lyzed separately. A two-way factorial analysis of variance (ANOVA) compared the main effects of treatment schedule (1-10 vs. 11-20 day) and group (cocaine vs. vehicle) for baseline activity. A three-way ANOVA compared the main effects of treatment schedule, group and dosage for the 90-min postamphetamine injection period. Student t-tests were used as post hoc tests when applicable to determine the significance of interactions between groups and individual dosages of amphetamine. The minimum accepted level of significance was p = 0 . 0 5 . Student t-tests were used to compare body weights and age at testing of cocaine- and vehicletreated animals. RESULTS
Body Weights and Baseline Activity No significant differences were observed in body weights at either 60 days or on day of testing between cocaine- and vehicleexposed rats within each treatment schedule. Also, the estrus cycle as determined by vaginal smearing beginning on day 56 did not differ in cocaine- and vehicle-treated rats (data not shown). No significant differences in total distance traveled were noted between treatment groups for either early or late exposed animals during the 15-min, preinjection baseline period (Fig. i). Repeated measures ANOVAs indicate that activity levels for cocaine- and vehicle-treated rats diminished significantly over time for early, F(2,116)=8.88, p0.10. At 0.25 mg/kg amphetamine, V ~1-2° rats demonstrated a peak increase in activity at about 30 min followed by a gradual decline, just as seen in the early treated animals. However, C 11-z° subjects at this dosage traveled significantly less than V j 1-2o rats, F(1,20)= 11.48, p
