Neurotoxicologyand Teratology,Vol. 14, pp. 337-342, 1992

0892-0362/92$5.00 + .00 Copyright©1992PergamonPress Ltd.

Primedin the U.S.A.All rightsreserved.

Prenatal Exposure to Cocaine I: Effects on Gestation, Development, and Activity in Sprague-Dawley Rats J O S E P H I N E M. J O H N S , L A R R Y W . M E A N S , M I C H A E L J. M E A N S A N D B R I A N A . M c M I L L E N ~

Departments o f Pharmacology and Psychology, East Carolina University, Greenville, N C 27858-4354 Received 21 J u n e 1991; Accepted 23 J u n e 1992 JOHNS, J. M., L. W. MEANS, M. J. MEANS AND B. A. McMILLEN. Prenatalexposure to cocaine I: Effects on gestation, development, and activity in Sprague-Dawleyrats. NEUROTOXICOL TERATOL 14(5) 337-342, 1992.-Spermpositive Sprague-Dawley rats received one of four treatments for 20 days beginning within 24 hours of conception. One group received subcutaneous injections of 15 mg/kg cocaine twice daily (Cocaine-D); a second group received 15 mg/kg cocaine twice daily for two consecutive days at 5-day intervals (Cocaine-I); a third group received normal saline twice daily (Saline); and a fourth group received 1.5 mg/kg amfonelic acid (AFA), a dopamine reuptake inhibitor, once daily. Cocaine-D, Cocaine-I, and AFA dams were fed ad lib. An attempt was made to pair-feed the Saline dams with the Cocaine-D dams; however, the Saline dams did not eat as much as the Cocalne-D dams which resulted in dams in all groups essentially eating ad lib. The Cocaine-D pups showed a slightly delayed righting behavior and neophobia at 30 days of age, as evidenced by hypoactivity during the first 15 min of a 6-h activity test. The Cocaine-I pups were hypoactive during the 3-h dark phase of the 6-h activity test when tested at 30 days of age. These effects did not occur in the offspring exposed to AFA, a potent dopamine uptake inhibitor and CNS stimulant which indicate that one or more other sites for cocaine action may combine for its effects on the developing fetus. Cocaine

Gestation

Development

Activity

Prenatal

COCAINE use has increased dramatically in the last 15 years and is now a drug of widespread abuse (1) including the obstetric population (6). Clinical data indicate several adverse effects directly related to maternal cocaine abuse such as infant weight reduction (19), growth retardation, premature delivery (2), and fetal death (3). Polydrug abuse is often cited as a confounding variable for determining whether neonatal adverse outcomes are due to cocaine (4,5,26). However, assessment of 3-day-old infants with the Brazelton Neonatal Assessment Scale shows clusters of abnormalities peculiar to fetal cocaine exposure and are independent of other drugs (4). Recently, in order to control for extraneous factors, animal studies have been conducted on the effects of prenatal exposure to cocaine (8-10,12,13,15,17,22-24). Prior to starting these studies, little data had been reported on development and behavior of rats prenatally exposed to cocaine. This article focuses on the effects of cocaine on maternal and infant birth indices. In order to determine whether the observed changes were due to the CNS stimulating effect of cocaine, an additional group of pregnant rats received the potent CNS stimulant, amfonelic acid (AFA), which is a preferential inhib-

Stimulants

Amfonelic acid

itor of dopamine re-uptake (11,21). The offspring from the treated and control rats were subsequently used in behavioral and social interaction studies reported in a companion article. METHOD Male and female (275-325 g) Spragne-Dawley rats (Charles River, Raleigh, NC) were housed singly for a 10-day acclimation period, females in plastic rat cages and males in a battery of suspended stainless steel cages. Room lighting was on a 12 L : 12 D h cycle with the light period starting at 7:00 a.m. Animals had free access to food (Agway Lab Blox) and water. Following the acclimation period, females were placed singly with one male in the stainless steel breeding battery. The morning a sperm plug was found was designated as gestational day 0. Sperm positive females were removed and randomly assigned to one of four treatment groups or as a surrogate dam and housed singly in plastic rat cages. A cocaine daily group (Cocaine-D) and a cocaine "weekend users" group (Cocaine-I or intermittent) received injections of 15 mg/kg of cocaine-HCL (dose calculated as the free base; Sigma Chemi-

i Requests for reprims should be addressed to Brian A. McMillen, Ph.D., Department of Pharmacology, East Carolina University School of Medicine, Greenville, NC 27858--4354. 337

338

JOHNS ET AL.

cal Co., St. Louis, MO) in a distilled water solution. Saline controls received an equal volume of (0.9%) normal saline and the A F A group received 1.5 mg/kg o f amfonelic acid (Sterling-Winthrop Research Laboratories, Rensselaer, NY) in a p H 10 solution. All injections were given subcutaneously from gestational days 1-20 according to the following schedule: Cocaine-D and Saline dams received injections twice daily at approximately 0900 and 1630 hours; Cocaine-I dams received injections twice dally on two consecutive days at 5-day intervals (on days 2, 3, 8, 9, 14, 15, 19, and 20, the last interval shortened to occur before parturition); A F A dams received injections daily at 0900 hours. The once daily dosing with A F A produced about the same duration of hyperactivity as the twice daily dosing with cocaine (Henderson & McMillen, unpublished observation). As an inhibitor of rat striatal synaptosomal DA reuptake, A F A is about 80 times more potent than cocaine in vitro and at least 10 times more potent in vivo for stimulation of locomotor activity (16). Surrogates received no treatment. The schedules were chosen to approximate cocaine dosage and regimen used in the human population, which varies widely: 20 m g / k g SC cocaine in the rat produces concentrations greater than that reported for most addicts (23) and human cocaine use often follows sporadic patterns (14). Each Saline dam was matched to a Cocaine-D dam so as to receive the same amount of rat chow on each day of gestation as had been consumed by the Cocaine-D dam. For example, the grams o f chow consumed on gestational day 3 o f the third Cocaine-D dam was the amount of food provided for the third Saline dam on its gestational day 3. Surrogate, Cocaine-I, and A F A dams were fed ad lib. Dams were weighed daily (surrogates were weighed every 3 days) and food consumption was measured for Cocaine-D and Saline treated dams prior to morning injections. Dally weight gain, food consumption, and any unusual observations were recorded. Enough dams were assigned to each treatment group to insure 14 to 16 viable treatment litters and 45 dams were assigned as surrogates so that two surrogate dams were available for every three treatment dams. Following delivery, each litter was weighed, sexed, and examined for deformities and stillbirths. As soon as possible treatment litters were culled to approximately 8-10 male pups. If fewer than 8 male pups were born to a dam they were marked and placed with another small litter o f male pups from the same treatment group born on the same day and all pups were given to a surrogate who had given birth usually within 24 hours of the treatment dams. Though, in some cases, the rearing litter consisted o f pups from two same treatment birth litters, pups were marked with a Devon skin marker and litter data were always collected separately for

each of the two birth litters so that the data were not combined. Values in tables refer to the number of birth litters. Surrogate born pups were removed, weighed, and euthanized. Daily assessments were made of litter weight (until day 16), postnatal death, or abnormalities and developmental indices (fighting reflex for 100% of each litter, righting reflex for 75% o f the litter, eye opening for 100% of the litter). Righting reflex was achieved when each pup in the entire litter had righted themselves on two consecutive days within 6 s. Because there were several outliers, 75% righting reflex was assessed as the day when 75% or more of the litter had righted themselves within the 6-s interval. Eye opening was recorded as the day when all pups in a litter had opened both eyes entirely. Pups were weaned on postnatal day (PND) 21 and randomly separated into groups of three from the same litter to be tested on several behavioral measures. Only one pup per litter was tested on any one task at any age. Spontaneous locomotor activity described in this article, was measured at 30 and 60 days postnatal. To measure activity, one offspring was placed in a clean cage identical to the home cages located atop each activity meter just prior to the session. During activity monitoring, water was available ad lib. Antomex 2S activity meters (Columbus Instruments, Columbus, OH) were used to monitor activity for 6 hours between 1600 and 2200 hours: the first 3 h with lights on and the last 3 h in darkness. The animals were compared on activity during the first 15 rain, the first 3 h, the second 3 h, and for the total 6-h period. This allowed comparisons across groups of the rats' exploratory phase, light phase, and nocturnal phases of activity. Data were evaluated by independent t-tests, analyses of variance (ANOVA) and, when indicated, post-hoe Tukey HSD tests were used to assess significant differences between groups (25). Statistical significance was assumed at the probability level o f 0.05 or less (two-tailed). RESULTS

Maternal Variables There was no maternal mortality in any of the groups. Data in Table 1 indicate that no differences occurred among groups on gestation length. A correlated t test showed that pair fed Saline controls ate significantly less than Cocaine-D dams (tn = 3.77, p < 0.01). Thus, the Saline dams did not eat all the food that they were given. Becau~ the Saline dams were the only dams restricted on the amount of food they could eat and they did not even consume all the food that they were given, all groups essentially were eating ad lib. An A N O V A revealed a significant difference in gestational weight

TABLE 1 EFFECTS OF COCAINE OR AFA ON MATERNAL VARIABLES(MEAN + SEM) Group Variable(Numberof litters)

Surrogate (45)

Gestation(days) 20.9 + 0.2 Food consumption (total g) nm* Weight gain (g/dam) 159t ± 5

Saline (17) 21.3 + 0.1 406.6 + 1 0 . 1 109 + 4

*nm = not measured. tDifferent from both Cocaine-D and Saline control, p < 0.05.

Cocaine-D (17) 21.1 + 0.1 463.0t + 16.3 119 + 9

Cocaine-I (18) 21.2 + 0.2 nm 156" ± 9

AFA (15) 21.7 + 0.3 nm 142" + 7

PRENATAL COCAINE AND DEVELOPMENT: I

339

TABLE 2 EFFECTS OF COCAINE OR AFA ON LITI'ER VARIABLES(MEAN + SEM) Group Surrogate (45)

Variable(numberof litters) Litter size (pups/fitter) Maies/females (%) Litter weight~ (total g) Grams/pup(g) Still births (number/fitter)

14.7 + 0.4 68 97.61" + 2.7 6.5 + 0.1 .24

Saline (14) 12.9 -e 0.6 47 78.8 + 3.2 6.2 :l: 0.1 .36

Cocaine-D (15)

Cocaine-I (18)

13.4 ± 0.7 52 81.3 + 4.8 6.1 + 0.1 .38

16.0" ± 0.5 44 103.5t + 3.3 6.4 + 0.1 .38

AFA (14) 14.6 + 0.8 55 95.3 ± 4.2 6.7* :t: 0.3 .64

*Different from saline control, p < 0.05. tDifferent from saline control and Cocaine-D, p < 0.05. ~Litter weight was determined after culling and transferring to surrogate dams.

gain among the five groups, F(4, 101) = 10.70, p < 0.0001. Post-hoc Tukey tests showed that dams from the surrogate, Cocaine-I, and AFA groups all gained more weight than either the Cocaine-D or Saline dams.

Litter Variables Table 2 shows fitter variables that differed significantly across groups. There were significant differences in fitter size, F(4, 104) = 3.32, p < 0.01; fitter birth weight, F(4, 103) =' 7.39, p < 0.0001; and mean pup weight, F(4, 98) = 2.57, p < 0.04. Post-hoc Tukey tests showed (a) that Cocalne-I dams had larger litter sizes than Saline; Co) that Cocaine-I, AFA, and surrogate fitters weighed more at birth than Saline and Cocaine-D pups; (c) the AFA exposed pups weighed more than the Saline pups. No significant differences were found on the proportion of males or number of stillbirths per fitter.

Offspring Developmental Variables Data presented in Table 3 show no significant differences among groups on the developmental variables of eye opening, F(3, 52) = 2.37, or 100% fighting reflex, or 15-day weight gain. There was a delay of almost one day (but not quite significant, Tukey HSD, p < 0.055) in the 75% righting reflex measure in Cocaine-D offspring as compared to all other groups.

Spontaneous Activity Table 4 presents PND 30 activity data. ANOVA's showed significant group differences on activity during the first 15

min. F(3, 31) = 3.19,p < 0.03, and during the dark period, F(3, 31) = 3.70,p < 0.02. Post-hoc Tukey tests revealed that Cocaine-D pups were less active than Saline control pups during the first 15-rain epoch and Cocaine-I pups were less active than Saline pups during the dark period of the daily cycle. The overall activity of the Cocaine-I pups appeared less than Saline pups but the difference was not significant, F(3, 56) = 2.34. At 60 days of age, another pup from each fitter was taken for activity measurement (Table 5). An ANOVA showed group differences on the dark phase, F(3, 34) = 3.02, p < 0.04. Post-hoc tests showed that AFA pups were less active than Saline pups during that time period. DISCUSSION The results reveal a subtle pattern of detrimental effects on the pups of dams receiving twice daily or intermittent injections of 15 mg/kg cocaine throughout pregnancy. First, the Cocalne-D pups showed a possible delay in the development of the fighting reflex as evidenced by elevated 75% righting reflex score which just missed significance (p < 0.055). Presumably, the 100% righting reflex measure failed to show a significant effect because nearly all fitters have an outlier or two that increased variance and mask when the typical pup is capable of fighting itself. A previous report from this laboratory on a different group of rats exposed to this same dose of cocaine, also had more than a 2-day delay in gaining the righting reflex (12). Several previous studies have reported developmental delays in physical and behavioral maturation of cocaine exposed rat pups (8,9,12,15,22,24). During this period

TABLE 3 EFFECTS OF COCAINE OR AFA ON RAT OFFSPRINGDEVELOPMENT (MEAN + SEM) Croup Variable(Numberof fitters) 75% fighting ref. (days) 100e/e righting ref. (days) Eye opening (days) Weight gain 15 days (total g/pup)

Saline (13) 3.9 7.2 14.3 27.8

+ + + +

0.4 0.7 0.2 0.1

Cocaine-D (15) 4.8 6.9 14.8 31.4

+ + + +

0.4 0.7 0.3 0.2

Cocaine-I (15) 3.6 5.4 14.1 32.6

+ ± ± ±

0.2 0.5 0.2 0.2

AFA (13) 3.8 + 7.5 ± 14.2 + 31.7 +

0.4 0.6 0.2 0.1

RiOting reflex indicates the number of days until 75% or 100% of the fitter could each right themselves within 6 s.

340

JOHNS ET AL. TABLE 4 EFFECTS OF COCAINE OR AFA ON 30-DAY-OLDRAT OFFSPRINGACTIVITY* (MEAN + SEM) Group Saline (14)

Activity(Numberof litters) First 15rain Remaining light phase Dark phase Total activity

621 731 4004 5356

+ + + ±

Cocaine-D (16)

51 123 500 507

331t 926 3464 4721

+ ± + ±

Cocaine-I (16)

72 135 669 625

411 844 2216t 3470

+ + + ±

117 159 217 308

AFA (14) 639 + 1388 + 2286 + 4314 ±

89 956 395 584

*Rats were placed in cages on activity meters and motion recorded in 15 rain epochs for 6 hours, 3 hours with the lights on and 3 hours with the lights off. tDifferent from saline control, p < 0.05.

of time there were no differences in weight gain by the pups from different groups. In other reports cocaine exposed pups were able to catch up in postnatal weight gain (9,15), so the significance of this result is not clear. Second, the data suggest that dally fetal cocaine exposure results in neophobia. When Cocaine-D pups at PND 30 were tested for activity over a 6-h period with the first 3 h lighted and last 3 h dark, the Cocaine-D pups were significantly less active than Controls during the first 15 min but normal during the remainder of the fight period and during the dark period. In other words, whereas the Cocaine-D animals were normally active, they were hesitant to begin exploring a novel activity chamber. Others have reported hypoactivity during a 15 rain recording period in pups exposed to 30 mg/kg b.i.d, cocaine in utero during gestationai days 7-20 (7). Littermates of these PND 30 Cocaine-D pups showed normal activity during the first 15 rain and both the fight and dark phases of the same activity test given at PND 60. The present results could have been even more pronounced had the activity test chamber not been so similar to the home cage. For example, PND 60 and 180 Cocaine-D rats refused to leave a home cage and enter a novel cage (16). This suggests that the neophobia may become less pronounced with age. Church and Overbeck (8) also found hypoactivity in PND 20 rat pups prenatally exposed to higher doses of cocaine than those in the present study and for one group (100 mg/kg/day) the hypoactivity persisted until PND 49. The hypoactivity was not present at PND 80-90, which is consistent with activity data we have taken at PND

120 (16). There may not be a clear dose dependent trend in activity changes at younger ages as suggested (8), but there does seem to be an age dependent change in activity behavior patterns as found in this and several other studies which have reported hypoactivity (7,8,24), hyperactivity (13,15), or no changes (22). Whether a cocaine-exposed subject shows neophobic behavior is probably a function of both age and the relative novelty of the environment. Data from other work in our laboratory involving more novel environments suggests that older cocaine-exposed subjects are hesitant to enter novel environments (16). A novel environment may have the same basic effect as that seen in an experiment by Spear and co-workers (24) who observed that locomotor action was decreased when precipitated by footshock. Although dose, age, and method of testing all probably affect the observed result, our results suggest that the relative novelty of the test environment and duration of test are also important. An additional difficulty for comparing data from different laboratories is the duration of activity measurement. Measurements made over 15 min or less after placement in the activity chamber measure exploration of the new environment and there may be differences between initial exploration and overall spontaneous activity. Third, it is difficult to relate activity changes to brain dopamine receptor changes. Prenatal cocaine has not been reported to change striatal dopamine receptor binding (13) or dopamine metabolism (17) at these juvenile and adolescent ages. A small

TABLE 5 EFFECTS OF COCAINE OR AFA ON 60-DAY-OLDRAT OFFSPRINGACTVITY* (MEAN + SEM) Group Activity(Numberof litters) First 15rain Remaining light phase Dark phase Total activity

Saline (14) 780 + 860 + 5832 + 7472 +

94 209 394 554

Cocaine-D (16) 786 + 1132+ 5055 + 6971 +

109 211 633 746

Cocaine-I (16)

AFA (14)

571 + 144 424 + 1561 + 518 1521 + 4019 + 998 2833t + 6151 + 1448 4779 +

81 260 848 866

*Rats were placed in cages on activity meters and motion recorded in 15-rainepochs for 6 hours: 3 hours with the lights on and 3 hours with the lights off. tDifferent from saline control, p < 0.05.

P R E N A T A L COCAINE AND DEVELOPMENT: I

341

but significant increase in affinity of D 2 dopamine receptors for spiperone was reported for PND 21 cocaine exposed rat offspring (20). Fourth, the cocaine-induced effects may not be due to the dopamine reuptake blocking effects o f cocaine as the A F A pups showed none of the same effects. The differences in potency and duration of action could only be partially accounted for by the once dally dosing with A F A and prevent making a strong conclusion regarding the relative importance of the stimulant properties of cocaine versus its other pharmacological effects. Prenatal treatment with A F A seemed to have tittle deleterious effect on the maternal, litter, or developmental variables measured. This may reflect a lack of direct or indirect noradrenergic activity in contrast to cocaine (1 l). A F A was selected because like cocaine it blocks dopamine uptake and indirectly causes dopamine release from storage pools (21) but A F A is without significant effects on norepinephrine reuptake ( l l ) . Thus, if results found in cocaine and A F A offspring were similar, a similar causal mechanism might be proposed: increased dopamine release and CNS stimulation in either the fetus or the mother. This was not the case in our study, although A F A offspring were hypoactive as compared to controls during the dark cycle when tested at PND 60. Perhaps cocaine's effects on noradrenergic a n d / o r serotonergic systems or its vasoconstrictive effects with regard to uterine, umbilical, and placental blood flow and the related fetal hypoxia may account for the observed cocaine-induced effects. Of course, it may be that the above described cocaineinduced changes were the result of an interaction o f dopamine reuptake blocking and nondopamine synaptic effects of cocaine. Sporadic treatment with cocaine produced very little effect on either the dams or the offspring. On most gestational measures the Cocaine-I dams resembled the surrogate dams more than the dams o f any other group. If sporadic cocaine exposure had an effect on gestation, it was to increase the size and weight o f the litters. The only significant change noted in the Cocalne-I pups was that they were significantly less active during the dark phase o f the activity test when tested at 30 days of age. Other Cocaine-I offspring tested at 60 days still tended to be less active than controls but the difference was no longer significant. The Cocaine-D and Cocaine-I groups were very similar to Saline controls with respect to number o f stillbirths, males per litter, and deformities (none). Thus, 15 mg/kg b.i.d, cocaine failed to produce demonstrable effects on these parameters.

Though no deformities were seen in any of the groups, two dams from each cocaine group killed their titters before they could be assessed for abnormalities. The reason for the litter killings was not determined though pup abnormality or stillbirths could have been the cause. Higher doses than the dose used in this study produce more profound effects such as stillbirths, deformities, as well as maternal mortality (9,10). The Cocaine-I and A F A animals resembled one another on many measures, but this seems to be the result of neither treatment producing a detectable effect rather than due to a common underlying synaptic mechanism. Twice daily injections, of either cocaine or saline throughout pregnancy appears to have affected normal gestation independent of any cocaine effects. On several of the important gestational measures, the Cocaine-D and Saline dams, the two groups receiving twice daily injections during gestation, had scores which suggested that their gestation was impaired relative to the other groups. Specifically, the Cocaine-D and Saline groups had decreased gestational weight gain and delivered lighter litters with fewer pups than did dams receiving sporadic cocaine injections (Cocaine-I dams), once daily A F A injections or no injections (surrogate dams). Because the Saline and Cocaine-D dams received their food in ceramic bowls and the other dams in a regular hopper, it is possible that this difference may have affected food intake, but feeding in the ceramic bowl should have allowed easier access to food. In retrospect, it would have been better to have measured food consumption for all groups but this was not feasible at the time. Behavioral and neurochemical measures are now being assessed in our laboratories and as noted will be reported in subsequent articles. Conflicting reports in both clinical and animal research literature reflect the need for further assessment of risks to a fetus prenatally exposed to cocaine. Prenatal cocaine exposure chronically or on a "weekend user" schedule produce alterations in gestation and rat offspring. Differential effects from cocaine alone and when used in conjunction with other drugs of abuse should be determined to best prescribe future methods of treatment for problems caused by drug abuse in pregnant women. ACKNOWLEDGEMENT We thank Helen Williams, Deborah Anderson, and Laura Zimmerman for their technical assistance. This work was supported by USPHS grant DA 04895 awarded to B. A. McMillen.

REFERENCES 1. Abelson, H. I.; Miller,J. D. A decade of trends in cocaine use in the household population. In: Kozel, J.; Adams, E. H., eds. Cocaine use in America: Epidemiologic and clinical perspectives; NIDA Monograph 61. Rockville, MD: U.S. Dept. Health and Human Services; 1985: 35-49. 2. Bingol, N.; Fuchs, M.; Diaz, V.; Stone, R. K.; Gromisch, D. S. Teratogenicity of cocaine in humans. J. Pediatr. 110:93-96; 1987. 3. Byck, R.; Van Dyke, C. What are the effects of cocaine in man? In: Peterson, R. C.; Stillman, R. C., eds. Cocaine: 1977, NIDA Monograph 13. Washington, DC: DHEW; 1977: 97-118. 4. Chasnoff, I. J.; Bums, W. J.; Schnoll, S. H.; Bums, K. A. Cocaine use in pregnancy. N. Eng. J. Med. 313:666-669; 1985. 5. Chasnoff, I. J.; Burns, K. A.; Bums, W. J. Cocaine use in pregnancy: Perinatal morbidity and mortality. Neurotoxicol. Teratol. 9:291-293; 1987. 6. Chasnoff, I. J.; Landress, H. J.; Barrett, M. E. The prevalence

7.

8. 9.

10.

of illicit-drug or alcohol use during pregnancy and discrepancies in mandatory reporting in Pinellas County, Florida. N. Eng. J. Med. 322:1202-1206; 1990. Church, M. W.; Holmes, P. A.; Overbeck, G. W.; Tilak, J. P.; Zajac, C. S. Interactive effects of prenatal alcohol and cocaine exposures on postnatal mortality, development and behavior in the Long-Evans rat. Neurotoxicol. Teratol. 13:377-386, 1991. Church, M. W.; Overbeck, G. W. Prenatal cocaine exposure in the Long Evans rat: II. Dose-dependent effects on offspring behavior. Neurotoxicol. Teratol. 12:335-343; 1990. Church, M. W.; Overbeck, G. W.; Andrzejczak, A. L. Prenatal cocaine exposure in the Long Evans rat: I. Dose-dependent effects on gestation, mortality and postnatal maturation. Neurotoxicol. Teratol. 12:327-334; 1990. Fantel, A. C.; MacPhail, B. J. The teratogenicity of cocaine. Teratolngy 26:17-19; 1982.

342 II. Fuller, R. W.; Perry, K. W. Amfonelic acid antagonism of dopamine and norepinephrine depletion by methyl-m-tyrosine in rat brain. Biochem. Pharmacol. 30:2025-2026; 1981. 12. Henderson, M. G.; McMillen, B. A. Effects of prenatal exposure to cocaine or related drugs on rat development and neurological indices. Brain Res. Bull. 24:207-212; 1990. 13. Henderson, M. G.; McConnaughey, M. M.; McMillen, B. A. Long-term consequences of prenatal exposure to cocaine or related drugs: Effects on rat brain monoaminergic receptors. Brain Res. Bull. 26:941-945; 1991. 14. Hume, R. F.; O'Donnell, K. J.; Stanger, C. L.; Killiam, A. P.; C-ingras, J. L. In utero cocaine exposure: Observations of fetal behavioral state may predict neonatal outcome. Am. J. Obstet. Gyneeol. 161:685-690; 1989. 15. Hutchings, D. E.; Fico, T. A.; Dow-Edwards, D. L. Prenatal cocaine: Maternal toxicity, fetal effects and locomotor activity in rat offspring. Neurotoxieol. Teratol. 11:65-69; 1989. 16. Johns, J. M.; Mean, M. J.; Anderson, D. R.; Bass, E. W.; Means, L. W.; McMillen, B. A. Prenatal exposure to cocaine: II. Effects on open field activity and cognitive behavior in SpragueDawley rats. Neurotoxicol. Teratol. 14:343-349; 1992. 17. McMillen, B. A.; Henderson, M. G. Prenatal exposure to cocaine or related drugs causes age related alterations of brain monoamine concentrations in rat offspring. FASEB J. 5:A1561; 1991. 18. McMillen, B. A.; Shore, P. A. Species-dependent responsiveness to nonamphetamine central stimulants: Relationship between uptake and release of brain dopamine. Pharmacol. 19:261-266; 1979.

J O H N S E T AL. 19. Oro, A. S.; Dixon, S. D. Perinatal cocaine and methamphetamine exposure: Maternal and neonatal correlates. J. Pediatr. I l 1:571578; 1987. 20. Scalzo, F. M.; Ali, S. F.; Frambes, N. A.; Spear, L. P. Weanling rats exposed to cocaine exhibit an increase in striatal D2 dopamine binding associate with an increase in ligand affinity. Pharmacol. Biochem. Behav. 37:371-373; 1990. 21. Shore, P. A. Actions of amfonefic acid and other nonamphetamine stimulants on the dopamine neuron. J. Pharm. Pharmacol. 28:855-857; 1976. 22. Smith, R. F.; Mattran, K. M.; Kurkjian, M. F.; Kurtz, S. L. Alterations in offspring behavior induced by chronic prenatal cocaine dosing. Neurotoxicol. Teratol. 11:35-38; 1989. 23. Spear, L. P.; Frambes, N. A.; Kirstein, C. L. Fetal and maternal brain and plasma levels of cocaine and benzoylecgonine following chronic subcutaneous administration of cocaine during gestation in rats. Psychopharmacol. 97:427--431; 1989. 24. Spear, L. P.; Kirstein, C. L.; Bell, J.; Yoottanaumpun, V.; Greenhaum, R.; O'Shed, J.; Hnggman, H.; Spear, N. E. Effects of prenatal cocaine exposure on behavior during the early postnatal period. Neurotoxicol. Teratol. 11:57-63; 1989. 25. Zar, J. H. Biostatistical analysis, Englewood Cliffs, NJ: Prentice Hall; 1984. 26. Zuckerman, B.; Frank, D. A.; Hingson, R.; Amaro, H.; Levenson, S. M.; Kayne, H.; Parker, S.; Vinci, R.; Aboagye, K.; Fried, L. E.; Carbral, H.; Timperi, R.; Bauchner, H. Effects of maternal marijuana and cocaine use on fetal growth. N. Eng. 3. Med. 320:762-768; 1989.