Neurotoxicology and Teratology, Vol. 14, pp. 183-189, 1992
0892-0362/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
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Effects of Prenatal Cocaine on Behavioral Responses to a Cocaine Challenge on Postnatal Day 11 JERROLD
S. M E Y E R , 1 J O H N
D. SHERLOCK
AND
NATHAN
R. M A C D O N A L D
Department o f Psychology, Neuroscience and Behavior Program, University o f Massachusetts, Amherst, M A 01003 R e c e i v e d 19 A u g u s t 1991; A c c e p t e d 13 M a r c h 1992 MEYER, J. S., J. D. SHERLOCK AND N. R. MACDONALD . Effects of prenatal cocaine on behavioral responses to a cocaine challenge on postnatal day 11. NEUROTOXICOL. TERATOL. 14(3) 183-189, 1992.-The purpose of this study was to investigate the effects of prenatal cocaine treatment on behavioral responsivity to a cocaine challenge on postnatal day (PD) 11. Timed-pregnant Sprague-Dawley rats were injected s.c. with 40 mg/kg/day of cocaine (20 mg/kg twice daily) from gestational day l 1 to 20. Saline-control females received saline injections and were pair-fed to the cocainetreated females, whereas untreated-control females were undisturbed and were fed ad lib. Litters were culled to eight pups on PD1 and fostered to normal lactating dams. On PD 11, subjects were given either saline or cocaine (1.25, 2.5, or 5.0 mg/kg s.c.) and then tested 15 min later for isolation-induced ultrasonic vocalizations and other behaviors. Prenatal cocaine-treated pups showed a reduced sensitivity to the stimulating effect of postnatal cocaine on wall climbing, which may reflect an underlying alteration in central dopaminergic and/or noradrenergic systems. Most of the other behaviors studied, including ultrasonic vocalizations, were unaffected by prenatal cocaine administration. However, one other notable finding was an increase in postnfital mortality among the cocaine-exposed pups. We hypothesize that prenatal cocaine treatment may alter pup behavior so as to produce abnormal maternal-offspring interactions and impaired development in some individuals. Prenatal cocaine
Development
Ultrasonic vocalization
B E C A U S E o f current concerns related to cocaine use by pregnant women, several laboratories have developed rodent models to examine the possible neurobehavioral teratogenicity of this compound. Indeed, rats exposed to cocaine in utero exhibit a variety of biochemical and functional changes, including alterations in dopamine receptor binding (9,23), regional glucose utilization (9), sexual differentiation (21), brainstem auditory-evoked potentials (5), l o c o m o t o r activity (4,14,16, 25), and various learning tasks (15,25,28). In ascertaining the behavioral effects of prenatal cocaine exposure, one useful approach is to determine whether later responsiveness to cocaine itself or to other psychomotor stimulants such as amphetamine has been altered. For example, prenatal cocaine has been reported to reduce the locomotor stimulating effects o f both cocaine and amphetamine at postnatal day (PD) 15 (26), although this effect may be transient as it was not observed at later time points (12,26). The use o f psychomotor stimulants in the behavioral evaluation o f cocaine-exposed subjects is valuable partly because the activational properties o f these drugs have been traced primarily to their dopamine releasing and uptake blocking effects in the
Rat
Wall climbing
nucleus accumbens and corpus striatum (11). If behavioral changes are found in gestationally treated subjects, follow-up neurochemical studies can be targeted to specific neurochemical pathways. Moreover, the possibility that in utero cocaine exposure leads to either a sensitization or tolerance to subsequent drug administration has important implications for the offspring o f cocaine-abusing women. A sensitization effect, for example, would imply that repeated exposure during the early stages of pregnancy might produce increased responsiveness to an equivalent cocaine dose experienced later in development, thereby increasing the cumulative damage experienced by the fetus. The present study investigated the influence of prenatal cocaine treatment on subsequent behavioral responsivity to a cocaine challenge on P D 11. One focus o f the study was isolation-induced ultrasonic vocalizations, which are potently suppressed by acute cocaine administration (19) and which are influenced by a wide variety of other drugs as well (2,3,7, 8,13,17,18,20,29). We also looked for changes in the behavior patterns exhibited during vocalization testing, such as wall climbing, locomotion, digging, and grooming.
To whom requests for reprints should be addressed. 183
184
MEYER, SHERLOCK AND MACDONALD METHOD
TABLE 1 PUP INJECTION AND TEST SCHEDULE FOR EACH LITTER
Animals Timed breedings were carried out with nulliparous female and stud male rats approximately 3-months-old. The animals were obtained from our own colony, which is descended from Charles River Sprague-Dawley (CD) stock. They were given Purina Rodent Chow and water ad lib, and maintained under a 14L : 10D cycle (lights on at 6:00 a.m.) at a temperature of approximately 22°C. The day that a sperm plug was detected was designated gestational day 1 (GD 1).
Treatment After successful breeding, females were switched to powdered chow in commercially available food cups to avoid spillage and thereby facilitate pair-feeding. The pregnant dams were randomly divided into three treatment groups: cocainetreated (C), saline-treated controls pair-fed to the cocaine dams (SC), and untreated controls (UC). From GD 11 to GD 20, the C group was injected s.c. with 40 mg/kg/day of cocaine Hcl (20 mg/kg twice daily) in a volume of 3.0 ml/kg of saline. Injections were performed between 9 a.m. and 10 a.m. and between 6 p.m. and 7 p.m. and were moved around the back to avoid excessive necrotic reactions. The SC group was treated in the same manner except that it received injections only of physiological saline. UC females were fed powdered chow ad lib and were not handled prior to parturition, although their food intake was monitored daily. On the day after birth (PD 1), litters were sexed and then 8 pups (sex-balanced if possible) were fostered to untreated lactating dams. Foster mothers were maintained in standard plastic tubs with a bedding of pine shavings, and were given tap water and Purina Formulab Chow to maximize lactation.
Behavioral Testing On PD 1l, each male and female within a litter was randomly assigned to 1 of 4 postnatal treatment groups: Saline, 1.25 mg/kg cocaine, 2.5 mg/kg cocaine, and 5.0 mg/kg cocaine injected s.c. in a volume of 0.5 ml/100 g body weight. The four postnatal treatments were tested in a semirandomized order across litters to control for potential order effects. Consequently, within the 6 or 7 litters that were studied for each prenatal condition (cocaine-treated, saline-treated, and untreated-control), each of the 8 postnatal dose x gender combinations was run no more than once in a particular testing slot (first through eighth; see Table 1). Pups were tested individually in a different room than that in which the animals were housed. Testing began at approximately 1 p.m. and either one or (occasionally) two litters were tested on a given day. At the times shown in Table 1, pups were gently removed from the litter, weighed and injected, and then returned to the litter for 15 rain. Each subject was then removed once again and placed in a glass mason jar 16 cm high x 10 cm diameter containing wood chips to a depth of 3.5 cm. During a 5-min test session, ultrasonic vocalizations were detected by a bat detector (Ultra Sound Advice, Model S-25) and tape-recorded for later playback and scoring. At the same time, the frequency and duration of other behaviors were recorded on a portable microcomputer. Based on preliminary tests of untreated PD 11 pups, we devised an exhaustive and mutually exclusive scoring system containing the following six behavioral categories: wall climbing (both forepaws up on the side of the jar, usually accompanied by treading movements), locomotion (movement around the jar), digging
Pup Number
InjectionTime (min)
1
0
2 3 4 5 6 7 8
10 35 45 70 80 105 115
TestingTime (min) 15 25 50 60 85 95 120 130
A stopwatch was started at time 0 when the first pup was injected. Subsequent injections and testing were then carried out at the indicated time points.
(displacement of the woodchip substrate by the forepaws), grooming (face-washing), stationary with head in a low position, and stationary with head up (with or without sniffing). At the conclusion of testing, each pup was removed to a container kept in a location separate from the colony room so as to avoid disturbing the remainder of the litter. Bedding in the mason jar was changed between subjects.
Statistical Analysis Ultrasonic vocalizations (number per 5 min) and other behaviors (duration per 5 min) were initially subjected to 3-way analyses of variance (ANOVA) with prenatal condition, postnatal treatment, and gender as factors. Significant F values were followed up with Tukey tests to determine individual group differences. Other data were analyzed as indicated next. In all cases, a probability of < 0.05 was considered statistically significant. RESULTS
Maternal and Offspring Characteristics Over the period of GD 11 to GD 20, daily food consumption values were 21.1 _+ 0.6, 20.9 + 0.6, and 23.2 ___ 0.9 g (mean _+ SEM), respectively for the cocaine-treated, salinetreated, and untreated control females. The difference between the cocaine and untreated control group was marginally significant by Student's t test (t = 2.13, df = 11, p = 0.057). Because of the pair-feeding protocol, data from the salinetreated group were not independent of those from the cocaine-treated subjects and were not included in the statistical analysis. The cocaine group was also compared to the saline group with respect to percentage body weight gain over the period of injection (because the untreated controls were not handled during this time, weight data were not available for that group). The cocaine-treated females showed a significantly higher percentage body weight gain than their salinetreated counterparts (26.3 + 1.7°70 vs. 20.7 _+ 1.1070, t = 2.80, d f = 12, p < 0.02), despite the virtually identical food intake in the two groups. This may have been related at least partially to a modest, statistically nonsignificant difference between groups in mean body weight observed at the beginning of the measurement period (cocaine-treated: 287.7 +_ 14.3 g; saline-treated: 310.3 _+ 11.6 g; t = 1.23, d f = 12, ns).
PRENATAL COCAINE AND LATER BEHAVIOR
185
The initial groups consisted of 8 cocaine-treated, 7 salinecontrol, and 6 untreated control females. On PD 1 when litters were culled and fostered, all pups appeared healthy and to be of normal size (body weights were not recorded at this time). However, a total of 12 pups from the cocaine group died prior to PD 11, as these subjects were not found in the tub at the time of testing. Five pups were missing in one litter alone, as a result of which the remainder of the litter and its mother were also eliminated from the study. Three other cocainetreated litters out of the original 8 also had at least one mortality. One pup was missing in one of the saline-treated litters, whereas no deaths were recorded among the untreated controis. A chi-square test was performed to analyze the distribution of viable and dead subjects at PD 11. The results confirmed a nonrandom distribution of pup mortality across the three treatment groups (X2 = 17.68, d f = 2, p < 0.001). Also, note that as a consequence of severe runting, two other pups were eliminated from the same saline-treated litter that had the missing subject. The final subject complement along with postnatal treatment assignment was as follows: 49 cocaine-treated pups (6 males and 6 females given saline, 7 males and 6 females given 1.25 mg/kg cocaine, 7 males and 6 females given 2.5 mg/kg cocaine, and 5 males and 6 females given 5.0 mg/kg cocaine); 53 saline-treated controls (7 males and 6 females at each dose except for 5.0 mg/kg cocaine, which was given to 7 males and 7 females); and 48 untreated controls (6 males and 6 females at each dose). No more than 1 male and 1 female from a given litter received the same postnatal cocaine dose. At the time of testing on PD 11, males were heavier overall than females, F(1, 144) = 4.04, p < 0.05, however body weights did not differ as a function of prenatal drug exposure (males = 29.0
350 300 O
, m
250 N O
[] I~ F~ •
Saline 1.25 Cocaine 2.5 cocaine 5.0 cocaine
+ 0.6, 28.5 _+ 0.6, and 29.8 _+ 0.8 g; females = 27.9 + 0.5, 28.3 + 0.5, and 28.2 + 0.6 g for the cocaine-treated, salinecontrol, and untreated control groups respectively).
Behavioral Results As shown in Fig. 1, cocaine administered 15 min prior to behavioral testing suppressed the rate of ultrasonic vocalization in a potent and dose-dependent manner, F(3, 126) = 92.59; p < 0.001, for the postnatal treatment main effect. However, there was no shift in the dose-response curve as a function of prenatal condition, indicating that prior exposure to cocaine failed to alter the sensitivity of pups to subsequent cocaine-induced inhibition of vocalization. Postnatal administration of cocaine enhanced the amount of time pups spent wall climbing (Fig. 2). More important, the sensitivity of this response was influenced by prenatal condition, F(6, 126) = 2.62, p < 0.02, for the prenatal condition x postnatal treatment interaction. Individual mean comparisons indicated that for both control groups, all postnatal cocaine doses produced significantly more wall climbing than saline, with no difference between doses. In contrast, pups exposed to cocaine prenatally only showed a significant increase in wall climbing when injected with the 5.0 mg/kg postnatal dose. There was also a (nonsignificant) tendency for the baseline level of wall climbing to be elevated in the prenatal cocaine group. As in the case of ultrasonic vocalizations, several behaviors were influenced by the postnatal cocaine challenge but not by prenatal treatment (Fig. 3). For example, the duration of digging tended to increase following acute cocaine administration, although this change was not statistically significant due
I
L
I
200
O U
150
, m
O
100 &--
-x.
*
50 T
0 Untreated
Saline
T
Cocaine
FIG. 1. Mean ( _+SEM) number of ultrasonic vocalizations per 5 min as a function of prenatal condition (untreated, saline, and 40 mg/kg cocaine) and postnatal treatment (saline or cocaine at the indicated dose). As there was no significant gender difference, the results were pooled across gender far presentation purposes. *significantly different (at least p < 0.05) from the respective postnatal saline control group.
186
MEYER, SHERLOCK AND MACDONALD
250 225 200 0
[ ] Saline [ ] 1.25 Cocaine [ ] 2.5 Cocaine
•
-X-~
5.0 Cocaine
175
tD
150 °
tO
I
/ /
125
/ /
~
/ / / /
100
/
/
/ /
a
/
75
/
/ / / /
50
/ /
/
/
/
25
/
/
/
/ / /
/
/ /
0
Untreated
Saline
Cocaine
FIG. 2. Mean (+_SEM) duration of wall climbing as a function of prenatal condition and postnatal treatment. *significantly different from the respective postnatal saline control group; **significantly different from all other postnatal treatment groups within the prenatal cocaine condition.
to a high degree of variability. In contrast, grooming, F(3, 126) = 6.53, p < 0.001, stationary/head-low, F(3, 126) = 39.75, p < 0.001, and stationary/head-up behaviors, F(3, 126) = 14.09, p < 0.001, all showed significant dosedependent decreases in duration. Finally, locomotion exhibited a complex interaction between prenatal condition, postnatal treatment, and gender interaction, F(6, 126) = 2.22, p < 0.05, as shown in Fig. 4. Post-hoc Tukey tests comparing the various postnatal treatment groups within each gender and prenatal condition showed that for the untreated male condition, significant increases in locomotion occurred in the 2.5 and 5.0 mg/kg postnatal treatment groups compared to the corresponding saline-treated controls. The only difference among the females occurred in the prenatally cocaine-exposed condition, where the 5.0 mg/kg postnatal dose was associated with less locomotion than the 1.25 mg/kg dose. DISCUSSION One of the major goals of this study was to determine whether prenatal cocaine administration influenced isolationinduced ultrasonic vocalizations in PD 11 rat pups. Confirming previous work (19), acute cocaine treatment prior to testing markedly suppressed this behavior. However, neither the baseline vocalization rate nor the degree of cocaine-induced inhibition were affected by prior in utero cocaine exposure. Based on preliminary studies with untreated pups, we had anticipated a more graded range of vocalization suppression to the doses administered on the test day. Nevertheless, even the lowest cocaine dose of 1.25 mg/kg produced a nearmaximum response on this measure. A lower dose range might have permitted the detection of altered vocalization rates in the prenatal cocaine-treated group. Prenatal treatment vari-
ables such as dose, period of exposure, and mode of administration are undoubtedly also important in determining developmental outcome following gestational cocaine exposure. In contrast to the ultrasonic vocalization results, the doseresponse curve for cocaine stimulation of wall climbing behavior was shifted to the right in the animals exposed to cocaine in utero. This decreased responsiveness could be considered a kind of "tolerance", particularly in view of the fact that the baseline wall climbing rate was not significantly affected by prenatal treatment. Interestingly, Spear and her colleagues (28) observed a similar reduction in shock-elicited wall climbing in PD 12 subjects given cocaine prenatally. Although the neurochemical changes underlying these effects are not yet known, it is possible that dopaminergic and/or noradrenergic systems may be involved. Studies have shown that wall climbing in pups can be activated by the dopaminergic agonist apomorphine (24), the c~-noradrenergic agonist clonidine (22), and the general catecholaminergic agonists amphetamine and cocaine (1, this study). Conversely, wall climbing is inhibited by the dopaminergic antagonist haloperidol (1), the c~-adrenergic blocker phentolamine (1), and the catecholamine neurotoxin 6-hydroxydopamine (10). Because the animals in the present study received the same drug both pre- and postnatally, we cannot rule out the possibility of altered wall climbing due to changes in cocaine pharmacokinetics. However, this seems unlikely due to the specificity of the behavioral effect. If cocaine availability during the postnatal challenge test was reduced in the prenatally-exposed subjects, one might expect that ultrasonic vocalizations or other behaviors would likewise have shown evidence of such drug disposition tolerance. A variety of group differences were also noted for the other behavioral categories examined in this study. For example,
PRENATAL COCAINE AND LATER BEHAVIOR
187
3O
35
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Dig 25
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20
0
15
Groom
Saline [ ] 1.25 Cocaine [ ] 2.5 Cocaine • 5,0 Cocaine
30
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(3
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80 Stationary/Head-Low 70 t,J
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60
30
50
t--
o
0
40
20
30
(:3
I
40
20
10
10
i
0
il
0
FIG. 3. Mean (+ SEM) duration of digging, grooming, stationary/head-low, and stationary/head-up as a function of postnatal treatment. *significantlydifferent from the postnatal saline control group; #significantly different from the postnatal 1.25 mg/kg cocaine group.
a complex interaction between prenatal condition, postnatal treatment, and gender was found for locomotor behavior. Untreated control males but not females exhibited increased locomotion when given 2.5 or 5.0 mg/kg cocaine prior to testing. In addition, both male and female subjects in the
25O 225
t Male
2oo
prenatal cocaine group seemed to show an inverted U-shaped response to increasing postnatal cocaine doses, although this was not validated statistically for the males and only partially in the case of the females. What these findings mean, and even whether they are reliable, is not clear at the present time.
[ ] Saline [ ] 1.25 Cocaine
250 225
[ ] 2.5 Cocaine
200
Female
175 150
~
J_
125 100
,~
75
125 ~
100
,'~
75
r
~
50 25
25
0
0
Untreated
Saline
Cocaine
Untreated
Saline
Cocaine
FIG. 4. Mean (+ SEM) duration of locomotion as a function of prenatal condition, postnatal treatment, and gender. *significantly different from the postnatal saline control group within the prenatal untreated condition; **significantly different from the postnatal 1.25 mg/kg cocaine group within the prenatal cocaine condition.
188
MEYER, SHERLOCK AND MACDONALD
In contrast, g r o o m i n g , s t a t i o n a r y / h e a d - l o w , a n d s t a t i o n a r y / h e a d - u p were u n a f f e c t e d b y p r e n a t a l c o n d i t i o n or subject gender, b u t did show significant reductions as a f u n c t i o n o f postn a t a l cocaine t r e a t m e n t . Earlier studies by Spear a n d Brick (27) h a d f o u n d t h a t a single injection o f cocaine o n P D 14 increased l o c o m o t o r b e h a v i o r b u t h a d n o effect o n wall climbing. The differences b e t w e e n their findings a n d those o f the present study are p r o b a b l y related m a i n l y to the a p p a r a t u s used in each case. Spear a n d Brick o b s e r v e d their subjects in a relatively large open-field, whereas o u r a n i m a l s were tested in a small j a r because o f the need to confine t h e m spatially for vocalization m o n i t o r i n g . It is likely t h a t the enclosed space facilitated wall climbing b e h a v i o r at the expense o f locomotion. Finally, some m e n t i o n s h o u l d be m a d e o f the m o r t a l i t y data. C h u r c h et al. (6) previously f o u n d h e i g h t e n e d p o s t n a t a l m o r t a l i t y in rats prenatally exposed to daily cocaine doses o f 80 or 100 m g / k g . T o the best o f o u r knowledge, however, this is the first r e p o r t o f a similar effect at a m o r e m o d e r a t e d r u g dose. As the cocaine-treated pups a p p e a r e d healthy at the time o f litter culling, there was n o discernible evidence for drug-
related intrauterine g r o w t h r e t a r d a t i o n or congenital defects t h a t m i g h t have led to subject loss. M o r e o v e r , the fact t h a t all pups were fostered to n o r m a l d a m s implies t h a t s u b s e q u e n t fatalities c a n n o t be a t t r i b u t e d to a d i s r u p t i o n of m a t e r n a l beh a v i o r secondary to cocaine withdrawal. W e instead h y p o t h e size t h a t p r e n a t a l cocaine t r e a t m e n t m a y have altered the beh a v i o r o f some subjects so as to p r o d u c e a failure to thrive. This could have been a direct result o f p o o r feeding by the pups, or alternatively it is conceivable t h a t a b e r r a n t p u p beh a v i o r led to deficits in m a t e r n a l responsivity. Direct studies o f the b e h a v i o r a l interactions between d a m s a n d cocaineexposed pups are needed to test this hypothesis. ACKNOWLEDGEMENTS We thank Dr. Priscilla Kehoe for her advice in setting up the ultrasonic vocalization testing. The BASIC program used for behavioral event recording was written by J. H. Preston of the U.S. Fish and Wildlife Service. This research was supported by NIDA grant DA-06495 and by a Faculty Research Grant from the University of Massachusetts.
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14. Henderson, M. G.; McMillen, B. A. Effects of prenatal exposure to cocaine or related drugs on rat developmental and neurological indices. Brain Res. Bull. 24:207-212. 15. Heyser, C. J.; Chen, W.-J.; Miller, J.; Spear, N. E.; Spear, L. P. Prenatal cocaine exposure induces deficits in Pavlovian conditioning and sensory preconditioning among infant rat pups. Behay. Neurosci. 104:955-963; 1990. 16. Hutchings, D. E.; Fico, T. A.; Dow-Edwards, D. L. Prenatal cocaine: maternal toxicity, fetal effects and locomotor activity in rat offspring. Neurotoxicol. Teratol. l 1:65-69; 1989. 17. Insel, T. R.; Hill, J. L.; Mayor, R. B. Rat pup ultrasonic isolation calls: possible mediation by the benzodiazepine receptor complex. Pharmacol. Biochem. Behav. 24:1263-1267; 1986. 18. Kehoe, P.; Blass, E. M. Opioid-mediation of separation distress in 10-day-old rats: reversal of stress with maternal stimuli. Dev. Psychobiol. 19:385-398; 1986. 19. Kehoe, P.; Boylan, C. B. Cocaine-induced effects on isolation stress in neonatal rats. Behav. Neurosci. (in press); 1991. 20. Kehoe, P.; Harris, J. C. Ontogeny of noradrenergic effects on ultrasonic vocalizations in rat pups. Behav. Neurosci. 103:10991107; 1989. 21. Raum, W. J.; McGivern, R. F.; Peterson, M. A.; Shryne, J. H.; Gorski, R. A. Prenatal inhibition of hypothalamic sex steroid uptake by cocaine: effects on neurobehavioral sexual differentiation in male rats. Dev. Brain Res. 53:230-236; 1990. 22. Reinstein, D. K.; Isaacson, R. L. Clonidine sensitivity in the developing rat. Brain Res. 135:378-382; 1977. 23. Scalzo, F. M.; All, S. F.; Frambes, N. A.; Spear, L. P. Weanling rats exposed prenatally to cocaine exhibit an increase in striatal D2 dopamine binding associated with an increase in ligand affinity. Pharmacol. Biochem. Behav. 37:371-373; 1990. 24. Shalaby, I.; Spear, L. P. Psychopharmacological effects of low and high doses of apomorphine during ontogeny. Eur. J. Pharmacol. 67:451-459; 1980. 25. 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. 26. Sobrian, S. K.; Burton, L. E.; Robinson, N. L.; Ashe, W. K.; James, H.; Stokes, D. L.; Turner, L. M. Neurobehavioral and immunological effects of prenatal cocaine exposure in rat. Pharmacol. Biochem. Behav. 35:617-629; 1990. 27. Spear, L. P.; Brick, J. Cocaine-induced behavior in the developing rat. Behav. Neural. Biol. 26:401-415; 1979.
PRENATAL COCAINE AND LATER BEHAVIOR 28. Spear, L. P.; Kirstein, C. L.; Bell, J.; Yoottanasumpun, V.; Greenbaum, R.; O'Shea, J.; Hoffmann, H.; Spear, N. E. Effects of prenatal cocaine exposure on behavior during the early postnatal period. Neurotoxicol. Teratol. 11:57-63; 1989.
189 29. Winslow, J. T.; Insel, T. R. Serotonergic modulation of rat pup ultrasonic vocal development: studies with 3,4-methylenedioxymethamphetamine. J. Pharmacol. Exp. Ther. 254:212-219; 1990.
0892-0362/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
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Effects of Prenatal Cocaine on Behavioral Responses to a Cocaine Challenge on Postnatal Day 11 JERROLD
S. M E Y E R , 1 J O H N
D. SHERLOCK
AND
NATHAN
R. M A C D O N A L D
Department o f Psychology, Neuroscience and Behavior Program, University o f Massachusetts, Amherst, M A 01003 R e c e i v e d 19 A u g u s t 1991; A c c e p t e d 13 M a r c h 1992 MEYER, J. S., J. D. SHERLOCK AND N. R. MACDONALD . Effects of prenatal cocaine on behavioral responses to a cocaine challenge on postnatal day 11. NEUROTOXICOL. TERATOL. 14(3) 183-189, 1992.-The purpose of this study was to investigate the effects of prenatal cocaine treatment on behavioral responsivity to a cocaine challenge on postnatal day (PD) 11. Timed-pregnant Sprague-Dawley rats were injected s.c. with 40 mg/kg/day of cocaine (20 mg/kg twice daily) from gestational day l 1 to 20. Saline-control females received saline injections and were pair-fed to the cocainetreated females, whereas untreated-control females were undisturbed and were fed ad lib. Litters were culled to eight pups on PD1 and fostered to normal lactating dams. On PD 11, subjects were given either saline or cocaine (1.25, 2.5, or 5.0 mg/kg s.c.) and then tested 15 min later for isolation-induced ultrasonic vocalizations and other behaviors. Prenatal cocaine-treated pups showed a reduced sensitivity to the stimulating effect of postnatal cocaine on wall climbing, which may reflect an underlying alteration in central dopaminergic and/or noradrenergic systems. Most of the other behaviors studied, including ultrasonic vocalizations, were unaffected by prenatal cocaine administration. However, one other notable finding was an increase in postnfital mortality among the cocaine-exposed pups. We hypothesize that prenatal cocaine treatment may alter pup behavior so as to produce abnormal maternal-offspring interactions and impaired development in some individuals. Prenatal cocaine
Development
Ultrasonic vocalization
B E C A U S E o f current concerns related to cocaine use by pregnant women, several laboratories have developed rodent models to examine the possible neurobehavioral teratogenicity of this compound. Indeed, rats exposed to cocaine in utero exhibit a variety of biochemical and functional changes, including alterations in dopamine receptor binding (9,23), regional glucose utilization (9), sexual differentiation (21), brainstem auditory-evoked potentials (5), l o c o m o t o r activity (4,14,16, 25), and various learning tasks (15,25,28). In ascertaining the behavioral effects of prenatal cocaine exposure, one useful approach is to determine whether later responsiveness to cocaine itself or to other psychomotor stimulants such as amphetamine has been altered. For example, prenatal cocaine has been reported to reduce the locomotor stimulating effects o f both cocaine and amphetamine at postnatal day (PD) 15 (26), although this effect may be transient as it was not observed at later time points (12,26). The use o f psychomotor stimulants in the behavioral evaluation o f cocaine-exposed subjects is valuable partly because the activational properties o f these drugs have been traced primarily to their dopamine releasing and uptake blocking effects in the
Rat
Wall climbing
nucleus accumbens and corpus striatum (11). If behavioral changes are found in gestationally treated subjects, follow-up neurochemical studies can be targeted to specific neurochemical pathways. Moreover, the possibility that in utero cocaine exposure leads to either a sensitization or tolerance to subsequent drug administration has important implications for the offspring o f cocaine-abusing women. A sensitization effect, for example, would imply that repeated exposure during the early stages of pregnancy might produce increased responsiveness to an equivalent cocaine dose experienced later in development, thereby increasing the cumulative damage experienced by the fetus. The present study investigated the influence of prenatal cocaine treatment on subsequent behavioral responsivity to a cocaine challenge on P D 11. One focus o f the study was isolation-induced ultrasonic vocalizations, which are potently suppressed by acute cocaine administration (19) and which are influenced by a wide variety of other drugs as well (2,3,7, 8,13,17,18,20,29). We also looked for changes in the behavior patterns exhibited during vocalization testing, such as wall climbing, locomotion, digging, and grooming.
To whom requests for reprints should be addressed. 183
184
MEYER, SHERLOCK AND MACDONALD METHOD
TABLE 1 PUP INJECTION AND TEST SCHEDULE FOR EACH LITTER
Animals Timed breedings were carried out with nulliparous female and stud male rats approximately 3-months-old. The animals were obtained from our own colony, which is descended from Charles River Sprague-Dawley (CD) stock. They were given Purina Rodent Chow and water ad lib, and maintained under a 14L : 10D cycle (lights on at 6:00 a.m.) at a temperature of approximately 22°C. The day that a sperm plug was detected was designated gestational day 1 (GD 1).
Treatment After successful breeding, females were switched to powdered chow in commercially available food cups to avoid spillage and thereby facilitate pair-feeding. The pregnant dams were randomly divided into three treatment groups: cocainetreated (C), saline-treated controls pair-fed to the cocaine dams (SC), and untreated controls (UC). From GD 11 to GD 20, the C group was injected s.c. with 40 mg/kg/day of cocaine Hcl (20 mg/kg twice daily) in a volume of 3.0 ml/kg of saline. Injections were performed between 9 a.m. and 10 a.m. and between 6 p.m. and 7 p.m. and were moved around the back to avoid excessive necrotic reactions. The SC group was treated in the same manner except that it received injections only of physiological saline. UC females were fed powdered chow ad lib and were not handled prior to parturition, although their food intake was monitored daily. On the day after birth (PD 1), litters were sexed and then 8 pups (sex-balanced if possible) were fostered to untreated lactating dams. Foster mothers were maintained in standard plastic tubs with a bedding of pine shavings, and were given tap water and Purina Formulab Chow to maximize lactation.
Behavioral Testing On PD 1l, each male and female within a litter was randomly assigned to 1 of 4 postnatal treatment groups: Saline, 1.25 mg/kg cocaine, 2.5 mg/kg cocaine, and 5.0 mg/kg cocaine injected s.c. in a volume of 0.5 ml/100 g body weight. The four postnatal treatments were tested in a semirandomized order across litters to control for potential order effects. Consequently, within the 6 or 7 litters that were studied for each prenatal condition (cocaine-treated, saline-treated, and untreated-control), each of the 8 postnatal dose x gender combinations was run no more than once in a particular testing slot (first through eighth; see Table 1). Pups were tested individually in a different room than that in which the animals were housed. Testing began at approximately 1 p.m. and either one or (occasionally) two litters were tested on a given day. At the times shown in Table 1, pups were gently removed from the litter, weighed and injected, and then returned to the litter for 15 rain. Each subject was then removed once again and placed in a glass mason jar 16 cm high x 10 cm diameter containing wood chips to a depth of 3.5 cm. During a 5-min test session, ultrasonic vocalizations were detected by a bat detector (Ultra Sound Advice, Model S-25) and tape-recorded for later playback and scoring. At the same time, the frequency and duration of other behaviors were recorded on a portable microcomputer. Based on preliminary tests of untreated PD 11 pups, we devised an exhaustive and mutually exclusive scoring system containing the following six behavioral categories: wall climbing (both forepaws up on the side of the jar, usually accompanied by treading movements), locomotion (movement around the jar), digging
Pup Number
InjectionTime (min)
1
0
2 3 4 5 6 7 8
10 35 45 70 80 105 115
TestingTime (min) 15 25 50 60 85 95 120 130
A stopwatch was started at time 0 when the first pup was injected. Subsequent injections and testing were then carried out at the indicated time points.
(displacement of the woodchip substrate by the forepaws), grooming (face-washing), stationary with head in a low position, and stationary with head up (with or without sniffing). At the conclusion of testing, each pup was removed to a container kept in a location separate from the colony room so as to avoid disturbing the remainder of the litter. Bedding in the mason jar was changed between subjects.
Statistical Analysis Ultrasonic vocalizations (number per 5 min) and other behaviors (duration per 5 min) were initially subjected to 3-way analyses of variance (ANOVA) with prenatal condition, postnatal treatment, and gender as factors. Significant F values were followed up with Tukey tests to determine individual group differences. Other data were analyzed as indicated next. In all cases, a probability of < 0.05 was considered statistically significant. RESULTS
Maternal and Offspring Characteristics Over the period of GD 11 to GD 20, daily food consumption values were 21.1 _+ 0.6, 20.9 + 0.6, and 23.2 ___ 0.9 g (mean _+ SEM), respectively for the cocaine-treated, salinetreated, and untreated control females. The difference between the cocaine and untreated control group was marginally significant by Student's t test (t = 2.13, df = 11, p = 0.057). Because of the pair-feeding protocol, data from the salinetreated group were not independent of those from the cocaine-treated subjects and were not included in the statistical analysis. The cocaine group was also compared to the saline group with respect to percentage body weight gain over the period of injection (because the untreated controls were not handled during this time, weight data were not available for that group). The cocaine-treated females showed a significantly higher percentage body weight gain than their salinetreated counterparts (26.3 + 1.7°70 vs. 20.7 _+ 1.1070, t = 2.80, d f = 12, p < 0.02), despite the virtually identical food intake in the two groups. This may have been related at least partially to a modest, statistically nonsignificant difference between groups in mean body weight observed at the beginning of the measurement period (cocaine-treated: 287.7 +_ 14.3 g; saline-treated: 310.3 _+ 11.6 g; t = 1.23, d f = 12, ns).
PRENATAL COCAINE AND LATER BEHAVIOR
185
The initial groups consisted of 8 cocaine-treated, 7 salinecontrol, and 6 untreated control females. On PD 1 when litters were culled and fostered, all pups appeared healthy and to be of normal size (body weights were not recorded at this time). However, a total of 12 pups from the cocaine group died prior to PD 11, as these subjects were not found in the tub at the time of testing. Five pups were missing in one litter alone, as a result of which the remainder of the litter and its mother were also eliminated from the study. Three other cocainetreated litters out of the original 8 also had at least one mortality. One pup was missing in one of the saline-treated litters, whereas no deaths were recorded among the untreated controis. A chi-square test was performed to analyze the distribution of viable and dead subjects at PD 11. The results confirmed a nonrandom distribution of pup mortality across the three treatment groups (X2 = 17.68, d f = 2, p < 0.001). Also, note that as a consequence of severe runting, two other pups were eliminated from the same saline-treated litter that had the missing subject. The final subject complement along with postnatal treatment assignment was as follows: 49 cocaine-treated pups (6 males and 6 females given saline, 7 males and 6 females given 1.25 mg/kg cocaine, 7 males and 6 females given 2.5 mg/kg cocaine, and 5 males and 6 females given 5.0 mg/kg cocaine); 53 saline-treated controls (7 males and 6 females at each dose except for 5.0 mg/kg cocaine, which was given to 7 males and 7 females); and 48 untreated controls (6 males and 6 females at each dose). No more than 1 male and 1 female from a given litter received the same postnatal cocaine dose. At the time of testing on PD 11, males were heavier overall than females, F(1, 144) = 4.04, p < 0.05, however body weights did not differ as a function of prenatal drug exposure (males = 29.0
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Saline 1.25 Cocaine 2.5 cocaine 5.0 cocaine
+ 0.6, 28.5 _+ 0.6, and 29.8 _+ 0.8 g; females = 27.9 + 0.5, 28.3 + 0.5, and 28.2 + 0.6 g for the cocaine-treated, salinecontrol, and untreated control groups respectively).
Behavioral Results As shown in Fig. 1, cocaine administered 15 min prior to behavioral testing suppressed the rate of ultrasonic vocalization in a potent and dose-dependent manner, F(3, 126) = 92.59; p < 0.001, for the postnatal treatment main effect. However, there was no shift in the dose-response curve as a function of prenatal condition, indicating that prior exposure to cocaine failed to alter the sensitivity of pups to subsequent cocaine-induced inhibition of vocalization. Postnatal administration of cocaine enhanced the amount of time pups spent wall climbing (Fig. 2). More important, the sensitivity of this response was influenced by prenatal condition, F(6, 126) = 2.62, p < 0.02, for the prenatal condition x postnatal treatment interaction. Individual mean comparisons indicated that for both control groups, all postnatal cocaine doses produced significantly more wall climbing than saline, with no difference between doses. In contrast, pups exposed to cocaine prenatally only showed a significant increase in wall climbing when injected with the 5.0 mg/kg postnatal dose. There was also a (nonsignificant) tendency for the baseline level of wall climbing to be elevated in the prenatal cocaine group. As in the case of ultrasonic vocalizations, several behaviors were influenced by the postnatal cocaine challenge but not by prenatal treatment (Fig. 3). For example, the duration of digging tended to increase following acute cocaine administration, although this change was not statistically significant due
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150
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100 &--
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FIG. 1. Mean ( _+SEM) number of ultrasonic vocalizations per 5 min as a function of prenatal condition (untreated, saline, and 40 mg/kg cocaine) and postnatal treatment (saline or cocaine at the indicated dose). As there was no significant gender difference, the results were pooled across gender far presentation purposes. *significantly different (at least p < 0.05) from the respective postnatal saline control group.
186
MEYER, SHERLOCK AND MACDONALD
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FIG. 2. Mean (+_SEM) duration of wall climbing as a function of prenatal condition and postnatal treatment. *significantly different from the respective postnatal saline control group; **significantly different from all other postnatal treatment groups within the prenatal cocaine condition.
to a high degree of variability. In contrast, grooming, F(3, 126) = 6.53, p < 0.001, stationary/head-low, F(3, 126) = 39.75, p < 0.001, and stationary/head-up behaviors, F(3, 126) = 14.09, p < 0.001, all showed significant dosedependent decreases in duration. Finally, locomotion exhibited a complex interaction between prenatal condition, postnatal treatment, and gender interaction, F(6, 126) = 2.22, p < 0.05, as shown in Fig. 4. Post-hoc Tukey tests comparing the various postnatal treatment groups within each gender and prenatal condition showed that for the untreated male condition, significant increases in locomotion occurred in the 2.5 and 5.0 mg/kg postnatal treatment groups compared to the corresponding saline-treated controls. The only difference among the females occurred in the prenatally cocaine-exposed condition, where the 5.0 mg/kg postnatal dose was associated with less locomotion than the 1.25 mg/kg dose. DISCUSSION One of the major goals of this study was to determine whether prenatal cocaine administration influenced isolationinduced ultrasonic vocalizations in PD 11 rat pups. Confirming previous work (19), acute cocaine treatment prior to testing markedly suppressed this behavior. However, neither the baseline vocalization rate nor the degree of cocaine-induced inhibition were affected by prior in utero cocaine exposure. Based on preliminary studies with untreated pups, we had anticipated a more graded range of vocalization suppression to the doses administered on the test day. Nevertheless, even the lowest cocaine dose of 1.25 mg/kg produced a nearmaximum response on this measure. A lower dose range might have permitted the detection of altered vocalization rates in the prenatal cocaine-treated group. Prenatal treatment vari-
ables such as dose, period of exposure, and mode of administration are undoubtedly also important in determining developmental outcome following gestational cocaine exposure. In contrast to the ultrasonic vocalization results, the doseresponse curve for cocaine stimulation of wall climbing behavior was shifted to the right in the animals exposed to cocaine in utero. This decreased responsiveness could be considered a kind of "tolerance", particularly in view of the fact that the baseline wall climbing rate was not significantly affected by prenatal treatment. Interestingly, Spear and her colleagues (28) observed a similar reduction in shock-elicited wall climbing in PD 12 subjects given cocaine prenatally. Although the neurochemical changes underlying these effects are not yet known, it is possible that dopaminergic and/or noradrenergic systems may be involved. Studies have shown that wall climbing in pups can be activated by the dopaminergic agonist apomorphine (24), the c~-noradrenergic agonist clonidine (22), and the general catecholaminergic agonists amphetamine and cocaine (1, this study). Conversely, wall climbing is inhibited by the dopaminergic antagonist haloperidol (1), the c~-adrenergic blocker phentolamine (1), and the catecholamine neurotoxin 6-hydroxydopamine (10). Because the animals in the present study received the same drug both pre- and postnatally, we cannot rule out the possibility of altered wall climbing due to changes in cocaine pharmacokinetics. However, this seems unlikely due to the specificity of the behavioral effect. If cocaine availability during the postnatal challenge test was reduced in the prenatally-exposed subjects, one might expect that ultrasonic vocalizations or other behaviors would likewise have shown evidence of such drug disposition tolerance. A variety of group differences were also noted for the other behavioral categories examined in this study. For example,
PRENATAL COCAINE AND LATER BEHAVIOR
187
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FIG. 3. Mean (+ SEM) duration of digging, grooming, stationary/head-low, and stationary/head-up as a function of postnatal treatment. *significantlydifferent from the postnatal saline control group; #significantly different from the postnatal 1.25 mg/kg cocaine group.
a complex interaction between prenatal condition, postnatal treatment, and gender was found for locomotor behavior. Untreated control males but not females exhibited increased locomotion when given 2.5 or 5.0 mg/kg cocaine prior to testing. In addition, both male and female subjects in the
25O 225
t Male
2oo
prenatal cocaine group seemed to show an inverted U-shaped response to increasing postnatal cocaine doses, although this was not validated statistically for the males and only partially in the case of the females. What these findings mean, and even whether they are reliable, is not clear at the present time.
[ ] Saline [ ] 1.25 Cocaine
250 225
[ ] 2.5 Cocaine
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175 150
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FIG. 4. Mean (+ SEM) duration of locomotion as a function of prenatal condition, postnatal treatment, and gender. *significantly different from the postnatal saline control group within the prenatal untreated condition; **significantly different from the postnatal 1.25 mg/kg cocaine group within the prenatal cocaine condition.
188
MEYER, SHERLOCK AND MACDONALD
In contrast, g r o o m i n g , s t a t i o n a r y / h e a d - l o w , a n d s t a t i o n a r y / h e a d - u p were u n a f f e c t e d b y p r e n a t a l c o n d i t i o n or subject gender, b u t did show significant reductions as a f u n c t i o n o f postn a t a l cocaine t r e a t m e n t . Earlier studies by Spear a n d Brick (27) h a d f o u n d t h a t a single injection o f cocaine o n P D 14 increased l o c o m o t o r b e h a v i o r b u t h a d n o effect o n wall climbing. The differences b e t w e e n their findings a n d those o f the present study are p r o b a b l y related m a i n l y to the a p p a r a t u s used in each case. Spear a n d Brick o b s e r v e d their subjects in a relatively large open-field, whereas o u r a n i m a l s were tested in a small j a r because o f the need to confine t h e m spatially for vocalization m o n i t o r i n g . It is likely t h a t the enclosed space facilitated wall climbing b e h a v i o r at the expense o f locomotion. Finally, some m e n t i o n s h o u l d be m a d e o f the m o r t a l i t y data. C h u r c h et al. (6) previously f o u n d h e i g h t e n e d p o s t n a t a l m o r t a l i t y in rats prenatally exposed to daily cocaine doses o f 80 or 100 m g / k g . T o the best o f o u r knowledge, however, this is the first r e p o r t o f a similar effect at a m o r e m o d e r a t e d r u g dose. As the cocaine-treated pups a p p e a r e d healthy at the time o f litter culling, there was n o discernible evidence for drug-
related intrauterine g r o w t h r e t a r d a t i o n or congenital defects t h a t m i g h t have led to subject loss. M o r e o v e r , the fact t h a t all pups were fostered to n o r m a l d a m s implies t h a t s u b s e q u e n t fatalities c a n n o t be a t t r i b u t e d to a d i s r u p t i o n of m a t e r n a l beh a v i o r secondary to cocaine withdrawal. W e instead h y p o t h e size t h a t p r e n a t a l cocaine t r e a t m e n t m a y have altered the beh a v i o r o f some subjects so as to p r o d u c e a failure to thrive. This could have been a direct result o f p o o r feeding by the pups, or alternatively it is conceivable t h a t a b e r r a n t p u p beh a v i o r led to deficits in m a t e r n a l responsivity. Direct studies o f the b e h a v i o r a l interactions between d a m s a n d cocaineexposed pups are needed to test this hypothesis. ACKNOWLEDGEMENTS We thank Dr. Priscilla Kehoe for her advice in setting up the ultrasonic vocalization testing. The BASIC program used for behavioral event recording was written by J. H. Preston of the U.S. Fish and Wildlife Service. This research was supported by NIDA grant DA-06495 and by a Faculty Research Grant from the University of Massachusetts.
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