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A fostering study of the effects of prenatal cocaine exposure: I. Maternal behaviors
Neurotaxicologyand Teratology,Voi. 14, pp. 415--421,1992
08924)362/92 $5.00 + .00 Copyright©1992PergamonPress Ltd.
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A Fostering Study of the Effects of Prenatal Cocaine Exposure: I. Maternal Behaviors CHARLES
J. H E Y S E R , V I C T O R A . M O L I N A
AND LINDA PATIA SPEAR 1
Department o f Psychology and the Center f o r Developmental Psychobiology, State o f University o f New York at Binghamton, 8inghamton, N Y 13902 R e c e i v e d 7 A p r i l 1992; A c c e p t e d 8 S e p t e m b e r 1992 HEYSER, C. J., V. A. MOLINA AND L. P. SPEAR. A fostering study of the effects of prenatal cocaineexposure: L Maternalbehaviors. NEUROTOX TERATOL 14(6) 415-421, 1992.- The effect of rearing condition and prenatal exposure to cocaine on maternal behaviors was examined. Sprague-Dawley dams were given SC injections of 40 mg/kg/3cc cocaine HCI (C40) or saline (LC) daffy from gestational days 8-20. Maternal behavior was assessed in treated dams rearing their biological pups (LC/LC; C40/C40), treated dams rearing untreated pups (LC/FOS; C40/FOS), and foster dams rearing treated pups (FOS/LC; FOS/C40). All dams were monitored for home cage behavior (time eating, drinking,and with pups) for 2 h during both the light and dark cycle on posmatal day 4 (P4), pup retrieval on P5-P9, and maternal aggression to a female intruder (latency to the first attack, number of attacks, boxing, pins, intruder time spent submissive and motionless) on P10. No differences were observed in nest behavior or in tests of pup retrieval among the six groups. Dams rearing their biological litter (LC/LC and C40/CA0) were significantly quicker to initiate the first attack when compared to all other groups. This increased aggression was maintained throughout the test session in the C40/CA0 dams who made significantly more intruder attacks than all other groups, with the intruder spending significantlymore time in a submissive posture (lying on back). In contrast, LC/LC dams did not exhibit an increased number of attacks during the test, apparently responding to an increased freezing in their intruders with a reduction in aggressive behavior. Taken together these findings suggest that prior cocaine exposure results in alterations in maternal aggression that is evident when these dams rear their own pups. Cocaine
Maternal behavior
Fostering
Prenatal drug exposure
A M A J O R objective o f research in neurobehavioral teratology is to determine whether offspring function is altered by exposure o f the developing organism to potential toxicants. However, prenatal exposure o f mammalian offspring to test substances essentially mandates concomitant maternal exposure. Thus, in addition to potentially altering offspring neurobehavioral development, the test substance may also influence behavior o f the dam both during and perhaps following the exposure period. Such drug-induced behavioral modifications in the dam may alter maternal/offspring interactions which in turn could influence subsequent offspring neurobehavioral maturation. Indeed, gestational exposure to a variety o f insults such as prepartum stress (14,29,30) and prenatal exposure to developmental toxicants (e.g., 27,42) has been shown to influence subsequent maternal behavior in the treated dams. The induction of maternal behavior is regulated by a complex series of neural and hormonal controls which include hormones such as estrogen, prolactin, placental lactogen, oxytocin, and beta-
Teratology
endorphin (see 40, for review) and neuronal circuitry involving the medial preoptic area, ventral tegmental area, and substantia nigra (e.g., 33,34,35). Presumably, insults during pregnancy that disrupt subsequent maternal behavior do so by altering the activity o f one or more of these systems. Note, however, that the offspring themselves serve as a regulator of maternal behavior. For instance, strain differences in maternal behavior seen between spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats have been shown to be dependent in part on the genetic background o f the offspring (8,9,31). Hence, it is possible that insult=induced alterations in pup behavior could also influence maternal/offspring interactions. Indeed, offspring prenatally exposed to stress elicit less maternal ticking (29) and offspring neonatally exposed to progestin or lead elicit more maternal ticking (3) and nursing behavior (I), respectively, than control offspring. As another example, rat pups treated prenatally with ethanol are less effective in eliciting dam retrieval and maternal behavior than nonethanol-exposed offspring (27,32).
Requests for reprints should be addressed to Linda Patia Spear, Department of Psychology, Box 6000, State University of New York, Binghamton, NY 13902-6000. 415
416 Alterations in maternal/offspring interactions have been shown to have a notable impact on later physiological and behavioral function of the offspring as illustrated by the following diverse examples. Pups from the SHR strain reared by normotensive WKY rats exhibit permanently lower blood pressures in adulthood than SHR offspring reared by hypertensive SHR dams (7). Male pups normally receive more maternal licking than female pups (28), and male pups that do not receive this extra stimulation exhibit a disruption in the later expression of sexual behavior and alterations in social play (4,29). Also, pups exposed neonataliy to 6-hydroxydopamine (6-OHDA) and reared in mixed litters with nontreated pups exhibit fewer behavioral deficits than 6-OHDA pups reared in homogenous litters (36). Given that potential insult-induced alterations in maternal/ offspring interactions may have an impact on offspring neurobehavioral development apart from the direct effects of the drug on the organism per se, developmental toxicologists have frequently used a fostering procedure. With this procedure, each litter of treated or control offspring is fostered to an untreated surrogate dam, thereby eliminating the possibility that offspring neurobehavioral alterations are a result of insuit-induced alterations in the maternal behavior of the dam per se. It has been argued that without fostering "interpretation of the data will be compromised in that one cannot rule out with any degree of confidence that offspring effects have not been maternally mediated during the postnatal period" (24, p. 640). However, note that fostering treated pups to untreated surrogate dams does not eliminate possible insultinduced alterations in pup behavior that could alter maternal behavior toward those pups, with consequent influences on subsequent neurobehavioral maturation in those offspring. Moreover, in at least one study where type of dam used for rearing was systematically varied, postnatal maternal factors were found to have only minimal effects on offspring neurobehavioral function, leading to the conclusion that these maternal factors axe not "sufficiently strong modifying variables on offspring development or behavior to be a source of experimental concern" (51, p. 115). Finally, note that surrogate fostering is costly with respect to animal usage. Using this procedure, twice as many litters are necessary to conduct the study as when litters are not fostered, with pups born to untreated surrogate dams typically being discarded prior to being replaced by experimental pups. Consequently, in programmatic studies of a particular developmental toxicant, it may be important to determine whether the routine use of surrogate fostering is warranted in studies with that substance. Several laboratories have examined the effects of prenatal cocaine exposure in the rat and have observed numerous neural and behavioral alterations in the exposed offspring (5,6, 10,11,20,22,23,25,37,41,43,44,46,47,49), although some essentially negative findings have also been reported (12,13, 15,17,38,39). Whereas in some instances offspring were reared by their biological dams (12,13,17,38,39,43,47), in much of this work litters were fostered to untreated surrogate dams shortly following parturition (5,6,10,11,15,20,22,23,25,37,41, 46,48,49). To date, there has been no systematic study of whether fostering is necessary with this developmental toxicant, although there is one brief report of the effects of gestational cocaine exposure on subsequent maternal behavior. In this study cocaine-exposed dams did not differ from control dams in assessments of maternal behavior when the dams were tested with their biological litters within the first four hours following parturition (44). The purpose of the present study and the following companion article (18) was to examine the necessity of surrogate
HEYSER ET AL. fostering in research with gestational cocaine exposure. In this experiment, maternal behavior was assessed in control dams and dams exposed to cocaine using a dosing regimen that previously has been shown to produce neurobehavioral teratogenic effects in both fostered (10,22,23,41,46,49) and nonfostered (47) offspring. Given that pup behavior has been shown to be an important determinant of the maternal care they receive (3,8,26,27,31,32), the behavior of cocaine-exposed and control dams was assessed both when rearing untreated pups and when rearing cocaine-exposed offspring. There are several different ways in which maternal behavior can be measured, including assessments of nest building, pup retrieval, time spent with the litter, nursing, pup licking, and maternal aggression (see 40, for review). These behaviors are subject to somewhat different neural, hormonal, and environmental controls (e.g., 16,19,29,35), and hence may be differentially influenced by gestational exposure to cocaine. For this reason, several different measures of maternal behavior were assessed. METHOD
Subjects, Breeding, and Chronic Drug Treatments Offspring were generated from Sprague-Dawley (Charles River-VAF/Plus, Kingston, NY) rats bred in our laboratory. Animals were housed in a temperature-controlled colony room on a 12L : 12D cycle with the lights on at 0700 h. All dams were housed in pairs and habituated to the colony room for two weeks after arrival. Prior to the onset of mating, dams randomly assigned to the cocaine treatment group or lab chow control group were handled for 5 min daily for 5 days, and received SC injections of 0.9% saline (3 cc/kg) the last three days of handling. All dams were individually housed in hanging cages prior to mating, with an adult male being placed in each cage daily at 1700 h and removed the following morning at approximately 0900 h. Day 1 of gestation (El) was defined as the day of detection of a copulatory plug at which time each dam was individually housed in Plexigias breeding cages. Dams assigned to the cocaine (C40) and lab chow (LC) treatment groups were given ad lib food (powdered lab chow) and water throughout the study, with dally weights and intake of food and water being recorded from El-E20. Dams were SC injected dally between 1000 and 1200 h with either 40 mg/kg/ 3cc cocaine hydrochloride (C40) or an equal volume of 0.9% saline (LC) from Eg-E20 (see 45). Dams to be used for surrogate fostering were left undisturbed throughout pregnancy except for routine feeding, watering, and cage cleaning. Gestational length was recorded for each gravid dam with the date of birth being designated as postnatal day 0 (P0). All pups were weighed and the total number of pups of each sex recorded. Each litter was culled to 8-10 pups. Rearing condition. The experimental design consisted of six different experimental groups designated by maternal treatment (untreated surrogate foster dam [FOS], LC, or C40) and pup origin (FOS, LC, or CA0), with 10-12 litters being tested in each of the six experimental groups. Group FOS/LC consisted of LC pups fostered by litter to untreated surrogate dams that had given birth to litters within the preceding 24--48 h. The pups from these surrogate foster dams were fostered by litter to the LC dams to form group LC/FOS. Similarly, group FOS/C40 consisted of CA0 pups fostered by litter to untreated surrogate foster dams, with the pups from these surrogate dams being fostered by litter to CA0 dams (group C40/FOS). Pups in the last two groups were not fostered but remained with the biological dam (LC/LC and CA0/CA0). Thus, within this study, the experimental groups consisted of
A FOSTERING STUDY OF THE EFFECTS: I
417
both the typical surrogate fostering (FOS/C40; F O S / L C ) as well as non-fostered (C40/C40; L C / L C ) conditions. In addition, dams from each treatment group rearing untreated foster pups (C40/FOS; LC/FOS) were included to determine whether any observed differences in maternal behavior between C40/C40 and L C / L C litters are related to the prior treatment of the dam per se or whether the prenatal treatment of the pups themselves contributed to these differences.
Maternal Behavior Tests Home cage behavior. On P3 each dam and her rearing litter were placed into a standard maternity cage (20 x 42 x 19 cm) with a ventilated Plexiglas lid to which a water bottle was attached; food pellets were placed inside the maternity cage. The following day (on P4), the behavior o f the dams was videotaped (Panasonic Time Lapse Video Recorder, model 6720A) for 2 h midcycle during both light and dark cycles. Normal colony room lighting was used for video recording during the light cycle and red light served as the illumination source during the night cycle. Videotapes were scored by two independent observers who were unaware of the prior treatment conditions of the dams or their litters. During both 2 h test sessions for each dam, the amount of time dams spent with the pups, as well as the amount of time they spent eating and drinking were determined. Pup retrieval. On P5, four pups (2 males and 2 females) were randomly selected and removed from the nest and placed in the maternity cage on the side opposite to the nest site. The
latency to retrieve each pup was recorded until all four pups were returned to the nest or 10 rain had elapsed. This test of pup retrieval was repeated on P6, 7, 8, and 9, with the four pups used for the retrieval test being randomly selected each day. The experimenters conducting these tests were unaware of the treatment condition o f the litters. Maternal aggression. Aggression toward a female intruder was assessed on P10. A naive adult female intruder was placed into the maternity cage with the resident dam and litter and their interactions were recorded via videotape for 10 rain. Testing was conducted in the colony room between 1000 and 1400 h. Video tapes were viewed by two independent observers who were unaware of prenatal history and rearing condition, with the behavior of the dam being scored for the latency to the first attack, number of attacks, boxing, and pins (dam on top and intruder rolled over on back). In addition, the time spent in submission, defined either as when the forepaws were extended toward the attacking animal with the ventral surface of the body facing the opponent, or when an animal was lying on its back and freezing (motionless for > 25 s) by the intruder female were also determined (see 50). RESULTS
Gestational Data Preliminary analyses of the maternal data revealed no differences with regard to subsequent rearing group assignment within each treatment group (i.e., C40/CA0 vs. CA0/FOS;
TABLE 1 MATERNAL-LiTIER SUMMARY DATA DERIVED FROM LITTER MEANS (SEM IN PARENTHESES) Maternal Data LC Dams
GestationalTreatment Typeof Litter Reared Percent gestational weight gain Food intake (g) Water intake (ml) Gestational length (d)
CA0 Dams
FOS Pups
LC Pups
FOS Pups
C40 Pups
41.79 (1.88) 29.49
41.21
30.68*
30.37*
(1.67)
(2.10)
(2.11)
30.14
25.85
28.67
(0.85)
(1.56)
(0.89)
(1.69)
57.73 (1.30) 22.72 (0.19)
56.81 (2.18) 23.18 (0.12)
55.62 (2.12) 22.91 (0.08)
58.64 (1.86) 22.75 (0.13)
Litter Data LC Pups Prenatal Treatment Reared by Litter size Number of male pups in litter Number of female pups in litter Offspring body weights on Postnatal day I (g) Males t Females
C40 Pups
FOS Dam
LC Dam
FOS Dam
C40 Dam
16.09 (0.65) 8.18 (0.53) 7.91 (0.86)
16.27 (0.65) 7.45 (0.79) 8.63 (0.75)
16.00 (0.80) 8.08 (0.78) 7.83 (0.85)
14.50 (0.80) 6.67 (0.63) 7.83 (0.61)
7.54 (0.20) 7.16 (0.19)
7.82 (0.25) 7.25 (0.23)
7.21 (0.21) 6.53 (0.37)
7.58 (0.21) 6.97 (0.27)
*Significant a t p < 0.05 from LC dams. tSignificant a t p < 0.05 from female pups.
418
I-IEYSER ET AL.
L C / L C vs. LC/FOS), therefore the data were collapsed across this variable prior to data analysis. An analysis o f variance (ANOVA) performed on percentage body weight gain during pregnancy revealed a significant main effect o f Prenatal Treatment, F(1, 44) = 32.40, p < 0.05, with dams in the LC condition gaining significantly more weight than C40 dams (Table 1). Moreover, a 2 x 20 (Prenatal Treatment x Days) repeated measures A N O V A across days on body weights revealed a significant main effect of Prenatal Treatment, F(1, 44) = 4.28, p < 0.05, and Days, F(19, 836) = 306.39, p < 0.05, along with a significant Prenatal Treatment × Days interaction, F(19, 836) = 6.78, p < 0.05. Subsequent Tukey's tests revealed that LC dams weighed more that C40 dams from E l 0 to E20 (Fig. 1A). A 2 × 20 (Prenatal Treatment × Days) repeated measure ANOVA across days on the food intake data revealed a significant main effect o f Days, F(19, 386) = 25.16, p < 0.05, along with a significant Prenatal Treatment × Days interaction, F(19, 836) = 5.58, p < 0.05. Tukey's tests revealed a significant decrease in CA0 dam food intake on ES, E9, El0, El8, and E l 9 compared to LC dams (Fig. 1B). A similar ANOVA conducted on daily water intake revealed a significant main effect of Days, F(19, 836) = 135.32, p < 0.05. Although the interaction was not significant, a trend toward a
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FIG. 1. Mean body weight (A: in grams), food intake (B: in grams), and water intake (C: milliliters) for gestational days 1-20. Error bars indicate SEM's. (LC = saline-injected controls; C40 = cocaine).
reduction in water intake was observed on E8 and E9 in C40 dams when compared to LC dams (Fig. IC). As shown in Table 1, no differences were observed between the two prenatal treatment groups in gestational length.
Litter Size, Composition, and Offspring Body Weight Litter data reflect data collected on P1 prior to fostering of the litters. Hence, data refer to the birth litters rather than rearing litters for C40/FOS and L C / F O S dams. A 2 x 2 (Prenatal Treatment x Sex) ANOVA conducted on the number of male and female pups in each litter failed to reveal any significant differences (Table 1). A similar A_NOVA performed on litter means of male and female body weights at P1 revealed only a significant main effect o f Sex, F(1, 44) = 40.49, p < 0.05, with male pups weighing more than females (Table 1).
Maternal Behavior Tests Data for each dependent measure collected from the six different groups defined by prenatal treatment and rearing condition were analyzed by a one-way ANOVA, with repeated measures where appropriate. Tukey's tests (26) were used to determine the locus o f significant main effects and interactions. A reciprocal transformation was appfied to all the latency data prior to analysis (26). A significance level of p < 0.05 was used for all statistical analyses. Home cage behavior. A 6 x 2 (Condition x Time [light vs. dark]) repeated measures ANOVA across time was conducted for each of the three measured behaviors of the dam in the home cage: time spent with pups and time spent eating and drinking. These ANOVAs revealed only significant main effects of time spent with pups, F ( I , 45) = 43.99, p < 0.05 and time spent drinking,/7(1, 45) = 5.717, p < 0.05. Subsequent Tukey's tests indicated that all dams spent significantly more time with the pups during the fight cycle when compared to the dark cycle and more time drinking in the dark cycle. A similar ANOVA conducted on the time spent eating failed to reveal any differences (all Fs < 1). Pup retrieval. The latency to retrieve the first pup and the total time required to retrieve all four pups were used as the dependent measures. A 6 x 5 (Condition x Days) repeated measures ANOVA, with days as a within-subject variable, conducted on the total time required to retrieve all four pups and reciprocal transformation of the latency to retrieve the first pup revealed no significant differences (all Fs < 1). No significant differences were observed among the various groups on any measure of pup retrieval. Maternal aggression. The latency to the first attack, number of attacks, boxing, and pins were used as dependent measures in the analysis of maternal aggression. An A N O V A performed on the number of attacks revealed a significant effect o f Group, F(5, 49) = 3.52. p < 0.05. Tukey's tests indicated that C40 dams rearing their own pups (C40/C40) made significantly more attacks to the female intruder than all other groups, which did not differ among themselves (Fig. 2A). A similar ANOVA conducted on the reciprocal transformation of the latency to the first attack revealed a significant effect o f Group, F(5, 49) = 3.05, p < 0.05. Tukey's test on the transformed data revealed that dams rearing their biological litters ( L C / L C and C40/C40) initiated the first attack significantly more rapidly than all other groups (Fig. 2B). Analyses conducted on the number of pins and boxing revealed no differences among the groups (all ps > .05). The amount of time spent in a submissive posture and
A FOSTERING STUDY OF THE EFFECTS: I
419
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they spent with their pups during either the fight or dark cycle. These results are consistent with previous reports of no differences in pup retrieval and nest activities in cocaine-exposed dams rearing their biological fitters (44). However, on the measures of maternal aggression, differences were observed among the treatment/rearing conditions. Dams rearing their biological pups (CAO/C40 and LC/LC) were quicker to attack the female intruder than all groups o f dams not rearing their own fitters (C40/FOS; LC/FOS; FOS/C40; FOS/LC). This aggressiveness was maintained in coeaine-exposed dams rearing their biological pups, with C40/C40 dams emitting significantly more aggressive attacks during the 10-rain test session than all other groups o f dams. This effect was not observed in control dams rearing their biological litters, in that LC/LC dams did not differ from dams not rearing their own litters in total number of attacks. Thus, these data provide evidence that prior cocaine exposure results in alterations in maternal aggression evident when these dams rear their own pups. Dams rearing their biological fitters, regardless of prenatal treatment condition, were quicker to attack a female intruder than dams not rearing their own fitters. Thus, it appeared that these dams respond differentially in terms of maternal aggression when rearing their own pups than when rearing a
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FIG. 2. Mean number of attacks (A) and the reciprocal transformation of the mean latency to the first attack-i.e., speed (B) for the test of maternal aggression. Error bars indicate SEM's. (FOS = Foster; LC = saline-injected controls; C40 = cocaine. Groups are designated by maternal treatment and pup origin [e.g., FOS/LC = LC pups reared by a FOS dam]).
freezing was used to analyze the response of the female intrnder. An ANOVA conducted on the time the intruder spent in a submissive posture revealed a significant main effect of Group, F(5, 49) = 2.42,p < 0.05, with intruders placed with C40 dams rearing their own pups (C40/C40) spending siguificandy more time in submission than all other groups (Fig. 3A). A similar ANOVA conducted on the amount of time the intruder spent freezing revealed a significant main effect of Group, F(5, 49) = 2.35,p < 0.05. Tukey's tests revealed that female intruders placed with LC dams rearing their own pups (LC/LC) spent significantly more time freezing than all other groups (Fig. 3B). No instances of freezing or submission were observed in the resident dam.
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DISCUSSION
In several respects, dams previously exposed to cocaine did not differ from control dams in maternal behavior. Cocaineexposed dams, regardless of whether they were rearing their own pups or untreated foster pups, did not differ from control dams in pup retrieval latencies nor in the amount of time that
FIG. 3. Mean time (in seconds) the intruder spent in a submissive posture (A) and the mean time (seconds) the intruder spent freezing (13) during the test of maternal aggression. Error bars indicate SEM's. (FOS = Foster; LC = saline-injected controls; C40 = cocaine. Groups are designated by maternal treatment and pup origin [e.g., FOS/LC = LC pups reared by FOS dams]).
420
HEYSER ET AL.
nonbiological litter, a distinction that is not disrupted by prior cocaine exposure. It has long been known that rat dams can recognize their offspring (2). Yet, seemingly it has been assumed that this recognition has little influence on subsequent maternal behavior, in that rat dams readily accept and rear foster pups, unlike females of many other species (see 21, for review). The results Of the present study provide initial evidence that the maternal behavior of dams rearing their biological pups is not identical to that of dams rearing nonbiological litters, a finding that may be of importance given the frequent use of surrogate fostering procedures in the field of developmental toxicology. Although both C40/C40 dams and L C / L C dams initiated the first attack more rapidly in the maternal aggression test, only the cocaine-exposed dams rearing their own litters exhibited an increased total number o f aggressive attacks. This was associated with an increased time spent by their intruders in a submissive "on back" posture, a behavior that is seen solely in response to attack. In contrast, although L C / L C dams also attacked rapidly, this aggression was not maintained throughout the test session, with these dams apparently responding to an increase in freezing behavior on the part of the intruders with a reduction in aggressive attacks. The behavior of the intruder was apparently influenced by the behavior of the resident dam, with a submissive posture being emitted in response to direct attack, and an increase in the time spent motionless (freezing) following an attack. Thus, the increased freezing behavior of intruders placed in with LC dams and their biological pups (LC/LC) may be related to the rapid onset of attack observed in these lactating dams. In contrast, intruders placed in with C40 dams rearing their biological litter (C40/C40) may have spent more time in a submissive posture due to the increased number of attacks directed toward them during the test session. The increase in aggressive attacks observed in cocaineexposed dams rearing their own pups was not observed in cocaine-exposed dams rearing foster pups. These findings are reminiscent of the results of previous work examining the effects of gestational and lactational exposure to ethanol on maternal behavior where ethanol-exposed dams were found to differ in maternal behavior from control dams when tested with their own pups but not with control pups (27). Together, these studies suggest that the characteristics of the previously drug-exposed pups themselves may contribute to observed treatment-induced alterations in maternal behavior in their dams. Note, however, that the extent to which these effects are related to the treatment status of the pup versus the genetic relationship of the pups to the dam cannot be determined from the designs of the Mathews and Jamison (27) study or the current experiment. However, in other work, ethanol-
exposed pups have been reported to be less effective than control pups in eliciting retrieval behavior from not only ethanol-exposed, but also control dams (32); these data argue for the critical importance of the treatment status of pups rather than their genetic relationship to the dam. Nevertheless, in future studies that are designed to specifically assess the relative contribution of dams versus the litter in contributing to treatment-induced alterations in maternal/offspring interactions, it would appear important to use a design where litters are systematically fostered both within and across treatment conditions (see 51, for an example). Whereas previously cocaine-exposed dams rearing their own litters exhibited more attacks than the other groups of dams in the maternal aggression test, no differences were observed with respect to pup retrieval latencies or the amount of time spent in the nest during either the light or dark cycle. Indeed, it is often the case that particular experimental treatments may disrupt some feature of maternal behavior, while leaving other aspects intact. For instance, several hypothalamic manipulations have been observed to disrupt maternal aggression toward a female intruder without influencing pup retrieval (19), and treatment with haloperidol has been reported to alter retrieval behavior and nest building but not nursing behavior or licking of the pups (16). These findings emphasize the importance of using multiple measures when assessing the effects of developmental toxicants on maternal behavior, and additionally raise the issue of whether other aspects of maternal behavior that were not assessed in this study might also be affected by prior cocaine treatment (e.g., nest building and maternal licking of the pups). It is not feasible, however, to design a study to investigate every possible aspect of maternal behavior that might potentially be disrupted by a developmental toxicant. Yet, treatment-induced alterations in maternal behavior are primarily of interest in developmental toxicology only to the extent that these alterations influence the subsequent neural and behavioral maturation of the offspring; thus, it is important to examine not only maternal behavior but also offspring outcome. Hence, the following companion article (18) examines the effects of the various treatments and rearing conditions on several behavioral measures in offspring of the dams examined for maternal behavior in the present study. ACKNOWLEDGEMENTS This research was supported by National Institute on Drug Abuse Grants R01 DA14478 and K02 DA00140 to Linda P. Spear. We gratefully acknowledge the assistance of George Ruggiero and Michael Bennenuto in the data collection and Yolanda Pinzas for technical support.
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A FOSTERING STUDY OF THE EFFECTS: I 9. Cierpial, M. A.; Shasby, D. E.; et al. Open-field behavior of spontaneously hypertensive and Wistar-Kyoto normotensive rats: effects of reciprocal cross-fostering. Beh. Neural Biol. 51:203210; 1989. 10. Clow, D. W.; Hammer, R. P.; Kirstein, C. L.; Spear, L. P. Gestational cocaine exposure increases opiate receptor binding in weanling offspring. Dev. Brain Res. 59:179-185; 1991. 11. Dow-Edwards, D. L.; Freed, L. A.; Fico, T. A. Structural and functional effects of prenatal cocaine exposure in adult rat brain. Dev. Brain Res. 57:263-268; 1990. 12. Foss, J. A.; Riley, E. P. Elicitation and modification of the acoustic startle reflex in animals prenatally exposed to cocaine. Neurotoxicol. Teratol. 13:541-546; 1991. 13. Foss, J. A.; Riley, E. P. Failure of acute cocaine administration to differentially affect acoustic startle and activity in rats prenatally exposed to cocaine. Neurotoxicol. Teratol. 13:547-551; 1991. 14. Fride, E.; Dan, Y.; Gavish, M.; Weinstock, M. Prenatal stress impairs maternal behavior in a conflict situation and reduces hippocampal benzodiazepine receptors. Life Sci. 36:2103-2109; 1985. 15. Fang, Y. K.; Reed, J. A.; Lau, Y. S. Prenatal cocaine exposure fails to modify neurobehavioral responses and the striatal dopaminergic system in newborn rats. Gen. Pharmacol. 20:689-693; 1989. 16. Giordano, A. L.; Johnson, A. E.; Rosenblatt, J. S. Haloperidolinduced disruption of retrieval behavior and reversal with apomorphine in lactating rats. Physiol. Behav. 48:211-214; 1990. 17. Giordano, M.; Moody, C. A.; Zubrycki, E. M.; Dreshfield, L.; Norman, A. B.; Sanberg, P. R. Prenatal exposure to cocaine in rats: Lack of long-term effects on locomotion and stereotypy. Bull. Psychonomic Soc. 28:51-54; 1990. 18. Goodwin, G. A.; Heyser, C. J.; Moody, C. A.; Rajachandran, L.; Molina, V. A.; Arnold, H. M.; McKinzie, D. L.; Spear, N. E.; Spear, L. P. A fostering study of the effects of prenatal cocaine exposure: II. Offspring behavioral measures. Neurotoxicol. Teratol. 14:423-432; 1992. 19. Hansen, S. Medial hypothalamic involvement in maternal aggression of rats. Behav. Neurosci. 103:1035-1046; 1989. 20. Henderson, M. G.; McMillen, G. A. Effects of prenatal exposure to cocaine or related drugs on rat developmental and neurological indices. Brain Res. Bull. 24:207-212; 1990. 21. Hepper, P. G. Parental recognition in the rat. Q. J. Exp. Psycol. [B] 38:151-160; 1986. 22. 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. Behav. Neurosci. 104:955-963; 1990. 23. Heyser, C. J.; Miller, J. S.; Spear, N. E.; Spear, L. P. Prenatal exposure to cocaine disrupts cocaine-induced conditioned place preference in rats. Neurotoxicol. Teratol. 14:57-64; 1992. 24. Hutchings, D. E. Issues of methodology and interpretation in clinical and animal behavioral teratology studies. Neurotoxicol. Teratol. 7:639-642; 1985. 25. Hutchings, D. E.; Fico, T. A.; Dow-Edwards, D. L. Prenatal cocaine: Maternal toxicity, fetal effects and locomotor activity in rat offspring. Neurotoxicol. Teratol. 11:65-69; 1989. 26. Kirk, R. E. Experimental design: Procedures for the behavioral sciences. (2nd ed.). Belmont, CA: Brooks/Cole; 1982. 27. Mathews, D.; Jamison, S. Effects of ethanol consumption on maternal behavior in the female rat. Physiol. Behav. 29:595-597; 1982. 28. Moore, C. L.; Morelli, G. A. Mother rats interact differentially with male and female offspring. J. Comp. Physiol. Psychol. 93: 123-129; 1979. 29. Moore, C. L.; Power, K. L. Prenatal stress affects mother-infant interaction in Norway rats. Dev. Psychobiol. 19:235-245; 1986. 30. Muir, J. L.; Pfister, H. P.; Ivinskis, A. Effects of prepartum stress and postpartum enrichment on mother-infant interaction and offspring problem-snlving ability in rattus norvegicus. J. Comp. Psychol. 99:468-478; 1985. 31. Myers, M. M.; Brnnelll, S. A.; Shalr, H. N.; Squire, J. M.; Hofer, M. A. Relationships between maternal behavior of SHR
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and WKY dams and adult blood pressures of cross-fostered FI pups. Dev. Psychobiol. 22:55-67; 1989. Ness, J. W.; Franchina, J. J. Effects of prenatal alcohol exposure on rat pups' ability to elicit retrieval behavior from dams. Dev. Psychobiol. 23:85-90; 1990. Numan, M. Neural basis of maternal behavior in the rat. Psychoneuroendocrinology 13:47-62; 1988. Numan, M.; Nagle, D. S. Preoptic area and substantia nigra interact in the control of maternal behavior in the rat. Behav. Neurosci. 97:120-139; 1983. Numan, M.; Smith, H. G. Maternal behavior in rats: Evidence for the involvement of preoptic projections to the ventral tegmental area. Behav. Neurosci. 98:712-727; 1984. Pearson, D. E.; Teicher, M. H.; Shaywitz, B. A.; Cohen, D. J.; Young, J. G.; Anderson, G. M. Environmental influences on body weight and behavior in developing rats after neonatal 6hydroxydopamine. Science 209:715-717; 1980. 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. Riley, E. P.; Foss, J. A. Exploratory behavior and locomotor activity: a failure to find effects in animals prenatally exposed to cocaine. Neurotoxicol. Teratol. 13:553-558; 1991. Riley, E. P.; Foss, J. A. The acquisition of passive avoidance, active avoidance, and spatial navigation tasks by animals prenatally exposed to cocaine. Neurotoxicol. Teratol. 13:559-564; 1991. Rosenblatt, J. S.; Mayer, A. D.; Giordano, A. L. Hormonal basis during pregnancy for the onset of maternal behavior in the rat. Psychoneuroendocrinology 13:29-46; 1988. Scalzo, F. M.; Ali, S. F.; Frambes, N. A.; Spear, L. P. Weanling rats exposed prenatally to cocaine exhibit an increase in striatal D2 binding associated with an increase ligand affinity. Pharmacol. Biochem. Bebav. 37:371-373; 1990. Sechzer, J. A.; Lieberman, K. W.; Alexander, G. J.; Weidman, D.; Stokes, P. E. Aberrant parenting and delayed offspring development in rats exposed to lithium. Biol. Psychiatry 21:1258-1266; 1986. 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. 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. 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. Psychopharmacology 97:427-431; 1989. Spear, L. P.; Heyser, C. J. Cocaine and the developing nervous system: Laboratory findings. In: Zagon, I. S.; Slotkin, T. A., eds. Maternal substance abuse and the developing nervous system. Orlando, FL: Academic Press 1992:155-175. Spear, L. P.; Kirstein, C. L.; Bell, J.; Yoottanasumpun, V.; Greenbanm, R.; O'Shea, J.; Hoffman, H.; Spear, N. E. Effects of prenatal cocaine exposure on behavior during the early postnatal period. Neurotoxicol. Teratol. 11:57-63; 1989. Spear, L. P.; Kirstein, C. L.; Frambes, N. A. Cocaine effects on the developing central nervous system: Behavioral, psychopharmacological, and neurochemical studies. Ann. NY Acad. Sci. 526:290-307; 1989. Spear, L. P.; Kirstein, C. L; Frambes, N. A.; Moody, C. A. Neurobehavinral teratogenicity of gestational cocaine exposure. NIDA Res. Monogr. 95:232-238; 1990. Vergnes, M.; Depaulis, A.; Boehrer, A. Parachiorophenylalanine-induced serotonin depletion increases offensive but not defensive aggression in male rats. Physiol. Behav. 36:653-658; 1986. Vorhees, C. V. A fostering/crossfostering analysis of the effects of prenatal ethanol exposure in a liquid diet on offspring development and behavior in rats. Neurotoxicol. Teratol. 11:115-120; 1989.
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A Fostering Study of the Effects of Prenatal Cocaine Exposure: I. Maternal Behaviors CHARLES
J. H E Y S E R , V I C T O R A . M O L I N A
AND LINDA PATIA SPEAR 1
Department o f Psychology and the Center f o r Developmental Psychobiology, State o f University o f New York at Binghamton, 8inghamton, N Y 13902 R e c e i v e d 7 A p r i l 1992; A c c e p t e d 8 S e p t e m b e r 1992 HEYSER, C. J., V. A. MOLINA AND L. P. SPEAR. A fostering study of the effects of prenatal cocaineexposure: L Maternalbehaviors. NEUROTOX TERATOL 14(6) 415-421, 1992.- The effect of rearing condition and prenatal exposure to cocaine on maternal behaviors was examined. Sprague-Dawley dams were given SC injections of 40 mg/kg/3cc cocaine HCI (C40) or saline (LC) daffy from gestational days 8-20. Maternal behavior was assessed in treated dams rearing their biological pups (LC/LC; C40/C40), treated dams rearing untreated pups (LC/FOS; C40/FOS), and foster dams rearing treated pups (FOS/LC; FOS/C40). All dams were monitored for home cage behavior (time eating, drinking,and with pups) for 2 h during both the light and dark cycle on posmatal day 4 (P4), pup retrieval on P5-P9, and maternal aggression to a female intruder (latency to the first attack, number of attacks, boxing, pins, intruder time spent submissive and motionless) on P10. No differences were observed in nest behavior or in tests of pup retrieval among the six groups. Dams rearing their biological litter (LC/LC and C40/CA0) were significantly quicker to initiate the first attack when compared to all other groups. This increased aggression was maintained throughout the test session in the C40/CA0 dams who made significantly more intruder attacks than all other groups, with the intruder spending significantlymore time in a submissive posture (lying on back). In contrast, LC/LC dams did not exhibit an increased number of attacks during the test, apparently responding to an increased freezing in their intruders with a reduction in aggressive behavior. Taken together these findings suggest that prior cocaine exposure results in alterations in maternal aggression that is evident when these dams rear their own pups. Cocaine
Maternal behavior
Fostering
Prenatal drug exposure
A M A J O R objective o f research in neurobehavioral teratology is to determine whether offspring function is altered by exposure o f the developing organism to potential toxicants. However, prenatal exposure o f mammalian offspring to test substances essentially mandates concomitant maternal exposure. Thus, in addition to potentially altering offspring neurobehavioral development, the test substance may also influence behavior o f the dam both during and perhaps following the exposure period. Such drug-induced behavioral modifications in the dam may alter maternal/offspring interactions which in turn could influence subsequent offspring neurobehavioral maturation. Indeed, gestational exposure to a variety o f insults such as prepartum stress (14,29,30) and prenatal exposure to developmental toxicants (e.g., 27,42) has been shown to influence subsequent maternal behavior in the treated dams. The induction of maternal behavior is regulated by a complex series of neural and hormonal controls which include hormones such as estrogen, prolactin, placental lactogen, oxytocin, and beta-
Teratology
endorphin (see 40, for review) and neuronal circuitry involving the medial preoptic area, ventral tegmental area, and substantia nigra (e.g., 33,34,35). Presumably, insults during pregnancy that disrupt subsequent maternal behavior do so by altering the activity o f one or more of these systems. Note, however, that the offspring themselves serve as a regulator of maternal behavior. For instance, strain differences in maternal behavior seen between spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats have been shown to be dependent in part on the genetic background o f the offspring (8,9,31). Hence, it is possible that insult=induced alterations in pup behavior could also influence maternal/offspring interactions. Indeed, offspring prenatally exposed to stress elicit less maternal ticking (29) and offspring neonatally exposed to progestin or lead elicit more maternal ticking (3) and nursing behavior (I), respectively, than control offspring. As another example, rat pups treated prenatally with ethanol are less effective in eliciting dam retrieval and maternal behavior than nonethanol-exposed offspring (27,32).
Requests for reprints should be addressed to Linda Patia Spear, Department of Psychology, Box 6000, State University of New York, Binghamton, NY 13902-6000. 415
416 Alterations in maternal/offspring interactions have been shown to have a notable impact on later physiological and behavioral function of the offspring as illustrated by the following diverse examples. Pups from the SHR strain reared by normotensive WKY rats exhibit permanently lower blood pressures in adulthood than SHR offspring reared by hypertensive SHR dams (7). Male pups normally receive more maternal licking than female pups (28), and male pups that do not receive this extra stimulation exhibit a disruption in the later expression of sexual behavior and alterations in social play (4,29). Also, pups exposed neonataliy to 6-hydroxydopamine (6-OHDA) and reared in mixed litters with nontreated pups exhibit fewer behavioral deficits than 6-OHDA pups reared in homogenous litters (36). Given that potential insult-induced alterations in maternal/ offspring interactions may have an impact on offspring neurobehavioral development apart from the direct effects of the drug on the organism per se, developmental toxicologists have frequently used a fostering procedure. With this procedure, each litter of treated or control offspring is fostered to an untreated surrogate dam, thereby eliminating the possibility that offspring neurobehavioral alterations are a result of insuit-induced alterations in the maternal behavior of the dam per se. It has been argued that without fostering "interpretation of the data will be compromised in that one cannot rule out with any degree of confidence that offspring effects have not been maternally mediated during the postnatal period" (24, p. 640). However, note that fostering treated pups to untreated surrogate dams does not eliminate possible insultinduced alterations in pup behavior that could alter maternal behavior toward those pups, with consequent influences on subsequent neurobehavioral maturation in those offspring. Moreover, in at least one study where type of dam used for rearing was systematically varied, postnatal maternal factors were found to have only minimal effects on offspring neurobehavioral function, leading to the conclusion that these maternal factors axe not "sufficiently strong modifying variables on offspring development or behavior to be a source of experimental concern" (51, p. 115). Finally, note that surrogate fostering is costly with respect to animal usage. Using this procedure, twice as many litters are necessary to conduct the study as when litters are not fostered, with pups born to untreated surrogate dams typically being discarded prior to being replaced by experimental pups. Consequently, in programmatic studies of a particular developmental toxicant, it may be important to determine whether the routine use of surrogate fostering is warranted in studies with that substance. Several laboratories have examined the effects of prenatal cocaine exposure in the rat and have observed numerous neural and behavioral alterations in the exposed offspring (5,6, 10,11,20,22,23,25,37,41,43,44,46,47,49), although some essentially negative findings have also been reported (12,13, 15,17,38,39). Whereas in some instances offspring were reared by their biological dams (12,13,17,38,39,43,47), in much of this work litters were fostered to untreated surrogate dams shortly following parturition (5,6,10,11,15,20,22,23,25,37,41, 46,48,49). To date, there has been no systematic study of whether fostering is necessary with this developmental toxicant, although there is one brief report of the effects of gestational cocaine exposure on subsequent maternal behavior. In this study cocaine-exposed dams did not differ from control dams in assessments of maternal behavior when the dams were tested with their biological litters within the first four hours following parturition (44). The purpose of the present study and the following companion article (18) was to examine the necessity of surrogate
HEYSER ET AL. fostering in research with gestational cocaine exposure. In this experiment, maternal behavior was assessed in control dams and dams exposed to cocaine using a dosing regimen that previously has been shown to produce neurobehavioral teratogenic effects in both fostered (10,22,23,41,46,49) and nonfostered (47) offspring. Given that pup behavior has been shown to be an important determinant of the maternal care they receive (3,8,26,27,31,32), the behavior of cocaine-exposed and control dams was assessed both when rearing untreated pups and when rearing cocaine-exposed offspring. There are several different ways in which maternal behavior can be measured, including assessments of nest building, pup retrieval, time spent with the litter, nursing, pup licking, and maternal aggression (see 40, for review). These behaviors are subject to somewhat different neural, hormonal, and environmental controls (e.g., 16,19,29,35), and hence may be differentially influenced by gestational exposure to cocaine. For this reason, several different measures of maternal behavior were assessed. METHOD
Subjects, Breeding, and Chronic Drug Treatments Offspring were generated from Sprague-Dawley (Charles River-VAF/Plus, Kingston, NY) rats bred in our laboratory. Animals were housed in a temperature-controlled colony room on a 12L : 12D cycle with the lights on at 0700 h. All dams were housed in pairs and habituated to the colony room for two weeks after arrival. Prior to the onset of mating, dams randomly assigned to the cocaine treatment group or lab chow control group were handled for 5 min daily for 5 days, and received SC injections of 0.9% saline (3 cc/kg) the last three days of handling. All dams were individually housed in hanging cages prior to mating, with an adult male being placed in each cage daily at 1700 h and removed the following morning at approximately 0900 h. Day 1 of gestation (El) was defined as the day of detection of a copulatory plug at which time each dam was individually housed in Plexigias breeding cages. Dams assigned to the cocaine (C40) and lab chow (LC) treatment groups were given ad lib food (powdered lab chow) and water throughout the study, with dally weights and intake of food and water being recorded from El-E20. Dams were SC injected dally between 1000 and 1200 h with either 40 mg/kg/ 3cc cocaine hydrochloride (C40) or an equal volume of 0.9% saline (LC) from Eg-E20 (see 45). Dams to be used for surrogate fostering were left undisturbed throughout pregnancy except for routine feeding, watering, and cage cleaning. Gestational length was recorded for each gravid dam with the date of birth being designated as postnatal day 0 (P0). All pups were weighed and the total number of pups of each sex recorded. Each litter was culled to 8-10 pups. Rearing condition. The experimental design consisted of six different experimental groups designated by maternal treatment (untreated surrogate foster dam [FOS], LC, or C40) and pup origin (FOS, LC, or CA0), with 10-12 litters being tested in each of the six experimental groups. Group FOS/LC consisted of LC pups fostered by litter to untreated surrogate dams that had given birth to litters within the preceding 24--48 h. The pups from these surrogate foster dams were fostered by litter to the LC dams to form group LC/FOS. Similarly, group FOS/C40 consisted of CA0 pups fostered by litter to untreated surrogate foster dams, with the pups from these surrogate dams being fostered by litter to CA0 dams (group C40/FOS). Pups in the last two groups were not fostered but remained with the biological dam (LC/LC and CA0/CA0). Thus, within this study, the experimental groups consisted of
A FOSTERING STUDY OF THE EFFECTS: I
417
both the typical surrogate fostering (FOS/C40; F O S / L C ) as well as non-fostered (C40/C40; L C / L C ) conditions. In addition, dams from each treatment group rearing untreated foster pups (C40/FOS; LC/FOS) were included to determine whether any observed differences in maternal behavior between C40/C40 and L C / L C litters are related to the prior treatment of the dam per se or whether the prenatal treatment of the pups themselves contributed to these differences.
Maternal Behavior Tests Home cage behavior. On P3 each dam and her rearing litter were placed into a standard maternity cage (20 x 42 x 19 cm) with a ventilated Plexiglas lid to which a water bottle was attached; food pellets were placed inside the maternity cage. The following day (on P4), the behavior o f the dams was videotaped (Panasonic Time Lapse Video Recorder, model 6720A) for 2 h midcycle during both light and dark cycles. Normal colony room lighting was used for video recording during the light cycle and red light served as the illumination source during the night cycle. Videotapes were scored by two independent observers who were unaware of the prior treatment conditions of the dams or their litters. During both 2 h test sessions for each dam, the amount of time dams spent with the pups, as well as the amount of time they spent eating and drinking were determined. Pup retrieval. On P5, four pups (2 males and 2 females) were randomly selected and removed from the nest and placed in the maternity cage on the side opposite to the nest site. The
latency to retrieve each pup was recorded until all four pups were returned to the nest or 10 rain had elapsed. This test of pup retrieval was repeated on P6, 7, 8, and 9, with the four pups used for the retrieval test being randomly selected each day. The experimenters conducting these tests were unaware of the treatment condition o f the litters. Maternal aggression. Aggression toward a female intruder was assessed on P10. A naive adult female intruder was placed into the maternity cage with the resident dam and litter and their interactions were recorded via videotape for 10 rain. Testing was conducted in the colony room between 1000 and 1400 h. Video tapes were viewed by two independent observers who were unaware of prenatal history and rearing condition, with the behavior of the dam being scored for the latency to the first attack, number of attacks, boxing, and pins (dam on top and intruder rolled over on back). In addition, the time spent in submission, defined either as when the forepaws were extended toward the attacking animal with the ventral surface of the body facing the opponent, or when an animal was lying on its back and freezing (motionless for > 25 s) by the intruder female were also determined (see 50). RESULTS
Gestational Data Preliminary analyses of the maternal data revealed no differences with regard to subsequent rearing group assignment within each treatment group (i.e., C40/CA0 vs. CA0/FOS;
TABLE 1 MATERNAL-LiTIER SUMMARY DATA DERIVED FROM LITTER MEANS (SEM IN PARENTHESES) Maternal Data LC Dams
GestationalTreatment Typeof Litter Reared Percent gestational weight gain Food intake (g) Water intake (ml) Gestational length (d)
CA0 Dams
FOS Pups
LC Pups
FOS Pups
C40 Pups
41.79 (1.88) 29.49
41.21
30.68*
30.37*
(1.67)
(2.10)
(2.11)
30.14
25.85
28.67
(0.85)
(1.56)
(0.89)
(1.69)
57.73 (1.30) 22.72 (0.19)
56.81 (2.18) 23.18 (0.12)
55.62 (2.12) 22.91 (0.08)
58.64 (1.86) 22.75 (0.13)
Litter Data LC Pups Prenatal Treatment Reared by Litter size Number of male pups in litter Number of female pups in litter Offspring body weights on Postnatal day I (g) Males t Females
C40 Pups
FOS Dam
LC Dam
FOS Dam
C40 Dam
16.09 (0.65) 8.18 (0.53) 7.91 (0.86)
16.27 (0.65) 7.45 (0.79) 8.63 (0.75)
16.00 (0.80) 8.08 (0.78) 7.83 (0.85)
14.50 (0.80) 6.67 (0.63) 7.83 (0.61)
7.54 (0.20) 7.16 (0.19)
7.82 (0.25) 7.25 (0.23)
7.21 (0.21) 6.53 (0.37)
7.58 (0.21) 6.97 (0.27)
*Significant a t p < 0.05 from LC dams. tSignificant a t p < 0.05 from female pups.
418
I-IEYSER ET AL.
L C / L C vs. LC/FOS), therefore the data were collapsed across this variable prior to data analysis. An analysis o f variance (ANOVA) performed on percentage body weight gain during pregnancy revealed a significant main effect o f Prenatal Treatment, F(1, 44) = 32.40, p < 0.05, with dams in the LC condition gaining significantly more weight than C40 dams (Table 1). Moreover, a 2 x 20 (Prenatal Treatment x Days) repeated measures A N O V A across days on body weights revealed a significant main effect of Prenatal Treatment, F(1, 44) = 4.28, p < 0.05, and Days, F(19, 836) = 306.39, p < 0.05, along with a significant Prenatal Treatment × Days interaction, F(19, 836) = 6.78, p < 0.05. Subsequent Tukey's tests revealed that LC dams weighed more that C40 dams from E l 0 to E20 (Fig. 1A). A 2 × 20 (Prenatal Treatment × Days) repeated measure ANOVA across days on the food intake data revealed a significant main effect o f Days, F(19, 386) = 25.16, p < 0.05, along with a significant Prenatal Treatment × Days interaction, F(19, 836) = 5.58, p < 0.05. Tukey's tests revealed a significant decrease in CA0 dam food intake on ES, E9, El0, El8, and E l 9 compared to LC dams (Fig. 1B). A similar ANOVA conducted on daily water intake revealed a significant main effect of Days, F(19, 836) = 135.32, p < 0.05. Although the interaction was not significant, a trend toward a
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FIG. 1. Mean body weight (A: in grams), food intake (B: in grams), and water intake (C: milliliters) for gestational days 1-20. Error bars indicate SEM's. (LC = saline-injected controls; C40 = cocaine).
reduction in water intake was observed on E8 and E9 in C40 dams when compared to LC dams (Fig. IC). As shown in Table 1, no differences were observed between the two prenatal treatment groups in gestational length.
Litter Size, Composition, and Offspring Body Weight Litter data reflect data collected on P1 prior to fostering of the litters. Hence, data refer to the birth litters rather than rearing litters for C40/FOS and L C / F O S dams. A 2 x 2 (Prenatal Treatment x Sex) ANOVA conducted on the number of male and female pups in each litter failed to reveal any significant differences (Table 1). A similar A_NOVA performed on litter means of male and female body weights at P1 revealed only a significant main effect o f Sex, F(1, 44) = 40.49, p < 0.05, with male pups weighing more than females (Table 1).
Maternal Behavior Tests Data for each dependent measure collected from the six different groups defined by prenatal treatment and rearing condition were analyzed by a one-way ANOVA, with repeated measures where appropriate. Tukey's tests (26) were used to determine the locus o f significant main effects and interactions. A reciprocal transformation was appfied to all the latency data prior to analysis (26). A significance level of p < 0.05 was used for all statistical analyses. Home cage behavior. A 6 x 2 (Condition x Time [light vs. dark]) repeated measures ANOVA across time was conducted for each of the three measured behaviors of the dam in the home cage: time spent with pups and time spent eating and drinking. These ANOVAs revealed only significant main effects of time spent with pups, F ( I , 45) = 43.99, p < 0.05 and time spent drinking,/7(1, 45) = 5.717, p < 0.05. Subsequent Tukey's tests indicated that all dams spent significantly more time with the pups during the fight cycle when compared to the dark cycle and more time drinking in the dark cycle. A similar ANOVA conducted on the time spent eating failed to reveal any differences (all Fs < 1). Pup retrieval. The latency to retrieve the first pup and the total time required to retrieve all four pups were used as the dependent measures. A 6 x 5 (Condition x Days) repeated measures ANOVA, with days as a within-subject variable, conducted on the total time required to retrieve all four pups and reciprocal transformation of the latency to retrieve the first pup revealed no significant differences (all Fs < 1). No significant differences were observed among the various groups on any measure of pup retrieval. Maternal aggression. The latency to the first attack, number of attacks, boxing, and pins were used as dependent measures in the analysis of maternal aggression. An A N O V A performed on the number of attacks revealed a significant effect o f Group, F(5, 49) = 3.52. p < 0.05. Tukey's tests indicated that C40 dams rearing their own pups (C40/C40) made significantly more attacks to the female intruder than all other groups, which did not differ among themselves (Fig. 2A). A similar ANOVA conducted on the reciprocal transformation of the latency to the first attack revealed a significant effect o f Group, F(5, 49) = 3.05, p < 0.05. Tukey's test on the transformed data revealed that dams rearing their biological litters ( L C / L C and C40/C40) initiated the first attack significantly more rapidly than all other groups (Fig. 2B). Analyses conducted on the number of pins and boxing revealed no differences among the groups (all ps > .05). The amount of time spent in a submissive posture and
A FOSTERING STUDY OF THE EFFECTS: I
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they spent with their pups during either the fight or dark cycle. These results are consistent with previous reports of no differences in pup retrieval and nest activities in cocaine-exposed dams rearing their biological fitters (44). However, on the measures of maternal aggression, differences were observed among the treatment/rearing conditions. Dams rearing their biological pups (CAO/C40 and LC/LC) were quicker to attack the female intruder than all groups o f dams not rearing their own fitters (C40/FOS; LC/FOS; FOS/C40; FOS/LC). This aggressiveness was maintained in coeaine-exposed dams rearing their biological pups, with C40/C40 dams emitting significantly more aggressive attacks during the 10-rain test session than all other groups o f dams. This effect was not observed in control dams rearing their biological litters, in that LC/LC dams did not differ from dams not rearing their own litters in total number of attacks. Thus, these data provide evidence that prior cocaine exposure results in alterations in maternal aggression evident when these dams rear their own pups. Dams rearing their biological fitters, regardless of prenatal treatment condition, were quicker to attack a female intruder than dams not rearing their own fitters. Thus, it appeared that these dams respond differentially in terms of maternal aggression when rearing their own pups than when rearing a
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freezing was used to analyze the response of the female intrnder. An ANOVA conducted on the time the intruder spent in a submissive posture revealed a significant main effect of Group, F(5, 49) = 2.42,p < 0.05, with intruders placed with C40 dams rearing their own pups (C40/C40) spending siguificandy more time in submission than all other groups (Fig. 3A). A similar ANOVA conducted on the amount of time the intruder spent freezing revealed a significant main effect of Group, F(5, 49) = 2.35,p < 0.05. Tukey's tests revealed that female intruders placed with LC dams rearing their own pups (LC/LC) spent significantly more time freezing than all other groups (Fig. 3B). No instances of freezing or submission were observed in the resident dam.
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DISCUSSION
In several respects, dams previously exposed to cocaine did not differ from control dams in maternal behavior. Cocaineexposed dams, regardless of whether they were rearing their own pups or untreated foster pups, did not differ from control dams in pup retrieval latencies nor in the amount of time that
FIG. 3. Mean time (in seconds) the intruder spent in a submissive posture (A) and the mean time (seconds) the intruder spent freezing (13) during the test of maternal aggression. Error bars indicate SEM's. (FOS = Foster; LC = saline-injected controls; C40 = cocaine. Groups are designated by maternal treatment and pup origin [e.g., FOS/LC = LC pups reared by FOS dams]).
420
HEYSER ET AL.
nonbiological litter, a distinction that is not disrupted by prior cocaine exposure. It has long been known that rat dams can recognize their offspring (2). Yet, seemingly it has been assumed that this recognition has little influence on subsequent maternal behavior, in that rat dams readily accept and rear foster pups, unlike females of many other species (see 21, for review). The results Of the present study provide initial evidence that the maternal behavior of dams rearing their biological pups is not identical to that of dams rearing nonbiological litters, a finding that may be of importance given the frequent use of surrogate fostering procedures in the field of developmental toxicology. Although both C40/C40 dams and L C / L C dams initiated the first attack more rapidly in the maternal aggression test, only the cocaine-exposed dams rearing their own litters exhibited an increased total number o f aggressive attacks. This was associated with an increased time spent by their intruders in a submissive "on back" posture, a behavior that is seen solely in response to attack. In contrast, although L C / L C dams also attacked rapidly, this aggression was not maintained throughout the test session, with these dams apparently responding to an increase in freezing behavior on the part of the intruders with a reduction in aggressive attacks. The behavior of the intruder was apparently influenced by the behavior of the resident dam, with a submissive posture being emitted in response to direct attack, and an increase in the time spent motionless (freezing) following an attack. Thus, the increased freezing behavior of intruders placed in with LC dams and their biological pups (LC/LC) may be related to the rapid onset of attack observed in these lactating dams. In contrast, intruders placed in with C40 dams rearing their biological litter (C40/C40) may have spent more time in a submissive posture due to the increased number of attacks directed toward them during the test session. The increase in aggressive attacks observed in cocaineexposed dams rearing their own pups was not observed in cocaine-exposed dams rearing foster pups. These findings are reminiscent of the results of previous work examining the effects of gestational and lactational exposure to ethanol on maternal behavior where ethanol-exposed dams were found to differ in maternal behavior from control dams when tested with their own pups but not with control pups (27). Together, these studies suggest that the characteristics of the previously drug-exposed pups themselves may contribute to observed treatment-induced alterations in maternal behavior in their dams. Note, however, that the extent to which these effects are related to the treatment status of the pup versus the genetic relationship of the pups to the dam cannot be determined from the designs of the Mathews and Jamison (27) study or the current experiment. However, in other work, ethanol-
exposed pups have been reported to be less effective than control pups in eliciting retrieval behavior from not only ethanol-exposed, but also control dams (32); these data argue for the critical importance of the treatment status of pups rather than their genetic relationship to the dam. Nevertheless, in future studies that are designed to specifically assess the relative contribution of dams versus the litter in contributing to treatment-induced alterations in maternal/offspring interactions, it would appear important to use a design where litters are systematically fostered both within and across treatment conditions (see 51, for an example). Whereas previously cocaine-exposed dams rearing their own litters exhibited more attacks than the other groups of dams in the maternal aggression test, no differences were observed with respect to pup retrieval latencies or the amount of time spent in the nest during either the light or dark cycle. Indeed, it is often the case that particular experimental treatments may disrupt some feature of maternal behavior, while leaving other aspects intact. For instance, several hypothalamic manipulations have been observed to disrupt maternal aggression toward a female intruder without influencing pup retrieval (19), and treatment with haloperidol has been reported to alter retrieval behavior and nest building but not nursing behavior or licking of the pups (16). These findings emphasize the importance of using multiple measures when assessing the effects of developmental toxicants on maternal behavior, and additionally raise the issue of whether other aspects of maternal behavior that were not assessed in this study might also be affected by prior cocaine treatment (e.g., nest building and maternal licking of the pups). It is not feasible, however, to design a study to investigate every possible aspect of maternal behavior that might potentially be disrupted by a developmental toxicant. Yet, treatment-induced alterations in maternal behavior are primarily of interest in developmental toxicology only to the extent that these alterations influence the subsequent neural and behavioral maturation of the offspring; thus, it is important to examine not only maternal behavior but also offspring outcome. Hence, the following companion article (18) examines the effects of the various treatments and rearing conditions on several behavioral measures in offspring of the dams examined for maternal behavior in the present study. ACKNOWLEDGEMENTS This research was supported by National Institute on Drug Abuse Grants R01 DA14478 and K02 DA00140 to Linda P. Spear. We gratefully acknowledge the assistance of George Ruggiero and Michael Bennenuto in the data collection and Yolanda Pinzas for technical support.
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