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Prenatal cocaine alters open-field behavior in young swine
Neurotoxicology and Teratology, Vol. 17, No. 2, pp. 81-87, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0892-0362/95 $9.50 + .OO
Pergamon 0892-0362(94)00056-5
Prenatal Cocaine Alters Open-Field Behavior in Young Swine ANDRB
LAFERRIGRE,
FATMA
ERTUG
AND
IMMANUELA
R. MOSS’
Department of Pediatrics, McGill University, The Montreal Children’s Hospital, Montreal, Quebec H3H IP3, Canada Received
18 January
1994; Accepted
3 August
1994
LAFERRIGRE, A., F. ERTUG AND I. R. MOSS. Prenatal cocaine alters open-field behavior in young swine. NEUROTOXICOL TERATOL 17(2) 81-87, 1995. -Yucatan minisows received 2 mg/kg cocaine IV 4 times daily during the last
third of gestation. Their piglets were fostered at birth to paired, unexposed sows with their litters, and studied at age 2 to 9 (young group) and 22 to 29 days (older group). Three to 5 exposed and unexposed piglets of each age group were videotaped together for 30 min on 5 consecutive days in an open-field environment. For each piglet, 41 behaviors were scored, timed, summed and clustered into 9 behavioral categories. With age, and independently of drug exposure, piglets spent more time in ingestion, immobility while alone and play/aggression, and less time in group locomotion. For the first 4 test days, the young exposed piglets spent more time in group immobility and less time in individual locomotion and rooting than their age-matched controls. In contrast, the older exposed and unexposed piglet groups did not differ in any of these behavioral clusters. These results suggest that prenatal cocaine exposure in neonatal swine may transiently affect responses to spatial novelty. Cocaine Neonate Swine Group activity Immobility
Behavior
Prenatal exposure
Open-field testing
Development
22,23,33,34). Tests of conditioning have demonstrated significant adverse effects of prenatal cocaine exposure (13,14,33,34) only when the conditioning takes place before maturity (13). Few other learning deficits have been found in active or passive avoidance, runway acquisition, schedule-controlled behavior, or spontaneous alternation even at that young age (7,25,34). It is noteworthy that the rodent is born at a relatively earlier gestational stage than is the human (9,21). For example, the spurt of brain growth, coupled with an enhanced sensitivity to pharmacological agents, takes place during the third trimester in the human and during 1 to 10 postnatal days in the rodent (8,9). Thus, to simulate the end-gestational cocaine exposure in the human, prenatal cocaine administration in rodents must be supplemented postnatally (6,10). We have, therefore, selected as our animal model the miniature swine, a species more comparable to the human in neurological development and general physiology at birth (8,9,36). In that model, we have undertaken to study the effects of prenatal cocaine exposure on behavior in an open-field environment during postnatal development.
PRENATAL cocaine in humans has been linked to a higher incidence of obstetrical, neonatal, developmental, and behavioral disturbances (3,4,5,30). Methodological problems associated with variable dosage of cocaine, multi-drug usage, and psychosocial factors make it difficult to assess the pure effect of cocaine on postnatal development and behavior in the human population (19,24,26,30,31). For these reasons, several laboratories have carried out animal studies, primarily in rodents, regarding the effects of prenatal cocaine exposure on development and behavior. Whereas prenatal cocaine has been shown to affect brain morphology, neurochemistry, and pharmacology in the offspring (1,11,15,16,28), studies of the effects of prenatal cocaine exposure on postnatal behavior have yielded more equivocal results. In measurements of open-field activity, rats exposed prenatally to moderate to high cocaine doses have been found to display either less activity (7,22), more activity (20,23), or the same level of activity as age-matched control animals (25,40). Furthermore, when differences between cocaine-exposed and control animals were observed, such differences were generally limited to the early postnatal period (2,7,13,14,15,20,
’ Requests for reprints should be addressed to Immanuela R. Moss, Pediatrics, McGill University, The Montreal Children’s Hospital, Suite BB-53, 2300 Tupper Street, Montreal, Quebec H3H lP3, Canada. 81
82
LAFERRIGRE,
in Sows
Paired Yucatan minisows (n = 10) were time-inseminated (Charles River, St-Constant, Quebec) and transported to our facilities at 0.64 gestation (1.0 gestation = 115 days) where each sow was housed in an individual, custom-designed farrowing cage (Godro Inc.). At 0.66 gestation, an axillary venous catheter was implanted under general anesthesia (Ketamine, 15 mg/kg and Atropine, 0.04 mg/kg im followed by inhaled Isoflurane, l%-5%) and sterile surgical conditions. The catheter was tunnelled to exit between the shoulders and was connected via an extension to infusion pumps (Harvard Apparatus 22 Basic Syringe Pump and 44 Programmable Syringe Pump). The IV line was protected by a metal tether which was fixed at the proximal end to a custom made jacket fitted to the pig at surgery and, at the distal end, to the pumps. The pumps were programmed to deliver 2 mg/kg cocaine hydrochloride (BDH Inc) as a 1% solution in heparinized saline IV over a lo-min period 4 times daily (at 9:00, ll:OO, 15:00, and 19:00 h) for the remainder of pregnancy. The rationale for this cocaine dose was that, in preliminary experiments, a higher dose (>3 mg/kg cocaine IV) had produced cardiac arrhythmias and seizures. Similar complications, as well as fetal malformations, were reported in response to 3 and 5 mg/kg cocaine IV in pregnant ewes (38). The present dose, 2 mg/kg IV, did not cause such complications, yet produced behavioral arousal as did a similar dose in humans (37). The behavioral activation was expressed by mild stereotypy (head shaking, pacing) on the first days of cocaine infusion and by alert immobility (sitting, perked ears, staring) thereafter. The IV line was programmed to be flushed with heparinized saline before and after each drug injection. The sows received topical and systemic antibiotics during surgery and for 3 days after surgery. The exposed and unexposed sows ingested the same amount of food and exhibited similar appetite, though, for the first few days of cocaine exposure, the exposed sows took longer than the usual 5 min to ingest their food.
Experimental Design and Assessment of Behavior The paired sows were expected to give birth on their due date and within 24 h of each other. When either did not occur,
TABLE DEFINITION
Category Grooming/reflexes Ingestion Elimination Rooting Individual locomotion Individual immobility Group locomotion Group immobility Play/aggression
OF BEHAVIORS
AND
MOSS
the tardy sows (n = 5, 2 unexposed, 3 exposed) were injected with a prostaglandin analog, cloprostenol (Planate; Coopers Agropharm Inc., Ajax, Ontario, Canada) to hasten delivery. Once the sows gave birth, the piglets from the cocaine-exposed dam were fostered immediately and successfully to the unexposed sow and her litter. Room temperature was maintained between 23OC and 25OC throughout the experiment supplemented by infrared lamps, and the sows and piglets were kept on a 12L : 12D cycle. All testing was performed during the early part of the light cycle (9:00 to 11:OO a.m.). The behavior of piglets was assessed in two age groups: young (2 to 9 days old) and older (22 to 29 days old). These age groups have been shown to represent distinct stages in the postnatal maturation of physiological systems such as cardiorespiration and sleep-wake states (27). Within each age-range, 2 groups were studied: Cocaine-exposed and cocaineunexposed piglets. Three to 5 unweaned piglets of the same age-range, and from both sexes and both experimental groups, were studied together on 5 consecutive days. Each piglet was used in one age-range and always tested with the same animals. Piglets were videotaped for 30 min daily while behaving in a fenced open field (1.22 x 2.44 m) with access to milk replacement identical to that available in the home cage ad lib. The floor of the enclosure was covered with paper mats and an infrared heat lamp was positioned over one of its corners for all groups. For every taped session, the occurrence of each of 41 behaviors, grouped into 9 categories (Table l), was recorded from each animal in the sequence of its appearance. The cumulated durations of individual behaviors were summed by category and expressed as a percentage of total session time. Averages of percent of session time allocated to each behavioral category were subjected to a three-way analysis of variance (ANOVA) (GLM-ANOVA, SAS Institute, Cary, NC) with repeated measures on one factor to assess the effect of cocaine exposure, age, and days of testing (the repeated factor). Probabilities related to tests of repeated measure were corrected by the Greenhouse-Geisser procedure. When significant interactions occurred, pairwise comparisons (Tukey’s HSD test) were made between the unexposed and the cocaineexposed groups at each age for each of the 5 test days. As the behavioral cluster scores over the 5 test days among agematched littermates did not exhibit a higher degree of correla-
METHOD
Prenatal Cocaine Administration
ERTUG
AND
1 BEHAVIORAL
CATEGORIES
Behavior
scratching, myoclonic jerking, stretching, yawning, wet-dog shakes, side rubbing, startle jump feeding, drinking, rooting at food defecation, urination rooting/sniffing and pawing at floor/bedding walking, trotting, running, climbing alone standing, sitting, lying down, sleeping alone walking, trotting, running, climbing while in contact with other piglets standing, sitting, lying down, sleeping while in contact with other piglets mouthing, mounting, biting, sniffing, circling, pushing with head, with shoulder, head thrusting, chasing, oral grasping, head shaking
PRENATAL
COCAINE
AND BEHAVIOR
83
IN SWINE
tion than that obtained from unrelated piglets, each piglet was regarded as an independent subject.
0
4.0 -
Measurement of Cocaine Levels
0
CONTROL COCAINE EXPOSED
3.5 -
Three ml blood samples were either drawn from an ear vein of sows just before and 20 min after the completion of the first daily cocaine infusion, or obtained from 3-day-old piglets immediately after sacrifice by decapitation. Each sample was placed immediately in an ice-cold glass tube containing 82 ~1 NaF and 16 ~1 acetic acid, then centrifuged for 20 min at 2,400 g (4OC) to remove cells. The plasma was passed through a centricon filter with a molecular weight cut-off of 10,000 Daltons. The filtered sample was analyzed for cocaine levels by high pressure liquid chromatography (Dionex, Mississauga, Ontario) using a Zorbax RP300-Cl8 column. The eluted peak was revealed by a UV detector at a 225 nm wavelength. The lower detection limit using this method was 43.3 ng/ml of plasma. The presence of cocaine metabolites in the urine of piglets was tested with a kit (Ontrak, Roche Diagnostics) using mouse monoclonal anti-benzoylecgonine antibody with a detection limit of 300 ng/ml.
3.0 2.5 2.0 1.5 1.0 0.5 0.0
L, 0
I
1
I
5
10
15
I
I
20
25
DAYS
RESULTS
Thirty-one piglets from 5 pairs of sows are included in this report. Seventeen were cocaine-exposed (9 young; 8 older) and 14 were unexposed (7 young; 7 older).
FIG. 1. Body weight of cocaine-exposed ing postnatal development.
Cocaine Levels
32) = 66.80, p < 0.0001, but no effect of drug exposure, or an interaction between time and drug exposure. Both the cocaine-treated and untreated sows delivered on gestational day 115 + 2 (mean + SD), the normal duration of gestation in swine. Cocaine-exposed sows had 4.8 +- 1.O (mean f SEM) offspring, 46% males and 54% females, and unexposed sows had 4.3 + 0.6 offspring, 50% males and 50070females. No gross malformations were found in the piglets. Thirty randomly selected piglets from all litters (14 control; 16 cocaine-exposed) and both sexes were weighed on postnatal days 1 to 7, 14 and 21 (Fig. 1). Cocaine-exposed piglets had lower birthweights than did unexposed animals (0.66 f 0.02 kg vs. 0.83 k 0.04 kg (mean + SEM), p < 0.005) and females weighed less than males (unexposed females 0.72 f 0.04 kg (n = 8) versus unexposed males 0.91 f 0.30 kg (n = 6); exposed females 0.62 f 0.03 kg(n = 8)versusexposedmales0.72 + 0.02 kg(n = 8)). A three-way ANOVA revealed effects of drug exposure, F(l,26) = 6.46,~ < 0.01, sex,F(l, 26) = 9.47,~ < 0.0001, and days, F(8, 208) = 388.04, p < 0.0001, on mean body weights, and no interaction between sex or days and drug exposure. Cocaine-exposed piglets, regardless of sex, thus gained weight at the same rate as their unexposed counterparts for the first 21 postnatal days.
Sow and Litter Weights cocaine-exposed sows weighed the same (Table 2) and weight at the same rate as did the unexposed sows the last 5 weeks of gestation: ANOVA performed on measured weights yielded a significant effect of time,
TABLE 2 BODY WEIGHT (KG, MEAN + SEM) OF COCAINE-EXPOSED AND UNEXPOSED SOWS DURING THE LAST 5 WEEKS OF GESTATION Weeks
piglets dur-
F(4,
Plasma cocaine levels between the 105th and 111th gestational day, i.e., following 27 + 6 days of exposure (mean + SEM) were undetectable immediately before, and 4,464 + 1,593 ng/ml (mean f SEM; n = 3) 20 min after the first morning dose. In 3-day-old exposed piglets, plasma cocaine levels were 457 f 133 ng/ml (mean f SEM; n = 2). At 2 and 3 days of age, i.e., the first and second days of behavioral assessment, cocaine metabolites were detected in the urine of all five tested exposed piglets. On the third and fourth test days, 3/4 and 2/4 of the same piglets tested positive, respectively. All tests performed on the last day of behavioral testing were negative.
The gained during weekly
and unexposed
Exposed’
1
54.7
f
3.0
2 3 4 5
59.3 61.8 63.9 67.9
+z + i rt
2.9 2.6 2.2 2.4
ns = not significant.
Unexposed*
51.9 56.3 59.5 63.1 66.8
i 2.8 f 3.0 f 2.6 f 2.2 f 2.5
ln = 5 in each group.
Difference
ns ns M M rls
Open-Field Behavior Effects of age alone were found in 4 of the 9 behavioral categories: Ingestion, immobility when alone, group locomotion, and play/aggression. Older piglets spent more time than young animals ingesting, F(l, 27) = 50.25, p < 0.001, immobile while alone, F( 1, 27) = 36.04, p < 0.0001, and in play/ aggressive interaction with other piglets, F(1, 27) = 5.30, p < 0.02, but less time in group locomotion, F(1,27) = 6.21, p < 0.01. Time allocation to these categories was not affected by either drug exposure or day of testing and there were no
84
LAFERRICRE,
interactions among these factors. These 4 categories together accounted for 6.7% to 49.7% of the mean total session time on any of the 5 test days in any of the four groups. No age, drug, or days-related differences were found in the proportion of time spent in 2 categories: grooming and elimination. These categories accounted for 0.3% to 6.38% of the total session time on any of the 5 test days. In the remaining 3 categories, interactions involving drug exposure were detected as well as effects of repeated testing. Time allocation to group immobility (Fig. 2) in young and older piglets was high initially and decreased over successsive test sessions as shown by an effect of days, F(4, 108) = 5.07, p < 0.0014, and varied depending on piglet age and drug exposure as shown by an interaction of drug exposure and age, F(1, 27) = 6.78, p < 0.01. During the first 4 days of testing, the young cocaine-exposed piglets allocated more time to group immobility than did the young control animals (HSD p < 0.05). Drug-related differences between the young piglet groups in this category were not detected on the 5th day of testing. In contrast, no such differences were found in the older group between exposed and control piglets. The sessional proportion of time spent locomoting alone (Fig. 3) also varied over days depending on age and drug exposure, as indicated by interactions of days and age, F(4, 108) = 5.40, p < 0.005, and drug exposure and age, F( 1, 27) = 6.46, p < 0.01. Thus, on the first 4 test days, young exposed piglets spent less time locomoting alone than unexposed ones, but increased the time spent in this category, reaching control levels by the 5th day of testing. Older exposed piglets did not differ in this category from their age-matched controls, nor did they alter the time spent locomoting alone over days. Rooting (Fig. 4) was the third category affected by drug
0
0 100
exposure, as shown by an interaction of drug, age, and days of testing, F(4, 108) = 4.12, p < 0.05. This interaction reflected the fact that young, but not older piglets gradually increased the proportion of session time engaged in rooting or sniffing at the floor bedding, and did so at a slower rate if they were drug exposed. Young exposed piglets thus spent less time rooting on test days 3 and 4 (HSDp < 0.05), but reached control levels by the 5th day of testing. DISCUSSION
The mean level of cocaine in the plasma of the sows was higher than that found in pregnant sheep 15 min after the same cocaine IV dose (38). This discrepancy can be explained by different methods of cocaine analysis, a species difference in cocaine pharmacokinetics (29), or by the fact that the ovine levels were measured after a single daily cocaine dose, whereas the porcine levels were measured after - 27 consecutive days of 4 daily cocaine doses which likely produced saturation of cocaine in tissues. The cocaine-exposed piglets in our study were smaller at birth than the unexposed piglets despite similar litter size, food intake, and gestational weight gains in their dams, a finding similar to that reported in humans (4). In rodents, only relatively high prenatal cocaine doses (> 60 mg/kg IP or SC) had this effect on pup birthweight (20) whereas moderate doses did not (22,32,33), suggesting that the present dosing in swine is comparable to the higher doses used in rodents. Postnatal malnutrition was likely not a factor in the persistent lower weight of the cocaine-exposed piglets, as both the exposed and unexposed groups gained weight at the same rate. The persistent smallness of the exposed piglets can perhaps be explained by a vasoconstrictive effect of cocaine on the placental
cocaine control
exposed
older
young *
r*
ERTUG AND MOSS
r
80 80
K
0
1
2345801
2
3
4
5
6
Days FIG. 2. Duration (expressed as % of session time) of group immobility in cocaine-exposed and -unexposed, young and older piglets during 5 consecutive days of open-field behavioral assessment. *Difference between results from exposed and unexposed animals.
PRENATAL
COCAINE
AND
BEHAVIOR
85
IN SWINE
cocaine control
0
0
exposed
r
older
Days FIG. 3. Duration (expressed as 070 of session time) of locomotion alone in cocaine-exposed and -unexposed, young and older piglets during 5 consecutive days of open-field behavioral assessment. *Difference between results from exposed and unexposed animals.
cocaine control
0 0
exposed
older F *
T
15 10 5 0 0123456 Days FIG. 4. Duration (expressed as Vo of session time) of rooting in cocaine-exposed and -unexposed, young and older piglets during 5 consecutive days of open-field behavioral assessment. *Difference between results from exposed and unexposed animals.
86
LAFERRIfiRE,
circulation, limiting fetal nutrition in the face of adequate maternal nutrition. Age differences in behavior were expressed by an increase in behaviors performed without contact with conspecifics and directed at environmental stimuli. Thus, standing/sitting alert (immobility) while alone and eating/drinking occupied relatively more session time in older animals. These, combined with relatively less time in group locomotion, appear to indicate an increase in behavioral autonomy or a decrease in herding, with age. In addition, the relatively greater proportion of time spent by the older age group in play/aggression indicates the beginning of social organization, reflecting maturation. The cocaine-related findings in this study were that (a) gestational exposure to cocaine affected only the young piglet group, (b) it enhanced a single behavioral category, i.e., immobility when in contact with conspecifics, and (c) this effect was transient. Thus, the time allocated to group immobility was elevated in the cocaine-exposed young animals for 4 consecutive test days before declining to control on the 5th day of testing. The increased time spent in group immobility in the young exposed group was compensated for by a decrease in time spent in locomotion and rooting. The group immobility category initially occupied the highest proportion of session time and declined gradually with repeated testing in all piglet groups. This suggests that the behaviors included in this category may be related to the reaction of the animals to novelty and/or stress, perhaps in the form of herding. On the assumption that the tendency to remain immobile while in contact with conspecifics expresses a neophobic reaction, the present observations suggest that the younger, cocaine-exposed piglets exhibited enhanced neophobia during the first 4 days of testing. As a corrollary to such neophobia, the piglets engaged less in exploratory behaviors, such as individual locomotion and rooting/sniffing (Table 1). Our findings confirm previous reports of neophobia and hypoactivity in cocaine-exposed rats (7,22,33). In view of the many unsolved controversies regarding the neurochemical changes induced by chronic cocaine administration (39), it seems premature to propose a specific mechanism which would fit with the present findings. If, however, enhanced group immobility is the behavioral correlate of depressed monoaminergic activity, then these findings are consistent with downregulation of dopaminergic systems by chronic, cocaine-induced inhibition of dopamine reuptake, release, or synthesis (e.g., 17,35).
ERTUG
AND
MOSS
The higher level of group immobility in the young cocaineexposed piglets coincided with a detectable presence of cocaine and/or its metabolites. Therefore, processes other than neophobia should be considered in explaining the increased group immobility of the cocaine-exposed piglets. For example, the smaller size of the young exposed animals might increase their sensitivity to the ambient temperature, causing them to huddle together during the first few test days. Such behavior could be accentuated by the peripheral vasoconstrictive effect of the lingering active cocaine, or by an effect of cocaine on the central thermoregulatory set-point (12). Alternatively, the piglets’ hypoactivity might depict an acute withdrawal from cocaine, as was described in human perinates (18). Note, however, that active cocaine levels were measured in our piglets only up until 3 days of age, and that the presence of systemic cocaine metabolites is not necessarily indicative of a continued activity of cocaine but rather may indicate a slow elimination of such metabolites from the circulation. These considerations would argue against an interpretation of the observed behavior as an effect of active systemic cocaine, and would support the notion that our findings reflect a true effect of prenatal cocaine on the development of behavior. Indeed, the fact that the exposed piglets gained weight at a rate identical to that of their unexposed counterparts (Fig. l), combined with the observation that they nursed as vigorously as their foster siblings, strongly suggests that, in the home cage environment, they were not hypoactive. This would support the notion that their increased group immobility in the test environment reflected neophobia. In summary, the present study suggests that the effect of prenatal cocaine on open-field behavior is limited to a transient alteration of the response to spatial novelty in neonatal swine. Inasmuch as our results are similar to those found in rodents during development, they also suggest that these effects of prenatal cocaine apply to a wide range of species. Therefore, further investigation of mechanisms underlying the reaction to novelty may provide a valid model for the subtle, cocaine-induced behavioral impairments reported in the Pediatric literature. ACKNOWLEDGEMENTS
This work was supported by Grant HD-29608 from the United States National Institute for Child Health and Human Development. We thank Cunxian Zhang for measurements of cocaine levels in plasma, and Jing-Ping Xie for computer programming.
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matic growth, and brain development. Neurotoxicol. Teratol. 15: 267-273; 1993. Church, M. W.; Overbeck, 0. W. Prenatal cocaine exposure in the Long-Evans rat: II. Dose-dependent effects on offspring behavior. Neurotoxicol. Teratol. 12:335-343; 1990. Dickerson, J. W. T.; Dobbing, J. Prenatal and postnatal growth and development of the central nervous system of the pig._ J. Physiol. (London) 166:384-395; 1967. Dabbing, J. The later development of the brain and its vulnerability. In: Da&, J. A.; Dobbing, J:, eds. Scientific foundations of paediatrics. Baltimore, MD: University Park Press; 1981:744-759. Dow-Edwards, D. L. Developmental effects of cocaine. NIDA Res. Monogr. 88:290-303; 1988. 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.
PRENATAL COCAINE AND BEHAVIOR IN SWINE 12. Gonzalez, L. P. Cocaine alters body temperature and behavioral thermoregulatory responses. NemoReport 4:106-108; 1993. 13. Goodwin, G. A.; Heyser, C. J.; Moody, C. A.; Rajachandran, L.; Molina, V. A.; Moore Arnold, H.; 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. 14. 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. 15. Heyser, C. J.; Goodwin, G. A.; Moody, C. A.; Spear, L. P. Prenatal cocaine exposure attenutates cocaine-induced odor preference in infant rats. Pharmacol. Biochem. Behav. 42:169-173; 1992. 16. Heyser, C. J.; Miller, J. S.; Spear, N. M.; Spear, L. P. Prenatal exposure to cocaine disrupts cocaine-induced conditioned place preference in rats. Neurotox. Teratol. 14:57-64; 1992. 17. Howard, S.; Blank, D.; Dao, R.; Blank, C. Chronic pre and postnatal exposure to cocaine produces an alteration in the release of dopamine and acetylcholine in the developing rat pup. Sot. Neurosci. Abstr. 18:368; 1992. 18. Hume, R. F.; O’Donnell, K. J.; Stanger C. L.; Killam, A. P.; Gingras, J. L. In utero cocaine exposure: observations of fetal behavioral state may predict neonatal outcome. Am. J. Obstet. Gynecol. 161:685-690; 1989. 19. Hutchings, D. E. The puzzle of cocaine’s effects following maternal use during pregnancy: Are there reconcilable differences? Neurotoxicol. Teratol. 15:281-286; 1993. 20. 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. 21. Jensen, F. E.; Applegate, C. D.; Holtzman, D.; Belin, T. R.; Burchfiel, J. L. Epileptogenic effect of hypoxia in the immature rodent brain. Ann. Neurol. 29:629-637; 1991. 22. Johns, J. M.; Means, L. W.; Means, M. J.; McMillen, B. A. Prenatal exposure to cocaine I: Effects on gestation, development and activity in Sprague-Dawley rats. Neurotoxicol. Teratol. 14: 337-342; 1992. 23. Johns, J. M.; Means, M. J.; Anderson, D. R; Means, L. W.; McMillen, B. A. Prenatal exposure to cocaine II. Effects on openfield activity and cognitive behavior in Sprague-Dawley rats. Neurotoxicol. Teratol. 14:343-349; 1992. 24. Mayes, L. C.; &anger, R. H.; Bornstein, M. H.; Zuckerman, B. The problem of prenatal cocaine exposure. JAMA 267:406-408; 1992. 25. Means, L. W.; Bass, E. W.; Fernandez, T. J.; McMillen, B. A. Prenatal cocaine has minimal effects on development and adult behavior in rats. Neurosci. Abstr. 18:368; 1992.
87 26. Neuspiel, D. R.; Hamel, S. C. Neurobehavioral sequelae of fetal cocaine exposure. Editorial correspondence. J. Pediatr. 120:661; 1992. 27. Scott, S.C.; Inman, J. D. G.; Moss, I. R. Ontogeny of sleep/ wake and cardiorespiratory behavior in unanesthetized piglets. Respir. Physiol. 80:83-102; 1990. 28. Seidler, F. J.; Slotkin, T. A. Fetal cocaine exposure causes persistent noradrenergic hypoactivity in rat brain regions: Effects on neurotransmitter turnover and receptors. J. Pharmacol. Exp. Ther. 263:413-421; 1992. 29. Shuster, L. S. Pharmacokinetics, metabolism, and disposition of cocaine. In: Lakoski, J. M.; Galloway, M. P.; White, F. J., eds. Cocaine: Pharmacology, physiology and clinical strategies. Boca Raton, FL: CRC Press; 1992:1-14. 30. Singer, L. T.; Garber, R.; Kliegman, R. Childhood medical and behavioral consequences of maternal cocaine use. J. Pediatr. Psychol. 17:389-406; 1992. 31. Slutsker, L. Risks associated with cocaine use during pregnancy. Obstet. Gynecol. 79:778-789: 1992. 32. Smith, RI 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. 33. Spear, L. P.; Kirstein, C. L.; Bell, J.; Yoottanasumpun, V.; Greenbaum, 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. 34. 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. 35. Trulson, M. E.; Ulissey, M. J. Chronic cocaine administration decreases dopamine synthesis rate and increases ‘H-spiroperidol binding in rat brain. Brain Res. Bull. 19:35; 1987. 36. Tumbleson, M. E. (Ed). Swine in biomedical research. New York: Plenum Press; 1986. 37. Van Dyke, C.; Jatlow, P.; Ungerer, J.; Barash, P. G.; Byck, R. Oral cocaine: Plasma concentrations and central effects. Science 200:211-213; 1978. 38. Woods, J. R.; Plesinger, M. A.; Scott, K.; Miller, R. K. Prenatal cocaine exposure to the fetus: A sheep model for cardiovascular function. Ann. NY Acad. Sci. 562~267-279; 1989. 39. Zahniser, N. R.; Peris, J. Neurochemical mechanisms of cocaineinduced sensitization. In: Lakoski, J. M.; Galloway, M. P.; White, F. J., eds. Cocaine: Pharmacology, physiology and clinical strategies. Boca Raton, FL: CRC Press; 1992:229-260. 40. Zmitrovich, A. C.; Hutchings, D. E.; Dow-Edwards, D. L.; Malowany, D.; Church, S. Effects of prenatal exposure to cocaine on the rest-activity cycle of the preweanling rat. Pharmacol. Biothem. Behav. 43:1059-1064; 1992.
Pergamon 0892-0362(94)00056-5
Prenatal Cocaine Alters Open-Field Behavior in Young Swine ANDRB
LAFERRIGRE,
FATMA
ERTUG
AND
IMMANUELA
R. MOSS’
Department of Pediatrics, McGill University, The Montreal Children’s Hospital, Montreal, Quebec H3H IP3, Canada Received
18 January
1994; Accepted
3 August
1994
LAFERRIGRE, A., F. ERTUG AND I. R. MOSS. Prenatal cocaine alters open-field behavior in young swine. NEUROTOXICOL TERATOL 17(2) 81-87, 1995. -Yucatan minisows received 2 mg/kg cocaine IV 4 times daily during the last
third of gestation. Their piglets were fostered at birth to paired, unexposed sows with their litters, and studied at age 2 to 9 (young group) and 22 to 29 days (older group). Three to 5 exposed and unexposed piglets of each age group were videotaped together for 30 min on 5 consecutive days in an open-field environment. For each piglet, 41 behaviors were scored, timed, summed and clustered into 9 behavioral categories. With age, and independently of drug exposure, piglets spent more time in ingestion, immobility while alone and play/aggression, and less time in group locomotion. For the first 4 test days, the young exposed piglets spent more time in group immobility and less time in individual locomotion and rooting than their age-matched controls. In contrast, the older exposed and unexposed piglet groups did not differ in any of these behavioral clusters. These results suggest that prenatal cocaine exposure in neonatal swine may transiently affect responses to spatial novelty. Cocaine Neonate Swine Group activity Immobility
Behavior
Prenatal exposure
Open-field testing
Development
22,23,33,34). Tests of conditioning have demonstrated significant adverse effects of prenatal cocaine exposure (13,14,33,34) only when the conditioning takes place before maturity (13). Few other learning deficits have been found in active or passive avoidance, runway acquisition, schedule-controlled behavior, or spontaneous alternation even at that young age (7,25,34). It is noteworthy that the rodent is born at a relatively earlier gestational stage than is the human (9,21). For example, the spurt of brain growth, coupled with an enhanced sensitivity to pharmacological agents, takes place during the third trimester in the human and during 1 to 10 postnatal days in the rodent (8,9). Thus, to simulate the end-gestational cocaine exposure in the human, prenatal cocaine administration in rodents must be supplemented postnatally (6,10). We have, therefore, selected as our animal model the miniature swine, a species more comparable to the human in neurological development and general physiology at birth (8,9,36). In that model, we have undertaken to study the effects of prenatal cocaine exposure on behavior in an open-field environment during postnatal development.
PRENATAL cocaine in humans has been linked to a higher incidence of obstetrical, neonatal, developmental, and behavioral disturbances (3,4,5,30). Methodological problems associated with variable dosage of cocaine, multi-drug usage, and psychosocial factors make it difficult to assess the pure effect of cocaine on postnatal development and behavior in the human population (19,24,26,30,31). For these reasons, several laboratories have carried out animal studies, primarily in rodents, regarding the effects of prenatal cocaine exposure on development and behavior. Whereas prenatal cocaine has been shown to affect brain morphology, neurochemistry, and pharmacology in the offspring (1,11,15,16,28), studies of the effects of prenatal cocaine exposure on postnatal behavior have yielded more equivocal results. In measurements of open-field activity, rats exposed prenatally to moderate to high cocaine doses have been found to display either less activity (7,22), more activity (20,23), or the same level of activity as age-matched control animals (25,40). Furthermore, when differences between cocaine-exposed and control animals were observed, such differences were generally limited to the early postnatal period (2,7,13,14,15,20,
’ Requests for reprints should be addressed to Immanuela R. Moss, Pediatrics, McGill University, The Montreal Children’s Hospital, Suite BB-53, 2300 Tupper Street, Montreal, Quebec H3H lP3, Canada. 81
82
LAFERRIGRE,
in Sows
Paired Yucatan minisows (n = 10) were time-inseminated (Charles River, St-Constant, Quebec) and transported to our facilities at 0.64 gestation (1.0 gestation = 115 days) where each sow was housed in an individual, custom-designed farrowing cage (Godro Inc.). At 0.66 gestation, an axillary venous catheter was implanted under general anesthesia (Ketamine, 15 mg/kg and Atropine, 0.04 mg/kg im followed by inhaled Isoflurane, l%-5%) and sterile surgical conditions. The catheter was tunnelled to exit between the shoulders and was connected via an extension to infusion pumps (Harvard Apparatus 22 Basic Syringe Pump and 44 Programmable Syringe Pump). The IV line was protected by a metal tether which was fixed at the proximal end to a custom made jacket fitted to the pig at surgery and, at the distal end, to the pumps. The pumps were programmed to deliver 2 mg/kg cocaine hydrochloride (BDH Inc) as a 1% solution in heparinized saline IV over a lo-min period 4 times daily (at 9:00, ll:OO, 15:00, and 19:00 h) for the remainder of pregnancy. The rationale for this cocaine dose was that, in preliminary experiments, a higher dose (>3 mg/kg cocaine IV) had produced cardiac arrhythmias and seizures. Similar complications, as well as fetal malformations, were reported in response to 3 and 5 mg/kg cocaine IV in pregnant ewes (38). The present dose, 2 mg/kg IV, did not cause such complications, yet produced behavioral arousal as did a similar dose in humans (37). The behavioral activation was expressed by mild stereotypy (head shaking, pacing) on the first days of cocaine infusion and by alert immobility (sitting, perked ears, staring) thereafter. The IV line was programmed to be flushed with heparinized saline before and after each drug injection. The sows received topical and systemic antibiotics during surgery and for 3 days after surgery. The exposed and unexposed sows ingested the same amount of food and exhibited similar appetite, though, for the first few days of cocaine exposure, the exposed sows took longer than the usual 5 min to ingest their food.
Experimental Design and Assessment of Behavior The paired sows were expected to give birth on their due date and within 24 h of each other. When either did not occur,
TABLE DEFINITION
Category Grooming/reflexes Ingestion Elimination Rooting Individual locomotion Individual immobility Group locomotion Group immobility Play/aggression
OF BEHAVIORS
AND
MOSS
the tardy sows (n = 5, 2 unexposed, 3 exposed) were injected with a prostaglandin analog, cloprostenol (Planate; Coopers Agropharm Inc., Ajax, Ontario, Canada) to hasten delivery. Once the sows gave birth, the piglets from the cocaine-exposed dam were fostered immediately and successfully to the unexposed sow and her litter. Room temperature was maintained between 23OC and 25OC throughout the experiment supplemented by infrared lamps, and the sows and piglets were kept on a 12L : 12D cycle. All testing was performed during the early part of the light cycle (9:00 to 11:OO a.m.). The behavior of piglets was assessed in two age groups: young (2 to 9 days old) and older (22 to 29 days old). These age groups have been shown to represent distinct stages in the postnatal maturation of physiological systems such as cardiorespiration and sleep-wake states (27). Within each age-range, 2 groups were studied: Cocaine-exposed and cocaineunexposed piglets. Three to 5 unweaned piglets of the same age-range, and from both sexes and both experimental groups, were studied together on 5 consecutive days. Each piglet was used in one age-range and always tested with the same animals. Piglets were videotaped for 30 min daily while behaving in a fenced open field (1.22 x 2.44 m) with access to milk replacement identical to that available in the home cage ad lib. The floor of the enclosure was covered with paper mats and an infrared heat lamp was positioned over one of its corners for all groups. For every taped session, the occurrence of each of 41 behaviors, grouped into 9 categories (Table l), was recorded from each animal in the sequence of its appearance. The cumulated durations of individual behaviors were summed by category and expressed as a percentage of total session time. Averages of percent of session time allocated to each behavioral category were subjected to a three-way analysis of variance (ANOVA) (GLM-ANOVA, SAS Institute, Cary, NC) with repeated measures on one factor to assess the effect of cocaine exposure, age, and days of testing (the repeated factor). Probabilities related to tests of repeated measure were corrected by the Greenhouse-Geisser procedure. When significant interactions occurred, pairwise comparisons (Tukey’s HSD test) were made between the unexposed and the cocaineexposed groups at each age for each of the 5 test days. As the behavioral cluster scores over the 5 test days among agematched littermates did not exhibit a higher degree of correla-
METHOD
Prenatal Cocaine Administration
ERTUG
AND
1 BEHAVIORAL
CATEGORIES
Behavior
scratching, myoclonic jerking, stretching, yawning, wet-dog shakes, side rubbing, startle jump feeding, drinking, rooting at food defecation, urination rooting/sniffing and pawing at floor/bedding walking, trotting, running, climbing alone standing, sitting, lying down, sleeping alone walking, trotting, running, climbing while in contact with other piglets standing, sitting, lying down, sleeping while in contact with other piglets mouthing, mounting, biting, sniffing, circling, pushing with head, with shoulder, head thrusting, chasing, oral grasping, head shaking
PRENATAL
COCAINE
AND BEHAVIOR
83
IN SWINE
tion than that obtained from unrelated piglets, each piglet was regarded as an independent subject.
0
4.0 -
Measurement of Cocaine Levels
0
CONTROL COCAINE EXPOSED
3.5 -
Three ml blood samples were either drawn from an ear vein of sows just before and 20 min after the completion of the first daily cocaine infusion, or obtained from 3-day-old piglets immediately after sacrifice by decapitation. Each sample was placed immediately in an ice-cold glass tube containing 82 ~1 NaF and 16 ~1 acetic acid, then centrifuged for 20 min at 2,400 g (4OC) to remove cells. The plasma was passed through a centricon filter with a molecular weight cut-off of 10,000 Daltons. The filtered sample was analyzed for cocaine levels by high pressure liquid chromatography (Dionex, Mississauga, Ontario) using a Zorbax RP300-Cl8 column. The eluted peak was revealed by a UV detector at a 225 nm wavelength. The lower detection limit using this method was 43.3 ng/ml of plasma. The presence of cocaine metabolites in the urine of piglets was tested with a kit (Ontrak, Roche Diagnostics) using mouse monoclonal anti-benzoylecgonine antibody with a detection limit of 300 ng/ml.
3.0 2.5 2.0 1.5 1.0 0.5 0.0
L, 0
I
1
I
5
10
15
I
I
20
25
DAYS
RESULTS
Thirty-one piglets from 5 pairs of sows are included in this report. Seventeen were cocaine-exposed (9 young; 8 older) and 14 were unexposed (7 young; 7 older).
FIG. 1. Body weight of cocaine-exposed ing postnatal development.
Cocaine Levels
32) = 66.80, p < 0.0001, but no effect of drug exposure, or an interaction between time and drug exposure. Both the cocaine-treated and untreated sows delivered on gestational day 115 + 2 (mean + SD), the normal duration of gestation in swine. Cocaine-exposed sows had 4.8 +- 1.O (mean f SEM) offspring, 46% males and 54% females, and unexposed sows had 4.3 + 0.6 offspring, 50% males and 50070females. No gross malformations were found in the piglets. Thirty randomly selected piglets from all litters (14 control; 16 cocaine-exposed) and both sexes were weighed on postnatal days 1 to 7, 14 and 21 (Fig. 1). Cocaine-exposed piglets had lower birthweights than did unexposed animals (0.66 f 0.02 kg vs. 0.83 k 0.04 kg (mean + SEM), p < 0.005) and females weighed less than males (unexposed females 0.72 f 0.04 kg (n = 8) versus unexposed males 0.91 f 0.30 kg (n = 6); exposed females 0.62 f 0.03 kg(n = 8)versusexposedmales0.72 + 0.02 kg(n = 8)). A three-way ANOVA revealed effects of drug exposure, F(l,26) = 6.46,~ < 0.01, sex,F(l, 26) = 9.47,~ < 0.0001, and days, F(8, 208) = 388.04, p < 0.0001, on mean body weights, and no interaction between sex or days and drug exposure. Cocaine-exposed piglets, regardless of sex, thus gained weight at the same rate as their unexposed counterparts for the first 21 postnatal days.
Sow and Litter Weights cocaine-exposed sows weighed the same (Table 2) and weight at the same rate as did the unexposed sows the last 5 weeks of gestation: ANOVA performed on measured weights yielded a significant effect of time,
TABLE 2 BODY WEIGHT (KG, MEAN + SEM) OF COCAINE-EXPOSED AND UNEXPOSED SOWS DURING THE LAST 5 WEEKS OF GESTATION Weeks
piglets dur-
F(4,
Plasma cocaine levels between the 105th and 111th gestational day, i.e., following 27 + 6 days of exposure (mean + SEM) were undetectable immediately before, and 4,464 + 1,593 ng/ml (mean f SEM; n = 3) 20 min after the first morning dose. In 3-day-old exposed piglets, plasma cocaine levels were 457 f 133 ng/ml (mean f SEM; n = 2). At 2 and 3 days of age, i.e., the first and second days of behavioral assessment, cocaine metabolites were detected in the urine of all five tested exposed piglets. On the third and fourth test days, 3/4 and 2/4 of the same piglets tested positive, respectively. All tests performed on the last day of behavioral testing were negative.
The gained during weekly
and unexposed
Exposed’
1
54.7
f
3.0
2 3 4 5
59.3 61.8 63.9 67.9
+z + i rt
2.9 2.6 2.2 2.4
ns = not significant.
Unexposed*
51.9 56.3 59.5 63.1 66.8
i 2.8 f 3.0 f 2.6 f 2.2 f 2.5
ln = 5 in each group.
Difference
ns ns M M rls
Open-Field Behavior Effects of age alone were found in 4 of the 9 behavioral categories: Ingestion, immobility when alone, group locomotion, and play/aggression. Older piglets spent more time than young animals ingesting, F(l, 27) = 50.25, p < 0.001, immobile while alone, F( 1, 27) = 36.04, p < 0.0001, and in play/ aggressive interaction with other piglets, F(1, 27) = 5.30, p < 0.02, but less time in group locomotion, F(1,27) = 6.21, p < 0.01. Time allocation to these categories was not affected by either drug exposure or day of testing and there were no
84
LAFERRICRE,
interactions among these factors. These 4 categories together accounted for 6.7% to 49.7% of the mean total session time on any of the 5 test days in any of the four groups. No age, drug, or days-related differences were found in the proportion of time spent in 2 categories: grooming and elimination. These categories accounted for 0.3% to 6.38% of the total session time on any of the 5 test days. In the remaining 3 categories, interactions involving drug exposure were detected as well as effects of repeated testing. Time allocation to group immobility (Fig. 2) in young and older piglets was high initially and decreased over successsive test sessions as shown by an effect of days, F(4, 108) = 5.07, p < 0.0014, and varied depending on piglet age and drug exposure as shown by an interaction of drug exposure and age, F(1, 27) = 6.78, p < 0.01. During the first 4 days of testing, the young cocaine-exposed piglets allocated more time to group immobility than did the young control animals (HSD p < 0.05). Drug-related differences between the young piglet groups in this category were not detected on the 5th day of testing. In contrast, no such differences were found in the older group between exposed and control piglets. The sessional proportion of time spent locomoting alone (Fig. 3) also varied over days depending on age and drug exposure, as indicated by interactions of days and age, F(4, 108) = 5.40, p < 0.005, and drug exposure and age, F( 1, 27) = 6.46, p < 0.01. Thus, on the first 4 test days, young exposed piglets spent less time locomoting alone than unexposed ones, but increased the time spent in this category, reaching control levels by the 5th day of testing. Older exposed piglets did not differ in this category from their age-matched controls, nor did they alter the time spent locomoting alone over days. Rooting (Fig. 4) was the third category affected by drug
0
0 100
exposure, as shown by an interaction of drug, age, and days of testing, F(4, 108) = 4.12, p < 0.05. This interaction reflected the fact that young, but not older piglets gradually increased the proportion of session time engaged in rooting or sniffing at the floor bedding, and did so at a slower rate if they were drug exposed. Young exposed piglets thus spent less time rooting on test days 3 and 4 (HSDp < 0.05), but reached control levels by the 5th day of testing. DISCUSSION
The mean level of cocaine in the plasma of the sows was higher than that found in pregnant sheep 15 min after the same cocaine IV dose (38). This discrepancy can be explained by different methods of cocaine analysis, a species difference in cocaine pharmacokinetics (29), or by the fact that the ovine levels were measured after a single daily cocaine dose, whereas the porcine levels were measured after - 27 consecutive days of 4 daily cocaine doses which likely produced saturation of cocaine in tissues. The cocaine-exposed piglets in our study were smaller at birth than the unexposed piglets despite similar litter size, food intake, and gestational weight gains in their dams, a finding similar to that reported in humans (4). In rodents, only relatively high prenatal cocaine doses (> 60 mg/kg IP or SC) had this effect on pup birthweight (20) whereas moderate doses did not (22,32,33), suggesting that the present dosing in swine is comparable to the higher doses used in rodents. Postnatal malnutrition was likely not a factor in the persistent lower weight of the cocaine-exposed piglets, as both the exposed and unexposed groups gained weight at the same rate. The persistent smallness of the exposed piglets can perhaps be explained by a vasoconstrictive effect of cocaine on the placental
cocaine control
exposed
older
young *
r*
ERTUG AND MOSS
r
80 80
K
0
1
2345801
2
3
4
5
6
Days FIG. 2. Duration (expressed as % of session time) of group immobility in cocaine-exposed and -unexposed, young and older piglets during 5 consecutive days of open-field behavioral assessment. *Difference between results from exposed and unexposed animals.
PRENATAL
COCAINE
AND
BEHAVIOR
85
IN SWINE
cocaine control
0
0
exposed
r
older
Days FIG. 3. Duration (expressed as 070 of session time) of locomotion alone in cocaine-exposed and -unexposed, young and older piglets during 5 consecutive days of open-field behavioral assessment. *Difference between results from exposed and unexposed animals.
cocaine control
0 0
exposed
older F *
T
15 10 5 0 0123456 Days FIG. 4. Duration (expressed as Vo of session time) of rooting in cocaine-exposed and -unexposed, young and older piglets during 5 consecutive days of open-field behavioral assessment. *Difference between results from exposed and unexposed animals.
86
LAFERRIfiRE,
circulation, limiting fetal nutrition in the face of adequate maternal nutrition. Age differences in behavior were expressed by an increase in behaviors performed without contact with conspecifics and directed at environmental stimuli. Thus, standing/sitting alert (immobility) while alone and eating/drinking occupied relatively more session time in older animals. These, combined with relatively less time in group locomotion, appear to indicate an increase in behavioral autonomy or a decrease in herding, with age. In addition, the relatively greater proportion of time spent by the older age group in play/aggression indicates the beginning of social organization, reflecting maturation. The cocaine-related findings in this study were that (a) gestational exposure to cocaine affected only the young piglet group, (b) it enhanced a single behavioral category, i.e., immobility when in contact with conspecifics, and (c) this effect was transient. Thus, the time allocated to group immobility was elevated in the cocaine-exposed young animals for 4 consecutive test days before declining to control on the 5th day of testing. The increased time spent in group immobility in the young exposed group was compensated for by a decrease in time spent in locomotion and rooting. The group immobility category initially occupied the highest proportion of session time and declined gradually with repeated testing in all piglet groups. This suggests that the behaviors included in this category may be related to the reaction of the animals to novelty and/or stress, perhaps in the form of herding. On the assumption that the tendency to remain immobile while in contact with conspecifics expresses a neophobic reaction, the present observations suggest that the younger, cocaine-exposed piglets exhibited enhanced neophobia during the first 4 days of testing. As a corrollary to such neophobia, the piglets engaged less in exploratory behaviors, such as individual locomotion and rooting/sniffing (Table 1). Our findings confirm previous reports of neophobia and hypoactivity in cocaine-exposed rats (7,22,33). In view of the many unsolved controversies regarding the neurochemical changes induced by chronic cocaine administration (39), it seems premature to propose a specific mechanism which would fit with the present findings. If, however, enhanced group immobility is the behavioral correlate of depressed monoaminergic activity, then these findings are consistent with downregulation of dopaminergic systems by chronic, cocaine-induced inhibition of dopamine reuptake, release, or synthesis (e.g., 17,35).
ERTUG
AND
MOSS
The higher level of group immobility in the young cocaineexposed piglets coincided with a detectable presence of cocaine and/or its metabolites. Therefore, processes other than neophobia should be considered in explaining the increased group immobility of the cocaine-exposed piglets. For example, the smaller size of the young exposed animals might increase their sensitivity to the ambient temperature, causing them to huddle together during the first few test days. Such behavior could be accentuated by the peripheral vasoconstrictive effect of the lingering active cocaine, or by an effect of cocaine on the central thermoregulatory set-point (12). Alternatively, the piglets’ hypoactivity might depict an acute withdrawal from cocaine, as was described in human perinates (18). Note, however, that active cocaine levels were measured in our piglets only up until 3 days of age, and that the presence of systemic cocaine metabolites is not necessarily indicative of a continued activity of cocaine but rather may indicate a slow elimination of such metabolites from the circulation. These considerations would argue against an interpretation of the observed behavior as an effect of active systemic cocaine, and would support the notion that our findings reflect a true effect of prenatal cocaine on the development of behavior. Indeed, the fact that the exposed piglets gained weight at a rate identical to that of their unexposed counterparts (Fig. l), combined with the observation that they nursed as vigorously as their foster siblings, strongly suggests that, in the home cage environment, they were not hypoactive. This would support the notion that their increased group immobility in the test environment reflected neophobia. In summary, the present study suggests that the effect of prenatal cocaine on open-field behavior is limited to a transient alteration of the response to spatial novelty in neonatal swine. Inasmuch as our results are similar to those found in rodents during development, they also suggest that these effects of prenatal cocaine apply to a wide range of species. Therefore, further investigation of mechanisms underlying the reaction to novelty may provide a valid model for the subtle, cocaine-induced behavioral impairments reported in the Pediatric literature. ACKNOWLEDGEMENTS
This work was supported by Grant HD-29608 from the United States National Institute for Child Health and Human Development. We thank Cunxian Zhang for measurements of cocaine levels in plasma, and Jing-Ping Xie for computer programming.
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