FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

19,474-477 (1992)

SHORT COMMUNICATION Behavioral Development

Following Daily Episodes of Mother-Infant Separation in the Rat’

handled daily as neonates and separated from their dams for 8 hr on Postnatal Days (PND) 8, 10, and 12 (Nadel and Willner, 1989) or in rats subjected to an early-life malnutrition procedure involving daily 12-hr separation from a lacUseof dermalor inhalation routesof maternalexposureduring tating female (Castro and Rudy, 1989). These reports raise the postnatal period in rodent developmental neurotoxicity the possibility that dermal or inhalation exposure protocols evaluationswould be most practical if damscould be separated involving repeated separation of rat dams from their pups from their pups during the exposureperiod. However, this pro- for an extended period of development would be inapprocedureraisesquestionsconcerning the effects of mother-infant priate for developmental neurotoxicity studies because of separationitself on neurotoxicity endpoints.In the presentstudy, inherent effects of maternal separation on behavioral endSprague-Dawley rat pups were either maternally deprived in points in their offspring (Kimmel and Francis, 1990). Howwarm incubators for 6 hr each day (7:00 AM-1:OO PM) or left ever, these protocols typically involve daily separation periods with their dams (control), from Postnatal Day 4-20 (PND4on the effects of this amount 20), and were testedon a range of endpointscommonly usedin of 6 hr and more information developmentalneurotoxicology. These included motor activity of separation on behavioral development is needed (Kimmel (PND13, 17, 19, 21, 29, 60), olfactory learning (PND18) and and Francis, 1990). The present study determined the effects retention (PND25), T-maze delayed alternation (PND23, 24), of such separation on a number of behavioral endpoints acousticstartle response(PND23,62), and auditory thresholds commonly used in developmental neurotoxicity evaluations.

BehavioralDevelopmentFollowing Daily Episodesof MotherInfant Separation in the Rat. STANTON, M. E., CROFTON, K. M., AND LAU, C. (1992). Fundam.Appl. Toxicol. 19,474-477.

(PND62). None of the behavioral measureswere affected by daily separation.Apparently, interrupting the mother-infant interaction for 6 hr/day haslittle or no effect by itself on behavioral development,as assessed by thesemeasures.o 1992 soci~tyofToxic01og~.

The effects of disruption of the mother-infant relationship on behavioral development may have both conceptual and practical implications for developmental neurotoxicology. Such disruption is, of course, one possible effect of developmental exposure to toxicants and could mediate or modulate neurobehavioral toxicity. At a practical level, motherinfant separation may, in some cases,be part of an exposure protocol. For example, developmental neurotoxicity studies submitted to government regulatory agenciesmay involve maternal separation in order to permit postnatal maternal exposure to an agent by dermal or inhalation routes (Kimmel and Francis, 1990). Given these implications, there is surprisingly little infor-

mation on the effects of mother-infant separation per se on behavioral development in the rat. There are reports of altered spatial learning and/or exploration in rats that were I This work has been reviewed by the Health EffectsResearch Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

0272-0590/92%5.00 Copyright All rights

0 1992 by the Society of Toxicology. of reproduction in any form reserved.

474

METHOD Time-pregnant female Sprague-Dawley rats were obtained from a local supplier (Charles River, Raleigh, NC) on GD2 and housed individually at the EPA animal colony in maternity cages under laboratory conditions that have been described more fully elsewhere (Stanton, 1991). On PND3, litters were culled to 10 pups with equal numbers of males and females (day of birth is PNDO). On PND4-20 different litters of rat pups were either maternally deprived as a litter in temperature- and humidity-controlled incubators (Servo-Care, Ohio Medical Products, Madison, WI) for 6 hr each day (7:00 AM-1:OO PM) or left alone with their dams. Incubators were maintained at 33°C from PND4-9,29”C from PNDlO-14, and 27°C thereafter. These temperature adjustments accommodate developmental increases in thermoregulatory function of rat pups and approximate nest temperature conditions (Leon, 1986). Humidity in the incubators was maintained at approximately 70% throughout the study. Offspring from each treatment group were evaluated on a number of tests at various stages of development, as summarized in Table 1. The study involved four separate cohorts of four litters. In each cohort two litters were assigned to the daily-separation condition and two to the control condition. The first two cohorts contributed subjects to the tests of olfactory conditioning and T-maze learning. One male and one female pup from each litter was assigned to each test, yielding sample sizes of 8 pups/ group per cohort of four litters. The other two cohorts contributed subjects again to the test of olfactory conditioning and to the tests of motor activity, acoustic startle response, and reflex modification audiometry. In these cohorts, one pup/sex/litter was assessedlongitudinally on the latter three tests. When testing occurred prior to PNDZO, procedures were carried out at the onset of the separation period. In this way, recent separation could not influence testing and testing could not extend separation beyond 6 hr.

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475

COMMUNICATION

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Postnatal

Age (days)

Postnatal

Age (days)

FIG. 1. Lack of effect of repeated maternal separation on the ontogeny of motor activity. Data points represent mean (*SE). Left, data from tests conducted on PND 13-2 1. Right, data from tests conducted on PND29 and 60. Control, open circles, open bars; deprived, closed circles, closed bars.

Details of the testing procedures can be found in previously published reports. Motor activity was monitored according to the method of Ruppert et al. (1984a, 1985). Rats were tested individually in figure-eight mazes for 30 min daily on PND13, 15, 17, 19, and 2 I and for 1 hr on PND29 and 60. Olfactory discrimination learning (PND18) and retention (PND25) were assessedaccording to Stanton’s ( 199 1) adaptation of the method of Kucharski and Spear ( 1984). Briefly, pups received four training trials involving exposure to two odors (peppermint and lemon) separately, one paired with footshock (CS+) and the other presented alone (CS-). Preference between the two odors was then determined in a 180-set test. Discrimination learning/retention consists of a reduction in preference for the CS+ odor relative to a 90set (chance) baseline. Spatial delayed alternation was tested in a T-maze, according to the procedure of Freeman and Stanton (199 1). Briefly, pups received five 12-trial blocks of training on discrete-trials alternation in a single day (PND23 for half the pups, PND24 for the other half). Trials consisted of a pair of runs, separated by a delay of 2 set: a “forced run,” in

TABLE 1 Scheduleof Treatment and Testing in Assessingthe Effects of Daily Maternal Separation on Offspring Development and Behavior 4%

Treatment/test

PND13, 15, 17, 19, 21 PNDl8/25 PND23-24 PND24 PND29 PND60 PND62

Deprivation (or no treatment) from 7:00 AM to I:00 PM Motor activity Olfactory learning/retention T-maze delayed alternation learning Acoustic startle response Motor activity Motor activity Reflex modification audiometry

PND4-20

Note. PND, postnatal day.

which only one arm could be entered in order to obtain light-cream reward, and a “choice run,” in which both arms were available and pups were rewarded only for entering the arm alternate to that entered on the preceding forced run. Finally, acoustic startle response (ASR) and auditory thresholds (reflex modification of ASR) were tested on PND24 and 62, according to the procedures of Ruppert et al. (1984b) and Young and Fechter (1983). For ASR testing, each rat was placed in a test apparatus and, following a IO-min adaptation period at a low background noise level [60 db(A)], received a total of 50 trials consisting of the presentation of the eliciting stimulus

i 30 g E

20 IO 0 L

CONTROL

DEPRIVED

FIG. 2. Mean (*SE) time spent over the CS+ odor during the IbO-set test of preference for the CS+ versus CS- odors, of normally reared rats (control) or of rats subjected to repeated maternal separation (deprived). The dashed line at 90 set indicates lack of preference whereas values below this level indicate aversion learning. Rat pups were trained and tested for acquisition on PNDll (open bars) and retested 1 week later for long-term retention (hatched bars). Both treatment groups showed normal acquisition and retention. Asterisks indicate mean differs from chance performance (p < 0.01).

476

SHORT COMMUNICATION all prepulse intensities were used to estimate auditory thresholds. Data were analyzed by between-within analysis of variance (ANOVA). No effects of gender were found so data from both sexeswere pooled in subsequent analysis and presentation of the results. 80 RESULTS

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FIG. 3. Mean (+SE) percentage of correct responses for 23-day-old rats trained on discrete trials delayed alternation as a function of repeated maternal separation (deprived, closed circles) or control treatment (control, open circles), and trial blocks. Performance of the two treatment groups improved similarly with training (chance performance is 50%).

only (120 dB, 40 msec, white noise burst), with an intertrial interval of 15 sec. Adults were additionally tested with reflex modification audiometry. Each rat was placed in the same apparatus and, following a lo-min adaptation period at a low background noise level [30 db(A)], received a total of 240 trials (IT1 = 10 set), consisting of 10 trials at each of 24 different sound pressure levels of a prepulse stimulus, (blank, and 6-90 dB SPL), followed 90 msec later by the eliciting stimulus. Data from blank control trials (e.g., eliciting stimulus only) were used to estimate the ASR for adults. Data from

AND

DISCUSSION

Daily episodes of mother-infant separation failed to produce any alterations in the normal ontogenetic profile of motor activity (Fig. 1). ANOVA revealed a significant effect of age (p < 0.000 1), reflecting increased activity at increasing ages, but no main effect or interaction involving the separation factor. This factor also did not alter within-sessions habituation of motor activity (data not shown). Maternal separation did not alter olfactory learning or retention (Fig. 2). Both the treatment groups acquired the olfactory discrimination, as indicated by preference scores that were reliably (p < 0.0 1) below chance. The two treatment groups did not differ between themselves during the acquisition test (PNDl8) or during a test of retention that was conducted 1 week later (PND25). ANOVA revealed no effect of retention interval and no main effect or interaction involving the deprivation factor. Pups from both treatment groups showed normal acquisition of delayed alternation (Fig. 3). Performance improved from 50 to 60% correct (chance) on the first block of training to 83-90% correct in the last training block. This was confirmed statistically by analysis of variance which revealed a

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Control

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40 Frequency

(kHz)

FIG. 4. Acoustic startle response (left) and auditory thresholds (right) following repeated maternal separation (deprived, closed bars) or normal rearing (control, open bars). Baseline startle amplitude is the mean on blank trials. Thresholds were derived as indicated in the text. Data points represent means (*SE).

477

SHORT COMMUNICATION

main effect of trial blocks (p < O.OOOl), but no main effect or interaction involving maternal separation. Performance of maternally separated pups was normal on tests of ASR (data not shown for PND23) and reflex modification audiometry (Fig. 4). ANOVA indicated that there were no significant main effects or interactions of separation on either ASR amplitudes or auditory thresholds (or withinsessions trends on these measures, data not shown). Daily separation of infant rats from their mothers for 6hr periods failed to alter postnatal neurobehavioral function, as assessedby the range of measures we employed. The absence of behavioral effects are consistent with the results of a recent study which found a “brain sparing” effect with this same separation protocol. Weight gain in the developing brain and the ontogeny of brain ornithine decarboxylase activity (an enzyme involved in growth and development) were not appreciably influenced by daily separation (C. Lau et al., in press). These findings suggest that the 6-hr separation period that we employed is not sufficiently adverse to alter development of brain or behavior. The conditions of maternal separation that have produced behavioral deficits in other studies (Nadel and Willner, 1989; Castro and Rudy, 1989) have been more severe and are of the type that could alter parameters of brain development, including brain weight (e.g., Tonkiss, Cohen, and Sparber, 1988). The design of the present study does not indicate whether maternal separation effects could interact with those of a neurotoxicant. The possibility of such interactions and their implications for toxicity assessment go beyond the narrow focus of the present study. The main implication of the present findings is that, in the laboratory rat, daily separations of 6 hr or less do not, by themselves, produce behavioral effects that would confound interpretation of toxicity studies involving maternal separation for purposes of inhalation or dermal exposure (Kimmel and Francis, 1990). ACKNOWLEDGMENTS The authors thank Craig Barry, Annie Cameron, Mary Coussons, Rebecca Hamrick, Bryant Murphy, Clark Spencer, and Dimitrios Tsoumbos for technical assistance; and Drs. James P. O’Callaghan and Mary E. Gilbert for reviewing an earlier draft of this manuscript.

REFERENCES Castro, C. A., and Rudy, J. W. (1989). Early-life malnutrition impairs the performance of both young and adult rats on visual discrimination learning tasks. Dev. Psychobiol. 22, 15-28.

Freeman, J. H., Jr., and Stanton, M. E. (199 I). Fimbria-fomix transections disrupt the ontogeny of delayed alternation but not position discrimination in the rat. Behav. Neurosci. 105, 386-395. Kimmel, C. A., and Francis, E. Z. (1990). Proceedings of the workshop on the acceptability and interpretation of dermal developmental toxicity studies. Fundam. Appl. Toxicol. 14, 386-398. Kucharski, D., and Spear, N. E. (1984). Conditioning of aversion to an odor paired with peripheral shock in the developing rat. Dev. Psychobiol. 17, 465-479. Lau, C., Cameron, A. M., Antolick, L. L., and Stanton, M. E. Influence of repeated maternal separation on cellular growth and development of the rat. J. Devel. Physiol., in press. Leon, M. (1986). Development of thermoregulation. In Handbook of Behavioral Neurobiology, Volume 8, Developmental Psychobiology and Developmental Neurobiology (E. M. Blass, Ed.), pp. 297-322. Plenum, New York. Nadel, L., and Willner, J. (1989). Some implications of postnatal maturation of the hippocampus. In The Hippocampus: New Vistas (V. Chan-Palay and C. Kohler, Eds.), pp. 17-31. A. R. Liss, New York. Ruppert, P. H., Dean. K. F., and Reiter, L. W. (1984a). Development of locomotor activity of rat pups in figure-eight mazes. Dev. Psychobiol. 18, 247-260. Ruppert, P. H.. Dean, K. F., and Reiter, L. W. (1984b). Trimethyltin disrupts acoustic startle responding in adult rats. Toxicol. Lett. 22, 33-38. Ruppert, P. H., Dean, K. F., and Reiter, L. W. (1985). Development of locomotor activity in rat pups exposed to heavy metals. Toxicol. Appl. Pharmacol. 78,69-77. Stanton, M. E. (199 1). Neonatal exposure to triethyltin (TET) disrupts olfactory learning during early development in the rat. Neurotoxicol. Teratol. 13,515-524. Tonkiss, J., Cohen, C. A., and Sparber, S. B. (1988). Different methods for producing neonatal undernutrition in rats cause different brain changes in the face of equivalent somatic growth parameters. Dev. Neurosci. 10, 141-151. Young. J. S., and Fechter, L. D. (1983). Reflex inhibition procedures for animal audiometry: A technique for assessingototoxicity. J. Acoust. Sot. Am. 73, 1686-1693. M. E. STANTON K. M. CROFTON Neurotoxicology Division U.S. EPA Research Triangle Park North Carolina 27711 CHRISTOPHER Developmental Toxicology Division U.S. EPA Research Triangle Park North Carolina 27711 Received August 7, 199 1; accepted March 6, 1992

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