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Behavioral toxicity of chronic postweaning lead exposure in the rat
TOXICOLOGY
AND
APPLIED
PHARMACOLOOY
47, 151-159 (1979)
Behavioral Toxicity of Chronic Postweaning Lead Exposure in the Rat’ DEBORAHA. CORY-SLECHTA* AND TRAVIS THOMPSON Department
of Psychology,
University of Minnesota,
Received April
Minneapolis,
Minnesota
55455
14, 1978; accepted July 5, 1978
Behavioral Toxicity of Chronic Postweaning Lead Exposure in the Rat. CORY-SLECHTA, D. A., AND THOMPSON, T. (1979). Toxicol. Appl. Pharmacol. 47, 151-159. Effects in the rat of chronic postweaning exposure to 50, 300, or 1000 ppm lead acetate in drinking water were assessed on responding maintained by a fixed-interval 30-s food reinforcement schedule. On this schedule, food reinforcement followed the first lever press .response occurring at least 30 s after the preceding food delivery. Exposure to 50 and 300 ppm lead acetate increased response rates and intersubject variability, while latency, or time to initiation of responding in the interval, decreased. Exposure to 1000 ppm initially decreased rates and increased latency values. Following termination of exposure to 50 ppm, response rates and latency values gradually returned to control levels. Behavioral effects produced by exposure to 50 and 300 ppm lead were similar in magnitude but varied in time to onset and decline, suggesting time-dependent, rather than concentration-dependent effects of lead.
Sustained exposure to current environmental levels of lead may be a health hazard, especially to children (Lead: Airborne Lead in Perspective, 1972). Elevated lead body burden r Submitted in partial fulfillment of the requirements for Ph.D., Department of Psychology, University of Minnesota. This work was supported in part by University of Minnesota Research Special State Appropriation 1975-1977, by Grant MH-11752 from the National Institute of Mental Health, by Grant ES-01247 from the National Institute of Environmental Health Sciences, and in part by a contract with the U.S. Energy Research and Development Administration at the University of Rochester Biomedical and Environmental Research Project for which the present report has been assigned Report No. UR 3490-1365. Special thanks to Neil Winston, Alice Young, Ronald W. Wood, Victor G. Laties, and Bernard Weiss. * Currently a Junior Staff Fellow of the National Center for Toxicological Research, Jefferson, Arkansas. Present address: Department of Radiation Biology and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, N.Y. 14642.
has been associated with hyperactivity (David, et al., 1972) and with deficits in fine motor function and behavior (de la Burde and Choate, 1972). Experimental models of chronic prenatal or preweaning lead exposures have produced inconsistent findings. Changes in motor activity of rodents have been reported (Silbergeld and Goldberg, 1973 ; Sauerhoff and Michaelson, 1973; Reiter et al., 1975). However, the direction of these changes is inconsistent. This may stem from the wide variety of response classes encompassed by activity measures, which include running, rearing, locomotion, exploratory behaviors, turning, and jumping (Robbins, 1977). The most sensitive developmental period during which lead exposure results in behavioral changes has yet to be determined. The behavioral toxicity of lead has been investigated following prenatal exposures (Brady 151 All
0041-008X/79/01015149$02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
152
CORY-SLECHTA
et al., 1975) pre- and postnatal exposures (Reiter et al., 1975), and postnatal exposures typically initiated at birth (Sauerhoff and Michaelson, 1973; Silbergeld and Goldberg, 1973). In children, however, peak lead exposure generally occurs postweaning, between 1 and 5 years of age (David et al., 1972). Only two reports have dealt with the behavioral effects of postweaning exposure in animals. Snowdon (1973) was unable to detect any performance effects in the HebbsWilliams maze series of postweaning ip injections of lead. Padich and Zenick (1977) reported no effect of chronic oral postweaning lead exposure on fixed-ratio performance in the rat. The absence of effects in these investigations could have resulted from the insensitivity of the performance selected for examination. The fixed-interval (FI) schedule has been found to be sensitive to a wide variety of pharmacologic agents (Dews, 1955; Kelleher and Morse, 1969). On this schedule, the first response occurring after a fixed interval of time elapsesis followed by a reinforcer, e.g., a food pellet; responseswhich occur before the interval elapses have no consequence. This schedule typically generates response rates which accelerate as the interval progresses.The effects of drugs on FI performance appear to have considerable species generality, being similar in mice, rats, pigeons, cats, and monkeys (Kelleher and Morse, 1969; Dews and Wenger, 1977; Richelle, 1968). Postweaning exposure of rats to lead in drinking water was initiated at 20 to 22 days of age, and lever pressing maintained by a fixed-interval food reinforcement schedule was examined in this study. The reversibility of lead-induced behavioral changes was also examined in some animals by terminating exposure to the 50 ppm concentration. METHODS Animals. Male Sprague-Dawley rats, 20 to 22 days of age and weighing between 30 and 45 g, were obtained from Bio Labs, St. Paul, Minnesota. They
AND THOMPSON were randomly assigned to lead-treated or control groups whose mean weights did not differ. Animals were caged as pairs or triplets for I week after arrival in the laboratory and subsequently housed individually for the remainder of the experiment. For the first 35 days postweaning, all animals had free access to food. Apparatus. The rat chamber (Model EIO-10 Coulbourn Instruments, Inc.) was housed in a soundattenuated stainless-steel enclosure. The chamber was 25.4 cm wide, 27.9 cm long, and 30.5 cm high with Plexiglass sides and back. Two response levers, separated by 11.4 cm, were each located 3.8 cm above the grid floor on the front panel. A combination dipper/pellet trough was located 6.3 cm between the levers. Continuous white noise was used to mask any extraneous sounds. Electromechanical scheduling and recording equipment were located in an adjoining room. Behavioral training procedure. Following 35 days of free access to food, all animals were food deprived for 23 h and trained over 3 days to press the right lever to deliver 45-mg Noyes food pellets. The animals were then given two consecutive daily sessions in which each response produced food delivery until 150 food pellets were delivered or 30 min elapsed. The FI 30-set schedule of food reinforcement was then imposed. On the FI schedule, the first lever press response occurring at least 30 set after the preceding food presentation resulted in food delivery. Responses occurring during the 30-set interval were recorded but had no consequence. Experimental sessions were 1 hr long and were conducted daily. Since the rats were juveniles (55-60 days of age) at the initiation of behavioral testing, each animal was fed an amount of Purina rat chow following each session that produced a daily weight gain of 1 to 3 g. The size of the food supplement was determined by the individual animal’s weight. Weight loss or failure to gain weight resulted in a I- or 2-g increase in the daily food supplement. Conversely, excessive weight gain or satiation prior to the end of the experimental session resulted in a I- or 2-g decrement in the daily food supplement. Body weights were recorded daily. There were no significant differences in body weight between lead-treated and corresponding control groups during the experiment. Any subject that failed to obtain 85% of the 120 possible food deliveries by the fifth FI session and that showed excessive pausing during the final 30 min of the session was eliminated from the experiment. Prior research had shown that such rats continued to earn a low proportion of available reinforcers despite continued growth and that their response rates could not be altered even by increased food deprivation. Two rats from the group exposed to 50 ppm lead and one from the corresponding control group, one rat
BEHAVIORAL
TOXICITY
each from the group exposed to 300 ppm lead and the corresponding control group, and one rat from the control group corresponding to the group exposed to 1000 ppm lead failed to meet these criteria and were consequently eliminated from the experiment. The reasons for the failures are unclear, but failures appeared to be equally frequent among control and lead-treated subjects. This produced sample sizes of four control animals and five animals exposed to 50 ppm lead from the previous experiment (Experiment I), four control animals and six animals exposed to 50 ppm lead from the present experiment (Experiment 2), three control animals and four animals exposed to 300 ppm lead, and four control and five animals exposed to 1000 ppm lead. Overall response rate was calculated by dividing the total number of responses during a session by total session time. Local response rate was calculated by dividing the total number of responses during each IO-set period of the interval by the total amount of time spent in that third of the interval. Latency or time to initiation of responding in the interval was converted to percentage of session time by dividing the total latency value by total session length and multiplying the result by 100. All group data points on figures represent the median of the means of five sessions. Differences in median response rates were assessed using the median test, while differences in variability of response rate distributions were determined by Cochran’s test (Winer, 1962). In all cases, the term significant is used only when p < 0.05. Exposure
protocol
and
blood
lead
determinations.
Exposures to 50, 300, or 1000 ppm lead acetate solution for drinking or tap-water solutions were initiated in 37 postweaning (2&22 days of age) rats.
OF POSTWEANING
LEAD
153
Thirty-five days of lead exposure preceded the beginning of behavioral training. Following 5 days of response shaping and 30 daily FI experimental sessions (after 70 days of lead exposure), the lead acetate solutions of three subjects from the 50 ppm group were replaced with tap water. All subjects were subsequently tested for an additional 60 daily sessions. Other investigators have reported that blood lead concentrations of rats return to control values within 60 days of termination of exposure to 21.5 pg/m3 atmospheric lead (Grillin et al., 1975). Data from a previous 50 ppm lead-exposure experiment (experiment I) are also included in the results. These subjects were treated exactly as described above except that several FI 45-set sessions preceded termination of lead exposure. For this original group, 30 FI 30-set sessions both preceded and followed cessation of lead exposure for all subjects. Sixty and eighty sessions were conducted with the groups exposed to 300 and 1000 ppm lead, respectively, with no termination of lead exposure. Blood lead determinations were made at 150 days of age, at 65 days postexposure for the three animals exposed to 50 ppm lead from experiment 2, and after 125 days of chronic exposure for the animals exposed to 50,300 and 1000 ppm lead. Five milliliters of blood per anesthetized rat was drawn by cardiac puncture at the termination of all behavioral testing and analyzed for lead content by atomic absorption spectrophotometry according to the method of Yaeger et al. (1971). Blood lead values increased linearly as a function of log of exposure concentration in drinking water (Fig. 1). Statistical analysis of the differences between all possible pairs of means (Newman-Keuls test, Winer, 1962) revealed that the blood lead values resulting from exposure to 300 and 1000 ppm lead acetate differed significantly
50
Pb Concentration
(PPM 1
FIG. 1. Blood lead concentrations in micrograms per 100 ml as a function of log of exposure concentration. Blood lead values were determined when the animals were approximately 150 days of age and increased linearly with log of lead acetate exposure concentration in drinking water.
154
CORY-SLECHTA
those resulting from exposure to all other concentrations. Blood lead concentrations of control animals, animals maintained on 50 ppm, and animals whose exposure to 50 ppm had been terminated did not differ. Kidney and liver tissue of two control animals and two animals treated with 1000 ppm lead were analyzed for lead content. Kidney lead values of control animals were 0.2 and 0.3 pg/g in contrast to 22.0 and 17.2pg/g for animals treated with 1000 ppm lead. Liver tissue determinations on control rats and 1000 ppm lead-treated rats resulted in values of 0.3 and 0.2 Dg/g and 1 .O and I .5 pg/g.
AND THOMPSON
sessions,however. responserates increasedto approximately 250 % of control values. Rates subsequently declined but remained at approximately 150% of control. The effects of exposure to 300 ppm lead were similar to those for exposure to 50 ppm in both direction and magnitude, but varied considerably in time course. Over the first 15 experimental sessions, median response rates of the group exposed to 300 ppm already ranged from 200 to 260% of control values. Overall rate declined sooner (after session 15) than did 50 ppm rates, eventually RESULTS approaching control values. Animals chronically exposed to 1000ppm Figure 2 summarizes the effects of chronic exposure to lead acetate on overall response lead acetate responded less frequently than rates during performance on the FI 30-set controls during the first 20 sessions.Rates schedule of reinforcement. Data for lead- subsequently increased to and then exceeded exposed groups are presented as percentage control values for the duration of the experiof their corresponding control group values. ment. Data from the final 40 sessions, Overall responserates for animals exposed to however, do not adequately represent changes 50 ppm lead were similar to control values in individual performances of animals exduring the initial 10 sessions(i.e., the first posed to 1000ppm lead. Three animals two 5-sessionblocks). Over the following 25 showed large increases in overall response rate, while two other animals intermittently exhibited decreasesin rate as a function of 3001 total cessation of responding during the final 30 min of the experimental session. This behavior pattern became increasingly more frequent during the final 20 sessions and was accompanied by a slight weight loss suggesting the onset of gross toxicosis. Statistical analysis revealed that the median overall rates of the groups exposed to 50 and 300 ppm lead during the first 30 sessionswere significantly higher than the corresponding control values. In addition, the median o’t, I I, I II, r I I,1 1 I, 6-10 26-30 46-50 66-70 86-90 responserates of animals exposed to 50 ppm SESSIONS lead on the final 60 sessionsand of animals exposed to 300 ppm on the final 30 sessions FIG. 2. Median overall response rates (as percentage were significantly greater than corresponding of corresponding control group) on a fixed-interval control values. Response’rates for the group 30-s reinforcement schedule. Each data point repreexposed to 1000ppm over the first 40 sessions sents the median of the mean rates of five sessions. Postweaning exposure to the lower two concentrations were significantly lower than corresponding (50 and 300 ppm) increased rates, which subsequently control values. declined but remained above control levels. Exposure A more detailed analysis revealed that to the 1000 ppm lead concentration decreased rates overall responserate increasesshown by the which (although not apparent here) became highly groups exposed to 50 and 300 ppm were due variable during the final 40 sessions, from
BEHAVIORAL
TOXICITY
primarily to significantly increased rates during the final 10 set of the 30-set fixed interval (Slechta, 1977). Rates also rose moderately during the middle third of the interval. Overall response rate decrements shown by the group exposed to 1000 ppm were primarily a function of significantly decreased rates during the final 10 set of the interval. Individual animals displayed large differences in susceptibility to lead-induced behavioral effects. The median overall response rates of individual animals treated with 50 and 300 ppm lead over the first 30 sessions are shown in Fig. 3. Data are plotted 5001
A
50 wm
0
I
6-10 16-20 SESSIONS
1
26-30
FIG. 3. Individual median overall response rates (as percentage of the corresponding control group median) on the fixed-interval 30-set schedule with (A) 50 ppm and (B) 300 ppm. The shaded area represents the control group semi-interquartile range. Each data point represents a median of five sessions. Exposure to all concentrations significantly increased intersubject variability, emphasizing the individual susceptibility to lead-induced behavioral effects.
OF POSTWEANING
155
LEAD
as percentage of the control group median rates. Although one animal from each of the groups treated with 50 and 300 ppm lead showed dramatic increases in response rate over the first 30 sessions, another exhibited response rates that differed little from control values. Remaining exposed animals showed moderate increases in response rate relative to control values. Statistical analysis confirmed that response rates of the groups treated with 50 and 300 ppm lead during the first 30 sessions were significantly more variable than corresponding control values. Response rates of the group exposed to 1000 ppm were significantly more variable than control values during the final 40 sessions. The effects of chronic exposure to lead acetate on latency or time to the first response in the interval are summarized in Fig. 4. Median latency values of the group exposed to 50 ppm were slightly greater than control values during the initial 10 sessions. Over the following 20 sessions, however, latency
I,
I\,,
6-10
,
26-30
,
,
(
,
46-50 SESSIONS
,
I
(
66-70
(
(
(
,
86-90
FIG. 4. Latency to the first response in the interval (as percentage of corresponding control group median) on the fixed-interval 30-set schedule. Each data point represents the median of the mean values for five sessions. Postweaning exposure to the lower two concentrations initially decreased latency. Values then increased, but remained below control levels. Exposure to the highest concentration, in general, increased latency values.
156
CORY-SLECHTA
values decreasedto a low of 72 % of controls. Values subsequently increased but remained lower than corresponding control values for the duration of the experiment. As with overall responserate, the effects of exposure to 300 ppm lead were similar to those of 50 ppm lead in both direction and magnitude, but varied in time course. Latency values were slightly lower than control values even during the initial 5 sessions, subsequently decreased, and ranged from 70 to 77% of control values over the following 30 sessions. Latency values then increased to near-control levels over the final 25 sessions.Statistical analysis confirmed a lower first 30-session median latency value for both the group exposed to 50 ppm lead and that exposed to 300 ppm. In general, median latency values for subjectschronically exposedto 1000 ppm lead were greater than corresponding control values. This effect, however, was more pronounced during the final 55 sessions,while differences during the intitial 25 sessionswere minimal. However, the 1000ppm median latency value of the first 40 sessions was significantly higher than that of controls. Lead treatment of the group exposed to 50 ppm lead from Experiment I and half of the group exposed to 50 ppm from Experiment 2 was terminated after the 30th experimental session. Figure 5 shows that animals maintained on the lead regimen (solid line) showed rate decreases after session 35. However, discontinuation of exposure led to reduction in overall rate of a greater magnitude (dashed lines), with median values of Experiment 2 animals reaching control levels approximately 30 sessionsafter cessation of treatment. Control groups from both experiments showed almost identical median responserates. Likewise, the effects of 50 ppm lead treatment were very similar in time course and magnitude in both exposure groups. Statistical analysis of the final 60 sessions of Experiment 2 revealed significant differences between median overall rates of the
AND THOMPSON
-,11,,,11, 4t:o,,,I 6-10
26-30
46-50
66-70
&
SESSIONS FIG. 5. Median overall response rates on the fixedinterval 30-set schedule following termination (I and 2 LEAD TERMINATED) and continuation (LEAD MAINTAINED 2) of SO ppm lead exposure. Data from a previous experiment are included for replication purposes and the numbers I and 2 refer to data from experiments I and 2, respectively. Each data point represents the median of the mean values for five sessions. Termination of exposure (dashed lines) resulted in rate decrements of a greater magnitude than exhibited by lead-maintained animals (solid lines). The two control groups showed very similar median response rates. The effects of 50 ppm lead treatment were similar in time course and magnitude in both experimental groups.
lead-maintained and corresponding control groups, between the lead-maintained and lead-terminated groups, and between the lead-terminated and corresponding control groups. Selected cumulative records of performance maintained by the FI schedule are shown in Fig. 6. The top records show the performance of a control animal during session 30 and again at session 90. These records illustrate the stability of response rates from minute to minute during the sessionand over the course of the experiment. The middle-left record shows the performance of a 50 ppm lead-exposed animal during session25. The overall rate of 38.5 responses/min was approximately four times greater than that of the control. The drinking solution of this animal was changed to tap water following session30. The middle-right
BEHAVIORAL
Session 30
TOXICITY
OF POSTWEANINGLEAD
157
50 PPM
I_ CONTROL
Session 28 ON LEAD
Session 90
CONTROL
,,,... ... . ,,,,/”
Session 90 ONLEAD
;_ .I
,,,J” ,’
FIG. 6. Selected cumulative records of responding maintained by the fixed-interval 30-set schedule for session numbers indicated. Each lever press stepped the pen vertically, while time and pausing are represented horizontally. Response rate is indicated by the slope of the line. The top records show the performance of a control animal. Middle- and bottom-right records are for animals exposed to 50 ppm lead. Rates were approximately four times greater than control rates. Termination of exposure resulted in rates decreasing to control levels (middle left). Despite moderate decrements, rates for animals maintained on 50 ppm lead remained above control levels (bottom left).
cumulative record shows the performance of this animal during the final session,60 days after the termination of lead exposure. By this time, response rates had decreased to control levels. Close inspection of the’record reveals an irregularity in minute-to-minute performance on the schedule resulting from alternation between very high and low response rates. The lower records show the performance of a lead-maintained animal during session 28 and again at session 90. Although a gradual decrement in response rate occurred between these sessions, it remained well above both control and leadterminated values. Within-session performance tended to be irregular. DISCUSSION Snowdon (1973) and Padich and Zenick (1977) were unable to detect effects of post-
weaning lead exposure on behavior. Brown (1975) found significant effects on T-maze performance only if rats were treated with lead before 10 days of age. There are at least two explanations for the absence of lead effects in these investigations. The neonatal rat may be sensitive to lead-induced behavioral changes only when exposed prior to 10 days of age. The results of the present experiment argue against such an interpretation. A more reasonable explanation for the absence of lead effects is the insensitivity of the tests used to measure performance. The T-maze, the Hebbs-Williams maze series,and certain fixed-ratio schedule parameters apparently produce types of behavior that are not influenced by lead exposure. Behavioral effects of chronic postweaning lead exposure were produced in the current experiment. Over the first 30 sessionsat 50
158
CORY-SLECHTA AND THOMPSON
and 300 ppm lead, overall and local response rates increased and responding was initiated earlier in the interval. Later, both groups exhibited moderate decreases in response rates and latency values increased. At the time of these later behavioral changes, the animals were 85 to 90 days of age. The decrements in response rate could reflect an interaction of lead effects with hormonal or other physiological changes associated with adolescence. Furthermore, gastrointestinal lead absorption declines gradually in the rat from a neonatal level of near 90% through postweaning and adolescence to the adult levels of approximately 15 % (Forbes and Reina, 1972). Thus, the animals probably had decreasing body burdens of lead. The fact that response rates remained above control levels for the duration of the behavioral observations may be accounted for by the continued exposure to lead, by the lengthy prior history of reinforcement following the lead-induced high rates of responding, or both. Exposure to the two lower concentrations of lead acetate also increased the variability of FI response rates between subjects (Fig. 3). Shapiro et a/. (1973) reported increases in both intersubject and intrasubject variability of performance of adult male rats on a multiple variable-interval 30-set extinction schedule of reinforcement following I6 ,ug/ 100 g lead acetate injections. In addition, Van Gelder and co-workers (1973) have reported that adult ewes dosed daily with 100 mg/kg lead showed significantly increased between-session variability on an auditory detection task. Lead-induced variability may be a genera1 effect and itself a reliable indicator of lead toxicity. Chronic exposure to 1000 ppm lead acetate initially resulted in decreased response rates. Over the final 40 sessions, latency values remained above control levels while response rates were highly variable among subjects; the behavior of two animals suggested the onset of toxicosis. The variability of response rates during the final 40 sessionsappears to
be another example of individual differences in susceptibility to lead. Differences produced by varying the concentration of lead acetate depended upon the time that the measurements were made. Behavioral effects produced by 50 and 300 ppm lead were very similar in magnitude but varied in time to onset and decline: while differences between animals exposed to 300 ppm lead and corresponding control animals were obvious even during the initial 5 sessions,animals exposed to 50 ppm and control animals did not differ until after 10 sessions.This suggeststime-dependent rather than concentration-dependent effects of lead. The reversibility of lead-induced behavioral changes was demonstrated by terminating exposure of several animals to 50 ppm lead. Responserates and time to the first response in the interval gradually returned to control values. This effect resemblesthat of chelation therapy in the human. Children with high blood leads, diagnosed as mentally retarded or having behavioral disorders, showed improvements in behavior and demeanor, and in one case in IQ score on the StanfordBinet test, after treatment with EDTA (Moncrieff et al., 1964). Thus both discontinuation of exposure or elimination of body lead burden via chelation (in certain circumstances) may facilitate behavioral recovery, depending on the level and duration of exposure. The range of types of behavior which might be improved by reversal of exposure and the extent of recuperation in each caseremains to be determined. Understanding the effects of lead on behavior will require knowledge of the exposure concentration and duration, the developmental period during which it occurs, the factors determining individual susceptibility, and the nature of the behavior. REFERENCES BRADY,
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D. R. (1975). Neonatal lead exposure in the rat: Decreased learning as a function of age and blood lead concentration. Toxicol. Appl. Pharmacol.
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32, 628-637. O., CLARK, J., AND VOELLER, K. (1972). Lead and hyperactivity. Lancet 2, 900-903. DE LA BURDE, B., AND CHOATE, M. S. (1972). Does asymptomatic lead in children have latent sequelae? J. Pediat. 81, 1088. DEWS, P. B. (1955). Studies on behavior: I, Differential sensitivity to pentobarbitol of pecking performance in pigeons depending on the schedule of reward. J. Pharmacol. Exp. Ther. 113, 393401. DEWS, P. B., and WENGER, G. (1977). Rate-dependency of the behavioral effects of amphetamine. In Advances in Behavioral Pharmacology (T. Thompson and P. B. Dews, eds.), pp. 167-227. Academic Press, New York. FORBES, G. B., AND REINA, J. C. (1972). Effect of age on gastrointestinal absorption (Fe, Sr, Pb) in the rat. J. Nufr. 102, 647-652. GRIFFIN, T. B., COULSTON, F., WILLS, H., AND RUSSEL, J. C. (1975). Biologic effects of airborne particulate lead on continuously exposed rats and rhesus monkeys. Environ. Qual. Saf. Suppl. Lead II, DAVID,
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behavior in the rat. Pharmacol. Biochem. Behav. 6, 371-375. REITER, L. W., ANDERSON, G. E., LASKEY, J. W., AND CAHILL, D. F. (1975). Developmental and behavioral changes in the rat during chronic exposure to lead. Environ. Health Perspect. 12, 119-123. RICHELLE, M. (1969). Combined action of diazepam and d-amphetamine on fixed-interval performance in cats. J. Exp. Anal. Behav. 12, 989-998. ROBBINS, T. W. (1977). A critique of the methods available for the measurement of spontaneous motor activity. In Handbook of Psychopharmacology (L. L. Iverson, S. D. Iverson, and S. H. Snyder, eds.), Vol. 7, pp. 37-77. Plenum, New York. SAUERHOFF, M. W., AND MICHAELSON, I. A. (1973). Hyperactivity and brain catecholamines in leadexposed developing rats. Science 182, 1022-1024. SHAPIRO, M. M., TRITSCHLER, J. M., AND ULM, R. A. (1973). Lead contamination: Chronic and acute behavioral effects in the albino rat. Bull. Psychon. Sot.
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Minnesota, Minneapolis. SNOWDON, C. T. (1973). Learning deficits in lead injected rats. Pharmacol. Biochem. Behav. 1, 599-603. B. A., CARSON, T., SMITH, R. M., AND B. (1973). Behavioral toxicologic assessment of the neurologic effect ,of lead in sheep. Clin. Toxicol. 6, 405-418.
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Design.
AND
APPLIED
PHARMACOLOOY
47, 151-159 (1979)
Behavioral Toxicity of Chronic Postweaning Lead Exposure in the Rat’ DEBORAHA. CORY-SLECHTA* AND TRAVIS THOMPSON Department
of Psychology,
University of Minnesota,
Received April
Minneapolis,
Minnesota
55455
14, 1978; accepted July 5, 1978
Behavioral Toxicity of Chronic Postweaning Lead Exposure in the Rat. CORY-SLECHTA, D. A., AND THOMPSON, T. (1979). Toxicol. Appl. Pharmacol. 47, 151-159. Effects in the rat of chronic postweaning exposure to 50, 300, or 1000 ppm lead acetate in drinking water were assessed on responding maintained by a fixed-interval 30-s food reinforcement schedule. On this schedule, food reinforcement followed the first lever press .response occurring at least 30 s after the preceding food delivery. Exposure to 50 and 300 ppm lead acetate increased response rates and intersubject variability, while latency, or time to initiation of responding in the interval, decreased. Exposure to 1000 ppm initially decreased rates and increased latency values. Following termination of exposure to 50 ppm, response rates and latency values gradually returned to control levels. Behavioral effects produced by exposure to 50 and 300 ppm lead were similar in magnitude but varied in time to onset and decline, suggesting time-dependent, rather than concentration-dependent effects of lead.
Sustained exposure to current environmental levels of lead may be a health hazard, especially to children (Lead: Airborne Lead in Perspective, 1972). Elevated lead body burden r Submitted in partial fulfillment of the requirements for Ph.D., Department of Psychology, University of Minnesota. This work was supported in part by University of Minnesota Research Special State Appropriation 1975-1977, by Grant MH-11752 from the National Institute of Mental Health, by Grant ES-01247 from the National Institute of Environmental Health Sciences, and in part by a contract with the U.S. Energy Research and Development Administration at the University of Rochester Biomedical and Environmental Research Project for which the present report has been assigned Report No. UR 3490-1365. Special thanks to Neil Winston, Alice Young, Ronald W. Wood, Victor G. Laties, and Bernard Weiss. * Currently a Junior Staff Fellow of the National Center for Toxicological Research, Jefferson, Arkansas. Present address: Department of Radiation Biology and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, N.Y. 14642.
has been associated with hyperactivity (David, et al., 1972) and with deficits in fine motor function and behavior (de la Burde and Choate, 1972). Experimental models of chronic prenatal or preweaning lead exposures have produced inconsistent findings. Changes in motor activity of rodents have been reported (Silbergeld and Goldberg, 1973 ; Sauerhoff and Michaelson, 1973; Reiter et al., 1975). However, the direction of these changes is inconsistent. This may stem from the wide variety of response classes encompassed by activity measures, which include running, rearing, locomotion, exploratory behaviors, turning, and jumping (Robbins, 1977). The most sensitive developmental period during which lead exposure results in behavioral changes has yet to be determined. The behavioral toxicity of lead has been investigated following prenatal exposures (Brady 151 All
0041-008X/79/01015149$02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
152
CORY-SLECHTA
et al., 1975) pre- and postnatal exposures (Reiter et al., 1975), and postnatal exposures typically initiated at birth (Sauerhoff and Michaelson, 1973; Silbergeld and Goldberg, 1973). In children, however, peak lead exposure generally occurs postweaning, between 1 and 5 years of age (David et al., 1972). Only two reports have dealt with the behavioral effects of postweaning exposure in animals. Snowdon (1973) was unable to detect any performance effects in the HebbsWilliams maze series of postweaning ip injections of lead. Padich and Zenick (1977) reported no effect of chronic oral postweaning lead exposure on fixed-ratio performance in the rat. The absence of effects in these investigations could have resulted from the insensitivity of the performance selected for examination. The fixed-interval (FI) schedule has been found to be sensitive to a wide variety of pharmacologic agents (Dews, 1955; Kelleher and Morse, 1969). On this schedule, the first response occurring after a fixed interval of time elapsesis followed by a reinforcer, e.g., a food pellet; responseswhich occur before the interval elapses have no consequence. This schedule typically generates response rates which accelerate as the interval progresses.The effects of drugs on FI performance appear to have considerable species generality, being similar in mice, rats, pigeons, cats, and monkeys (Kelleher and Morse, 1969; Dews and Wenger, 1977; Richelle, 1968). Postweaning exposure of rats to lead in drinking water was initiated at 20 to 22 days of age, and lever pressing maintained by a fixed-interval food reinforcement schedule was examined in this study. The reversibility of lead-induced behavioral changes was also examined in some animals by terminating exposure to the 50 ppm concentration. METHODS Animals. Male Sprague-Dawley rats, 20 to 22 days of age and weighing between 30 and 45 g, were obtained from Bio Labs, St. Paul, Minnesota. They
AND THOMPSON were randomly assigned to lead-treated or control groups whose mean weights did not differ. Animals were caged as pairs or triplets for I week after arrival in the laboratory and subsequently housed individually for the remainder of the experiment. For the first 35 days postweaning, all animals had free access to food. Apparatus. The rat chamber (Model EIO-10 Coulbourn Instruments, Inc.) was housed in a soundattenuated stainless-steel enclosure. The chamber was 25.4 cm wide, 27.9 cm long, and 30.5 cm high with Plexiglass sides and back. Two response levers, separated by 11.4 cm, were each located 3.8 cm above the grid floor on the front panel. A combination dipper/pellet trough was located 6.3 cm between the levers. Continuous white noise was used to mask any extraneous sounds. Electromechanical scheduling and recording equipment were located in an adjoining room. Behavioral training procedure. Following 35 days of free access to food, all animals were food deprived for 23 h and trained over 3 days to press the right lever to deliver 45-mg Noyes food pellets. The animals were then given two consecutive daily sessions in which each response produced food delivery until 150 food pellets were delivered or 30 min elapsed. The FI 30-set schedule of food reinforcement was then imposed. On the FI schedule, the first lever press response occurring at least 30 set after the preceding food presentation resulted in food delivery. Responses occurring during the 30-set interval were recorded but had no consequence. Experimental sessions were 1 hr long and were conducted daily. Since the rats were juveniles (55-60 days of age) at the initiation of behavioral testing, each animal was fed an amount of Purina rat chow following each session that produced a daily weight gain of 1 to 3 g. The size of the food supplement was determined by the individual animal’s weight. Weight loss or failure to gain weight resulted in a I- or 2-g increase in the daily food supplement. Conversely, excessive weight gain or satiation prior to the end of the experimental session resulted in a I- or 2-g decrement in the daily food supplement. Body weights were recorded daily. There were no significant differences in body weight between lead-treated and corresponding control groups during the experiment. Any subject that failed to obtain 85% of the 120 possible food deliveries by the fifth FI session and that showed excessive pausing during the final 30 min of the session was eliminated from the experiment. Prior research had shown that such rats continued to earn a low proportion of available reinforcers despite continued growth and that their response rates could not be altered even by increased food deprivation. Two rats from the group exposed to 50 ppm lead and one from the corresponding control group, one rat
BEHAVIORAL
TOXICITY
each from the group exposed to 300 ppm lead and the corresponding control group, and one rat from the control group corresponding to the group exposed to 1000 ppm lead failed to meet these criteria and were consequently eliminated from the experiment. The reasons for the failures are unclear, but failures appeared to be equally frequent among control and lead-treated subjects. This produced sample sizes of four control animals and five animals exposed to 50 ppm lead from the previous experiment (Experiment I), four control animals and six animals exposed to 50 ppm lead from the present experiment (Experiment 2), three control animals and four animals exposed to 300 ppm lead, and four control and five animals exposed to 1000 ppm lead. Overall response rate was calculated by dividing the total number of responses during a session by total session time. Local response rate was calculated by dividing the total number of responses during each IO-set period of the interval by the total amount of time spent in that third of the interval. Latency or time to initiation of responding in the interval was converted to percentage of session time by dividing the total latency value by total session length and multiplying the result by 100. All group data points on figures represent the median of the means of five sessions. Differences in median response rates were assessed using the median test, while differences in variability of response rate distributions were determined by Cochran’s test (Winer, 1962). In all cases, the term significant is used only when p < 0.05. Exposure
protocol
and
blood
lead
determinations.
Exposures to 50, 300, or 1000 ppm lead acetate solution for drinking or tap-water solutions were initiated in 37 postweaning (2&22 days of age) rats.
OF POSTWEANING
LEAD
153
Thirty-five days of lead exposure preceded the beginning of behavioral training. Following 5 days of response shaping and 30 daily FI experimental sessions (after 70 days of lead exposure), the lead acetate solutions of three subjects from the 50 ppm group were replaced with tap water. All subjects were subsequently tested for an additional 60 daily sessions. Other investigators have reported that blood lead concentrations of rats return to control values within 60 days of termination of exposure to 21.5 pg/m3 atmospheric lead (Grillin et al., 1975). Data from a previous 50 ppm lead-exposure experiment (experiment I) are also included in the results. These subjects were treated exactly as described above except that several FI 45-set sessions preceded termination of lead exposure. For this original group, 30 FI 30-set sessions both preceded and followed cessation of lead exposure for all subjects. Sixty and eighty sessions were conducted with the groups exposed to 300 and 1000 ppm lead, respectively, with no termination of lead exposure. Blood lead determinations were made at 150 days of age, at 65 days postexposure for the three animals exposed to 50 ppm lead from experiment 2, and after 125 days of chronic exposure for the animals exposed to 50,300 and 1000 ppm lead. Five milliliters of blood per anesthetized rat was drawn by cardiac puncture at the termination of all behavioral testing and analyzed for lead content by atomic absorption spectrophotometry according to the method of Yaeger et al. (1971). Blood lead values increased linearly as a function of log of exposure concentration in drinking water (Fig. 1). Statistical analysis of the differences between all possible pairs of means (Newman-Keuls test, Winer, 1962) revealed that the blood lead values resulting from exposure to 300 and 1000 ppm lead acetate differed significantly
50
Pb Concentration
(PPM 1
FIG. 1. Blood lead concentrations in micrograms per 100 ml as a function of log of exposure concentration. Blood lead values were determined when the animals were approximately 150 days of age and increased linearly with log of lead acetate exposure concentration in drinking water.
154
CORY-SLECHTA
those resulting from exposure to all other concentrations. Blood lead concentrations of control animals, animals maintained on 50 ppm, and animals whose exposure to 50 ppm had been terminated did not differ. Kidney and liver tissue of two control animals and two animals treated with 1000 ppm lead were analyzed for lead content. Kidney lead values of control animals were 0.2 and 0.3 pg/g in contrast to 22.0 and 17.2pg/g for animals treated with 1000 ppm lead. Liver tissue determinations on control rats and 1000 ppm lead-treated rats resulted in values of 0.3 and 0.2 Dg/g and 1 .O and I .5 pg/g.
AND THOMPSON
sessions,however. responserates increasedto approximately 250 % of control values. Rates subsequently declined but remained at approximately 150% of control. The effects of exposure to 300 ppm lead were similar to those for exposure to 50 ppm in both direction and magnitude, but varied considerably in time course. Over the first 15 experimental sessions, median response rates of the group exposed to 300 ppm already ranged from 200 to 260% of control values. Overall rate declined sooner (after session 15) than did 50 ppm rates, eventually RESULTS approaching control values. Animals chronically exposed to 1000ppm Figure 2 summarizes the effects of chronic exposure to lead acetate on overall response lead acetate responded less frequently than rates during performance on the FI 30-set controls during the first 20 sessions.Rates schedule of reinforcement. Data for lead- subsequently increased to and then exceeded exposed groups are presented as percentage control values for the duration of the experiof their corresponding control group values. ment. Data from the final 40 sessions, Overall responserates for animals exposed to however, do not adequately represent changes 50 ppm lead were similar to control values in individual performances of animals exduring the initial 10 sessions(i.e., the first posed to 1000ppm lead. Three animals two 5-sessionblocks). Over the following 25 showed large increases in overall response rate, while two other animals intermittently exhibited decreasesin rate as a function of 3001 total cessation of responding during the final 30 min of the experimental session. This behavior pattern became increasingly more frequent during the final 20 sessions and was accompanied by a slight weight loss suggesting the onset of gross toxicosis. Statistical analysis revealed that the median overall rates of the groups exposed to 50 and 300 ppm lead during the first 30 sessionswere significantly higher than the corresponding control values. In addition, the median o’t, I I, I II, r I I,1 1 I, 6-10 26-30 46-50 66-70 86-90 responserates of animals exposed to 50 ppm SESSIONS lead on the final 60 sessionsand of animals exposed to 300 ppm on the final 30 sessions FIG. 2. Median overall response rates (as percentage were significantly greater than corresponding of corresponding control group) on a fixed-interval control values. Response’rates for the group 30-s reinforcement schedule. Each data point repreexposed to 1000ppm over the first 40 sessions sents the median of the mean rates of five sessions. Postweaning exposure to the lower two concentrations were significantly lower than corresponding (50 and 300 ppm) increased rates, which subsequently control values. declined but remained above control levels. Exposure A more detailed analysis revealed that to the 1000 ppm lead concentration decreased rates overall responserate increasesshown by the which (although not apparent here) became highly groups exposed to 50 and 300 ppm were due variable during the final 40 sessions, from
BEHAVIORAL
TOXICITY
primarily to significantly increased rates during the final 10 set of the 30-set fixed interval (Slechta, 1977). Rates also rose moderately during the middle third of the interval. Overall response rate decrements shown by the group exposed to 1000 ppm were primarily a function of significantly decreased rates during the final 10 set of the interval. Individual animals displayed large differences in susceptibility to lead-induced behavioral effects. The median overall response rates of individual animals treated with 50 and 300 ppm lead over the first 30 sessions are shown in Fig. 3. Data are plotted 5001
A
50 wm
0
I
6-10 16-20 SESSIONS
1
26-30
FIG. 3. Individual median overall response rates (as percentage of the corresponding control group median) on the fixed-interval 30-set schedule with (A) 50 ppm and (B) 300 ppm. The shaded area represents the control group semi-interquartile range. Each data point represents a median of five sessions. Exposure to all concentrations significantly increased intersubject variability, emphasizing the individual susceptibility to lead-induced behavioral effects.
OF POSTWEANING
155
LEAD
as percentage of the control group median rates. Although one animal from each of the groups treated with 50 and 300 ppm lead showed dramatic increases in response rate over the first 30 sessions, another exhibited response rates that differed little from control values. Remaining exposed animals showed moderate increases in response rate relative to control values. Statistical analysis confirmed that response rates of the groups treated with 50 and 300 ppm lead during the first 30 sessions were significantly more variable than corresponding control values. Response rates of the group exposed to 1000 ppm were significantly more variable than control values during the final 40 sessions. The effects of chronic exposure to lead acetate on latency or time to the first response in the interval are summarized in Fig. 4. Median latency values of the group exposed to 50 ppm were slightly greater than control values during the initial 10 sessions. Over the following 20 sessions, however, latency
I,
I\,,
6-10
,
26-30
,
,
(
,
46-50 SESSIONS
,
I
(
66-70
(
(
(
,
86-90
FIG. 4. Latency to the first response in the interval (as percentage of corresponding control group median) on the fixed-interval 30-set schedule. Each data point represents the median of the mean values for five sessions. Postweaning exposure to the lower two concentrations initially decreased latency. Values then increased, but remained below control levels. Exposure to the highest concentration, in general, increased latency values.
156
CORY-SLECHTA
values decreasedto a low of 72 % of controls. Values subsequently increased but remained lower than corresponding control values for the duration of the experiment. As with overall responserate, the effects of exposure to 300 ppm lead were similar to those of 50 ppm lead in both direction and magnitude, but varied in time course. Latency values were slightly lower than control values even during the initial 5 sessions, subsequently decreased, and ranged from 70 to 77% of control values over the following 30 sessions. Latency values then increased to near-control levels over the final 25 sessions.Statistical analysis confirmed a lower first 30-session median latency value for both the group exposed to 50 ppm lead and that exposed to 300 ppm. In general, median latency values for subjectschronically exposedto 1000 ppm lead were greater than corresponding control values. This effect, however, was more pronounced during the final 55 sessions,while differences during the intitial 25 sessionswere minimal. However, the 1000ppm median latency value of the first 40 sessions was significantly higher than that of controls. Lead treatment of the group exposed to 50 ppm lead from Experiment I and half of the group exposed to 50 ppm from Experiment 2 was terminated after the 30th experimental session. Figure 5 shows that animals maintained on the lead regimen (solid line) showed rate decreases after session 35. However, discontinuation of exposure led to reduction in overall rate of a greater magnitude (dashed lines), with median values of Experiment 2 animals reaching control levels approximately 30 sessionsafter cessation of treatment. Control groups from both experiments showed almost identical median responserates. Likewise, the effects of 50 ppm lead treatment were very similar in time course and magnitude in both exposure groups. Statistical analysis of the final 60 sessions of Experiment 2 revealed significant differences between median overall rates of the
AND THOMPSON
-,11,,,11, 4t:o,,,I 6-10
26-30
46-50
66-70
&
SESSIONS FIG. 5. Median overall response rates on the fixedinterval 30-set schedule following termination (I and 2 LEAD TERMINATED) and continuation (LEAD MAINTAINED 2) of SO ppm lead exposure. Data from a previous experiment are included for replication purposes and the numbers I and 2 refer to data from experiments I and 2, respectively. Each data point represents the median of the mean values for five sessions. Termination of exposure (dashed lines) resulted in rate decrements of a greater magnitude than exhibited by lead-maintained animals (solid lines). The two control groups showed very similar median response rates. The effects of 50 ppm lead treatment were similar in time course and magnitude in both experimental groups.
lead-maintained and corresponding control groups, between the lead-maintained and lead-terminated groups, and between the lead-terminated and corresponding control groups. Selected cumulative records of performance maintained by the FI schedule are shown in Fig. 6. The top records show the performance of a control animal during session 30 and again at session 90. These records illustrate the stability of response rates from minute to minute during the sessionand over the course of the experiment. The middle-left record shows the performance of a 50 ppm lead-exposed animal during session25. The overall rate of 38.5 responses/min was approximately four times greater than that of the control. The drinking solution of this animal was changed to tap water following session30. The middle-right
BEHAVIORAL
Session 30
TOXICITY
OF POSTWEANINGLEAD
157
50 PPM
I_ CONTROL
Session 28 ON LEAD
Session 90
CONTROL
,,,... ... . ,,,,/”
Session 90 ONLEAD
;_ .I
,,,J” ,’
FIG. 6. Selected cumulative records of responding maintained by the fixed-interval 30-set schedule for session numbers indicated. Each lever press stepped the pen vertically, while time and pausing are represented horizontally. Response rate is indicated by the slope of the line. The top records show the performance of a control animal. Middle- and bottom-right records are for animals exposed to 50 ppm lead. Rates were approximately four times greater than control rates. Termination of exposure resulted in rates decreasing to control levels (middle left). Despite moderate decrements, rates for animals maintained on 50 ppm lead remained above control levels (bottom left).
cumulative record shows the performance of this animal during the final session,60 days after the termination of lead exposure. By this time, response rates had decreased to control levels. Close inspection of the’record reveals an irregularity in minute-to-minute performance on the schedule resulting from alternation between very high and low response rates. The lower records show the performance of a lead-maintained animal during session 28 and again at session 90. Although a gradual decrement in response rate occurred between these sessions, it remained well above both control and leadterminated values. Within-session performance tended to be irregular. DISCUSSION Snowdon (1973) and Padich and Zenick (1977) were unable to detect effects of post-
weaning lead exposure on behavior. Brown (1975) found significant effects on T-maze performance only if rats were treated with lead before 10 days of age. There are at least two explanations for the absence of lead effects in these investigations. The neonatal rat may be sensitive to lead-induced behavioral changes only when exposed prior to 10 days of age. The results of the present experiment argue against such an interpretation. A more reasonable explanation for the absence of lead effects is the insensitivity of the tests used to measure performance. The T-maze, the Hebbs-Williams maze series,and certain fixed-ratio schedule parameters apparently produce types of behavior that are not influenced by lead exposure. Behavioral effects of chronic postweaning lead exposure were produced in the current experiment. Over the first 30 sessionsat 50
158
CORY-SLECHTA AND THOMPSON
and 300 ppm lead, overall and local response rates increased and responding was initiated earlier in the interval. Later, both groups exhibited moderate decreases in response rates and latency values increased. At the time of these later behavioral changes, the animals were 85 to 90 days of age. The decrements in response rate could reflect an interaction of lead effects with hormonal or other physiological changes associated with adolescence. Furthermore, gastrointestinal lead absorption declines gradually in the rat from a neonatal level of near 90% through postweaning and adolescence to the adult levels of approximately 15 % (Forbes and Reina, 1972). Thus, the animals probably had decreasing body burdens of lead. The fact that response rates remained above control levels for the duration of the behavioral observations may be accounted for by the continued exposure to lead, by the lengthy prior history of reinforcement following the lead-induced high rates of responding, or both. Exposure to the two lower concentrations of lead acetate also increased the variability of FI response rates between subjects (Fig. 3). Shapiro et a/. (1973) reported increases in both intersubject and intrasubject variability of performance of adult male rats on a multiple variable-interval 30-set extinction schedule of reinforcement following I6 ,ug/ 100 g lead acetate injections. In addition, Van Gelder and co-workers (1973) have reported that adult ewes dosed daily with 100 mg/kg lead showed significantly increased between-session variability on an auditory detection task. Lead-induced variability may be a genera1 effect and itself a reliable indicator of lead toxicity. Chronic exposure to 1000 ppm lead acetate initially resulted in decreased response rates. Over the final 40 sessions, latency values remained above control levels while response rates were highly variable among subjects; the behavior of two animals suggested the onset of toxicosis. The variability of response rates during the final 40 sessionsappears to
be another example of individual differences in susceptibility to lead. Differences produced by varying the concentration of lead acetate depended upon the time that the measurements were made. Behavioral effects produced by 50 and 300 ppm lead were very similar in magnitude but varied in time to onset and decline: while differences between animals exposed to 300 ppm lead and corresponding control animals were obvious even during the initial 5 sessions,animals exposed to 50 ppm and control animals did not differ until after 10 sessions.This suggeststime-dependent rather than concentration-dependent effects of lead. The reversibility of lead-induced behavioral changes was demonstrated by terminating exposure of several animals to 50 ppm lead. Responserates and time to the first response in the interval gradually returned to control values. This effect resemblesthat of chelation therapy in the human. Children with high blood leads, diagnosed as mentally retarded or having behavioral disorders, showed improvements in behavior and demeanor, and in one case in IQ score on the StanfordBinet test, after treatment with EDTA (Moncrieff et al., 1964). Thus both discontinuation of exposure or elimination of body lead burden via chelation (in certain circumstances) may facilitate behavioral recovery, depending on the level and duration of exposure. The range of types of behavior which might be improved by reversal of exposure and the extent of recuperation in each caseremains to be determined. Understanding the effects of lead on behavior will require knowledge of the exposure concentration and duration, the developmental period during which it occurs, the factors determining individual susceptibility, and the nature of the behavior. REFERENCES BRADY,
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AND
ZENICK,
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D. R. (1975). Neonatal lead exposure in the rat: Decreased learning as a function of age and blood lead concentration. Toxicol. Appl. Pharmacol.
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32, 628-637. O., CLARK, J., AND VOELLER, K. (1972). Lead and hyperactivity. Lancet 2, 900-903. DE LA BURDE, B., AND CHOATE, M. S. (1972). Does asymptomatic lead in children have latent sequelae? J. Pediat. 81, 1088. DEWS, P. B. (1955). Studies on behavior: I, Differential sensitivity to pentobarbitol of pecking performance in pigeons depending on the schedule of reward. J. Pharmacol. Exp. Ther. 113, 393401. DEWS, P. B., and WENGER, G. (1977). Rate-dependency of the behavioral effects of amphetamine. In Advances in Behavioral Pharmacology (T. Thompson and P. B. Dews, eds.), pp. 167-227. Academic Press, New York. FORBES, G. B., AND REINA, J. C. (1972). Effect of age on gastrointestinal absorption (Fe, Sr, Pb) in the rat. J. Nufr. 102, 647-652. GRIFFIN, T. B., COULSTON, F., WILLS, H., AND RUSSEL, J. C. (1975). Biologic effects of airborne particulate lead on continuously exposed rats and rhesus monkeys. Environ. Qual. Saf. Suppl. Lead II, DAVID,
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behavior in the rat. Pharmacol. Biochem. Behav. 6, 371-375. REITER, L. W., ANDERSON, G. E., LASKEY, J. W., AND CAHILL, D. F. (1975). Developmental and behavioral changes in the rat during chronic exposure to lead. Environ. Health Perspect. 12, 119-123. RICHELLE, M. (1969). Combined action of diazepam and d-amphetamine on fixed-interval performance in cats. J. Exp. Anal. Behav. 12, 989-998. ROBBINS, T. W. (1977). A critique of the methods available for the measurement of spontaneous motor activity. In Handbook of Psychopharmacology (L. L. Iverson, S. D. Iverson, and S. H. Snyder, eds.), Vol. 7, pp. 37-77. Plenum, New York. SAUERHOFF, M. W., AND MICHAELSON, I. A. (1973). Hyperactivity and brain catecholamines in leadexposed developing rats. Science 182, 1022-1024. SHAPIRO, M. M., TRITSCHLER, J. M., AND ULM, R. A. (1973). Lead contamination: Chronic and acute behavioral effects in the albino rat. Bull. Psychon. Sot.
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A. M. (1973). A
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VAN GELDER, BUCK, W.
WINER,
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Design.