ENVIRONMENTAL RESEARCH 58, 236-252 (1992)
Postnatal Lead Exposure Affects Motor Skills and Exploratory Behavior in Rats J O H A N L U T H M A N , * ' t A G N E T A OSKARSSON,~ LARS OLSON,* AND BARRY H O F F E R t
*Department of Histology and Neurobiology, Box 60400, Karolinska Institute, S-104 O1 Stockholm, Sweden; ?Departments of Pharmacology and Psychiatry, University of Colorado Health Sciences Center, Denver, Colorado; and SToxicology Laboratory, National Food Administration, Box 622, S-751 26 Uppsala, Sweden Received September 3, 1991 The present study was undertaken to investigate the behavioral effects of postnatal lead exposure. Newborn male Sprague-Dawley rats were given l or 8 mg/kg lead acetate intraperitoneally daily for 20 days. Control rats received 1 mg/kg sodium acetate, or 8 mg/kg sodium acetate in oversized litters. The high dose lead acetate group and the high dose, oversized sodium acetate group showed impaired weight and length increment during the end of the treatment. Rats treated with the higher dose of lead showed delayed eye opening. The time required to turn in a negative geotaxis test was transiently longer in rats treated with the higher dose of lead. A tendency of reduced forepaw grasping ability was seen in lead-treated rats during the end of the lead exposure. Ambulation and rearing in an open field were lower for the rats treated with the higher dose of lead acetate during certain periods of development. Impaired performance in a balancing rod test was also seen in the rats treated with the higher dose of lead at the adult stage, while no difference was seen in ambulation or gnawing activity during tail pinch-induced stress. Thus, lead intoxication in rats during the early postnataI period, with doses that approximate those in children, induced transient as well as persistent dysfunctions in exploratory behavior and motor skills. These observed actions of lead may be related to impaired maturation of sensitive brain regions which develop postnatally. © 1992AcademicPress, Inc.
INTRODUCTION
Exposure to lead is a well-known health problem, which may constitute an environmental pandemic (Grandjean, 1978; Needleman, 1980; Singhal and Thomas, 1980). It appears that some of the most profound effects of lead intoxication are found in the central nervous system (CNS), which seems to be especially vulnerable during development (Bornschein et al., 1980; Bjfrklund et al., 1980; Needleman, 1981, 1983; Bull et al., 1983; Winder, 1984). The effects of lead on the developing CNS may partly be due to the fact that immature organisms absorb lead to a larger extent than adults (Forbes and Reina, 1972; Ziegler et al., 1978; Mykkanen et al., 1979). However, the developing CNS is also more susceptible to the toxic effects of lead (Brown, 1975) and, even after lead exposure has ceased, functional effects of lead persist (Fox et al., 1977; Palmer et al., 1981; Bjfrklund et al., 1983; Munoz et al., 1986). Thus, intoxication with lead during critical periods of brain development appears to induce not only a developmental delay but also a permanent deficit (McMichael et al., 1988). 236 0013-9351/92 $5.00 Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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In humans, exposure to sufficiently high concentrations of lead during development has been reported to induce impairments in functions related to both cognition and motor performances (David et al., 1972; David, 1974; Needleman et al., 1980; Singhal and Thomas, 1980, Winneke et al., 1982). Human lead intoxication is known to occur as a consequence of various forms of exposure. Thus, the developing organism can be exposed to lead during gestation, during lactation, or later, due to a more general environmental distribution, e.g., air or drinking water (EPA, 1986). Another form of lead intoxication that may appear during development is derived from a more short term lead exposure from specific sources, e.g., ingestion of leaded-paint chips (ATSDR, 1988). The effects of lead exposure have been thoroughly studied in experimental animals in order to obtain a more comprehensive knowledge of behavioral, morphological, and biochemical actions. Combined pre- and postnatal exposure by administration of lead via the dam's drinking water has been the most commonly used model experimentally (Pentschew and Garro, 1966). However, this model is not devoid of drawbacks because of indirect actions on the pups due to effects of lead on the dams (Bornschein et al., 1979; Michaelson, 1980). It has also become clear that exposure to lead at later stages of development may induce detrimental effects, although these effects may differ from the actions of constant and early exposure during development (e.g., see Bull et al., 1983). In fact, postnatal exposure of lead has been shown to induce electrophysiological (Bjrrklund et al., 1983), neurochemical (see SheUenberg, 1984), and behavioral, i.e., food reinforcement (Cory-Slechta et al., 1985), changes that may last long after exposure has ceased. Thus, lead appears to induce a variety of neurotoxic actions at developmental stages later than those frequently regarded as being most susceptible. However, even though behavioral studies generally are sensitive indices of subtle damages (Riley and Vorhees, 1986), only a few studies have been performed on behavioral effects of postnatal lead exposure (see Cory-Slechta et al., 1985), and no comprehensive study has been done on the development of motor functions after postnatal lead exposure. In the present study we describe behavioral effects in rats during a limited postnatal period of direct exposure to lead. This treatment protocol, mimicking one form of more restricted lead exposure that may occur in humans during development, has previously been shown to induce alterations in the electrophysiological activity in adult cerebellum (Bjrrklund et al., 1983). The analysis was focused on lead-induced changes in physical development, sensorimotor skills, and exploratory activity during development. MATERIAL AND METHODS L e a d Treatments and A n i m a l Care
Twenty pregnant Sprague-Dawley rats were obtained at Day 18 of pregnancy from the breeder (Harlan). Immediately after delivery, the male offspring were randomly allocated to litters of 10 pups der dam, except for three litters that consisted of 15 pups/dam. The litters were divided into four different treatment
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groups: (1) two litters received I mg/kg sodium acetate (NaAc 1; 99.9% purity; Sigma) dissolved in distilled water (freshly boiled), given in daily intraperitoneal (ip) injections for 20 days; (2) three litters received 1 mg/kg lead acetate (PbAc 1) ip daily for 20 days; (3) three litters received 8 mg/kg lead acetate (PbAc 8) ip daily for 20 days; and (4) two oversized litters received 8 mg/kg sodium acetate (NaAc 8) ip daily for 20 days to control for nutritional changes. Treatments, physical examinations, and behavioral tests were performed at 12.00-16.00 hr in a small room with white artificial light and a fan producing a background noise of approximately 35 dB. The dams were removed to a separate cage before physical and behavioral measurements and injections of the pups were performed. The pups were kept in their home cage after handling and blood at the injection site was removed before the dam was taken back to the cage. Water and standard laboratory-quality food pellets (R3) were provided ad libitum. A small number of pups died (approximately 10% per litter) during the first 2-week period postnatally; thus the different groups consisted of 9-10 rats, or 13-15 rats (oversized group), per dam. Animals that died during the treatment period were briefly examined for general pathological changes, including an examination of the injection sites in the peritoneum. The rats were weaned at 35 days postpartum and kept at 4 rats per cage.
Physical Development Weight and length of the pups were measured daily during the first 20 days after birth. The weight was thereafter measured at 5-day intervals following behavioral tests. The day of complete fur development (fur at the back and the belly) was recorded as well as the day when both eyes were fully open.
Neurological Examination Righting ability was tested at Days 1-10 postnatally on a sandpaper-covered (extrafine grade) fiat testing surface. The rat was placed in a supine position and then released. The duration from the release in supine position to righting with all four legs stretched out was recorded. Negative geotaxis was tested on a sandpaper covered ramp with an 15° inclination. Each rat was placed in the middle of the ramp with the head facing down and the ability to successfully turn 180° was recorded. Time until complete and successful turning was measured for a maximum period of 30 sec. Lack of turning during the 30 sec period, or falling, terminated the test. The ability of rats to hang with the forelimbs was tested at Days 16, 19, and 20 postnatally. The rats were allowed to grasp a 6.5-mm-thick plastic rod, which was kept at 20 cm above a padded surface. Time between release and falling was recorded.
Open Field Activity The open field behavior was recorded at regular intervals at Postnatal Days 23-50. The open field consisted of a black box with an interior size of 100 x 100
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cm and a height of 30 cm, and with the floor divided into 25 segments of 25 x 25 cm. The rats were placed in the corner to the right of the observer, with the head of the rat facing into the corner. Each rats was studied separately for a 2-min period and the box was cleaned with water and soap between tests. Ambulation was recorded every time the rat passed, with all four limbs, from one segment into another segment, while rearing was recorded when the rat raised without any of the forepaws in contact with the floor. Balancing R o d Balance rod performance was tested at Day 48 postnatally on a round beam as adapted from Wallace et al. (1980). The apparatus was a 60-cm-long wooden dowel rod of 5 cm diameter that was grooved and suspended between two 20 x 20-cm safety platforms 60 cm above a padded surface. Each rat was placed in the center of the horizontal rod and the time required to reach either of the safety platforms was recorded for a maximum of 60 sec. The testing was halted if the rat failed to reach the platform within the 60-sec time period or fell off the rod. Tail Pinch At Day 50, the activity of the rats during tail pinch was recorded. The rats were studied separately in their plastic home cages for 2 min. A wooden pin was placed in the cage. Tail pinch was induced by applying a paper clip on the lower third of the tail. The cage was divided into two halves and ambulations were recorded each time the rat passed from one side of the cage to the other. Gnawings were recorded when the rat started to chew on the wooden pin or on its tail. Lead Determinations At 21 and 51 days of age, seven animals per group were sacrificed for brain lead level determinations. The lead levels were determined with atomic absorption spectrophotometry utilizing an electrothermal flameless atomization unit (Oskarsson et al., 1986). Brains were analyzed after dry-ashing at 450°C and the ash was dissolved in 10 ml 0.1 M nitric acid (Jorhem et al., 1984). The lead concentration was determined by Zeeman-corrected ETA-AAS, using Livov's platform and the method of standard addition. The results were blank corrected. Bovine liver (Nist 1577a) was analyzed in duplicate three times in order to control the accuracy of the method. The result was 0.146 +- 0.012 mg/kg (mean - SD) compared to the certified level of 0.135 +-- 0.015 mg/kg (mean -+ 95%/95% tolerance limit). Statistical Analysis The results of the weight and length determinations, as well as the results from the behavioral tests, were compared using ANOVA followed by post hoc, comparisons by the Scheffe F test. The fur and eye development, as well as the percentage success in the negative geotaxis test and the percentage failure in the balancing rod test, was compared using the Kruskal-Wallis test, followed by post hoc comparisons using nonparametric Tukey-type multiple comparisons.
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RESULTS
Physical Development The mean weights of the rats in the various treatment groups did not differ significantly until 20 days postnatally. Thus, the high dose lead acetate group and the high dose, oversized sodium acetate group showed a slightly lower weight increment than the other two groups [F(3,92) = 9.99] (Fig. 1A). Thereafter, a gradual difference was seen in the mean weights, the high dose lead acetate group and the high dose, oversized sodium acetate group still showing a slightly lower weight increment than the other two groups. Thus, at Postnatal Day 30 the high dose lead acetate group and the high dose, oversized sodium acetate group were significantly lower in weight (59 --+ 3 and 62 -_+ 2 g) compared to the low lead acetate group and the control group (73 -+ 3 and 72 --+ 2 g). At Postnatal Day 40 the high dose lead acetate group and the high dose, oversized sodium acetate group weighed 105 + 4 and 108 --- 3 g, respectively, and the low lead acetate group and A 35
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BEHAVIORAL EFFECTS OF POSTNATAL LEAD
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the control group 123 _+ 4 and 126 + 4 g, respectively. At the end of the experiment, Day 50 postnatally, these differences were still evident; the high dose lead acetate group and the high dose, oversized sodium acetate group were significantly lower in weight (171 -+ 7 and 180 -+ 4 g) compared to the low lead acetate group and the control group (193 -+ 7 and 194 + 6 g). The mean lengths of the rats followed a similar pattern. Thus, at 18 and 20 days of age a significant difference was seen [F(3,91) = 6.768]. The rats in the groups treated with the higher dose of lead acetate and the higher dose of sodium acetate (oversized group) were slightly shorter than the rats treated with the lower sodium acetate and lead acetate doses (Fig. 1B). The lengths of the rats were not measured after 20 days postnatally. There were no detrimental effects on fur development produced by the lead acetate treatments. In fact, the fur development was slightly more rapid in the rats treated with the lower dose of lead acetate, compared to the other groups (data not shown). Fur development was completed for all rats by Day 12. On the other hand, a delay in eye opening was observed in the rats treated with the higher lead acetate dose compared to both control groups. The eye opening was completed in all rats by Day 19 (Fig. 2). The pups in the group treated with the higher dose of lead acetate that died during the treatment period showed minor deposits in the peritoneum, while no deposits could be found in pups that died in the other groups. No obvious inflammatory reaction was seen at the injection sites in the different groups.
Neurological Development No difference was seen between the different groups in the percentage of rats that were able to turn in the negative geotaxis test at Days 1 to 12 postnatalty (Fig. 3A). However, at Day 12, when all rats successfully completed this test, a significant group difference was seen in turning time [F(3,92) = 7.416]. In the group
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FIG. 3. Negative geotaxis performance as studied using the development of turning behavior (A) and the time required at the age when all rats were able to turn (B). There is no difference in the development of the turning behavior (Kruskal-Wallis test); however, the lead-treated rats required a longer time to complete the turning at the age when all rats were able to turn at Day 12. In A, the percentage of rats that completed the turning successfully is presented for Days 1 to 12 (n = 18-27/ group). The data in B are expressed as mean - SEM (n = 18-27) of the time required in seconds. (a) P < 0.05, as compared to the sodium acetate, l mg/kg group (NaAc 1). (b) P < 0.05, as compared to the sodium acetate, 8 mg/kg group (NaAc 8). (c) P < 0.05, as compared to the lead acetate, 1 mg/kg group (PbAc 1). Statistical comparisons performed using analysis of variance (ANOVA) with the Scheffe F test.
treated with the higher dose of lead acetate, the turning time was longer compared to the two control groups (Fig. 3B). At Day 14, however, no significant difference was seen between the different groups [F(3,90) = 0.409] (data not shown). The time required for the righting reflex was not different for the different treatment groups when tested at Days 1 to 10 postnatally. In fact, a faster righting was seen at Day 2 in the group treated with the lower dose of lead acetate (Fig. 4). The ability to hang by the forepaws was tested at Days 16, 19, and 20 postnatally. A tendency toward reduced ability was seen at both Day 19 [F(3,91) = 1.387] and Day 20 [F(3,91) = 1.596] in the lead acetate-treated groups compared to the control groups (Fig. 5).
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Open Field Activity The activity of the rats in the large open field arena was tested at regular intervals from Days 23 to 50 postnatally. A significant increase in the main effect was seen on ambulation [F(9,333) = 27.699]. The group treated with the higher dose of lead acetate demonstrated significantly lower ambulatory activity compared to the other groups at Day 26, while no other significant differences were seen between the different groups at the various time points (Fig. 6A). At Day 27, as well as at Day 35, the high lead acetate dose group also demonstrated less rearing activity compared to the other groups. A significant group interaction was seen on the rearing activity [F(3,342) = 5.892] (Fig. 6B). 18-
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FIG. 6. Ambulation (A) and rearing (B) activity in an open field at Postnatal Days 23 to 50. The data are expressed as mean (n = 8-13); SEM was less than 15% of means. (a) P < 0.05, as compared to the sodium acetate, 1 mg/kg group (NaAc 1). (b) P < 0.05, as compared to the sodium acetate, 8 mg/kg group (NaAc 8). (c) P < 0.05, as compared to the lead acetate, 1 mg/kg group (PbAc 1). Statistical comparisons performed using analysis of variance (ANOVA) with the Scheffe F test.
Balancing Rod At Day 48, the rats were tested at the balancing rod. The majority of the rats managed to successfully walk to the platforms located at either end of the rod. However, a significantly higher degree of failure was seen in the group of rats treated with the higher dose of lead acetate (Fig. 7). No difference was seen in the time to reach either platform in the rats that successfully completed the test (data not presented). Tail Pinch
Ambulation and gnawing behavior during tail pinch were tested in the home cage environment at Day 50. N o significant differences were seen in the ambulatory behavior during the 2-rain tail pinch between the different treatment groups
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[F(3,39) = 1.363]. Furthermore, gnawing behavior did not differ after tail pinch [F(3,38) = 0.425] (Fig. 8). Lead Levels The brain lead levels were measured at the end of exposure (Day 21) and at 51 days postnatally. Significant group effects were seen at both time points [F(3,27) = 121.18 and F(3,27) = 173.56]. At Day 21, the brain lead levels were more than 13 times higher in the group treated with 1 mg/kg lead acetate and almost 50 times higher in the group treated with 8 mg/kg lead acetate. At 51 days, the brain lead levels in the lead acetate-treated animals were approximately a third of the levels seen at the end of exposure. In the 1 mg/kg lead acetate group the lead levels were almost three times the levels of controls, while in the 8 mg/kg lead acetate group the lead levels were more than 10 times higher than those in the control group. No difference in brain lead levels were seen in the two groups receiving sodium acetate (Table 1).
DISCUSSION It is apparent that lead intoxication at later stages of brain development can induce functional deficits that are qualitatively different from chronic lead exposure started during pregnancy (see Bull et al., 1983). Since one form of lead intoxication that may appear during development is a more short term postnatal lead exposure from specific sources (see ATSDR, 1988), it is of importance to further characterize the effects of postnatal lead exposure in experimental animals. The present study demonstrates several behavioral effects of lead exposure during the early postnatal period. The major findings document deficiencies in negative geotaxis during early development after lead exposure, as well as altered activity in the open field. Furthermore, a less efficient performance in the horizontal rod
246
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balance test was seen at a later stage. All these changes were fairly modest, although clearly dose-dependent and consistant. It has been argued that lead-induced malnutrition may influence behavioral changes observed in lead-treated animals (Michaelson, 1980) and nutritional deficiency during development can affect locomotor activity (Loch et al., 1978). In many experiments lead exposure has been effected via the food or drinking water. This method could cause reduction in feeding of treated nursing mothers, which in turn could result in malnourished pups. To minimize nutritional deficiencies due to maternal intoxication, the rats were directly injected ip with lead acetate in the present study. Another possible route of direct administration of lead is per os (p.o.). However, this administration technique presents some technical difficulties in newborn rats. Thus, it is hard to give p.o. administrations consistantly in newborn rats. Also, the lead acetate has to be given in higher concentrations with p.o. administrations, which may lead to a higher degree of precipitation than that observed with the ip injections, and disturbed food intake. To further avoid nonspecific effects of the treatment on behavioral parameters
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TABLE 1 CONCENTRATIONS OF LEAD IN THE BRAINS OF RATS TREATED POSTNATALLY FOR 20 DAYS WITH LEAD ACETATE OR SODIUM ACETATE Brain lead level (~g/kg wet weight) At 21 days postnatally S o d i u m acetate, 1 mg/kg L e a d acetate, 1 mg/kg L e a d acetate, 8 mg/kg Sodium acetate, 8 mg/kg A t 51 days postnatally Sodium acetate, 1 mg/kg L e a d acetate, 1 mg/kg L e a d acetate, 8 mg/kg Sodium acetate, 8 mg/kg
35 351 1312 34
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due to any lead-induced reduction in body weight, one control group was reared oversized. The rats in this group manifested weight, length, and fur development that were comparable to those of the group of rats treated with the higher dose of lead acetate. Even though no differences were seen in general body development in the group treated with the higher dose of lead acetate, compared to the oversized control group, a delay in one parameter of physical development studied was observed, i.e., eye opening, concomitant with various neurological and behavioral effects, discussed below. It is therefore evident that lead intoxication can affect specific aspects of somatic development. This finding may be due to the fact that certain processes occur under a limited time period, cf. eye opening to fur development, rendering them more sensitive to lead. Thus, by studying several different physical parameters, sensitive processes or structures can be detected. The observed impairment in some functional tests during development, but not in others, suggests that postnatal lead exposure affects specific subsets of motor skills. For instance, the success rate in the negative geotaxis test was not altered by the lead treatment, whereas the rats treated with the higher dose of lead needed more time to complete the turning. Furthermore, the success rate or the time needed for the righting reflex was not affected. Neither was the performance in the forepaw hanging test significantly affected. It is therefore tempting to speculate that these selective impairments may be related to lead-induced dysfunctions in specific neuronal regions, which may show up as transient dysfunctions. In the present study the rats were tested at regular intervals in a large open field arena for a short period (2 min). Although this test has been criticized because of the influence of emotionality factors (Renner, 1990), it provides at least a crude estimate of exploratory behavior. Rats treated with the higher dose of lead had a significant deficiency in the exploratory behavior during a specific period after the lead treatment, as evidenced by lower ambulation as well as rearing scores. It was
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originally suggested that lead exposure during development induces hyperactivity in rodents (Silbergeld and Goldberg, 1973). However, further experimentation has indicated a situation and measure specificity, as well as a dose dependency, of lead-induced effects on spontaneous motor activity (Driscoll and Stegner, 1978; Zimering et al., 1982). Indeed, hyperactivity is not a uniform consequence of lead intoxication and hyperactive behavior may indicate a failure to habituate rather than a true alteration in motor performance (Driscoll and Stegner, 1978). As mentioned above, the decreased activity of the lead-treated rats in the open field test may have been related to a higher degree of emotional stress. However, exploratory behavior in rats does not appear to be directly related to emotionality (Maier et al., 1988). Furthermore, stress induced by tail pinch (Antelman et al., 1975) did not induce any apparent differences. It has previously been shown that lead exposure during postnatal life leads to greater response to stress in rats (Barrett and Liversey, 1985). Differences in the experimental design may account for the lack of any specific stress-induced alterations in the lead-treated rats studied here, e.g., direct injection of lead (this study) or exposure via the dams. However, tail pinch is a rather nonspecific test for stress (cf. Barrett and Liversey, 1985). Thus, alterations in the sensory transmission may occur following lead exposure (see Winder, 1984), which could affect the interpretation of the tail pinch test. The rats treated with the higher dose of lead showed an impairment in the balancing rod test. This test appears to be an especially sensitive method to detect alterations in motor coordination skills (see Zion et al., 1990). In this test, impaired performance has been shown to occur in senescent rats, an effect that has been suggested to relate to dysfunction in the cerebellum (Bickford et aI., 1992; see also Friedeman and Gerhardt, 1992). The deficiency in equilibrium behavior as well as the longer time needed to complete the negative geotaxis task suggests that the postnatal lead exposure in fact induces an impairment in motor coordination skills. Furthermore, it appears that this deficiency persists after termination of the lead exposure. The sites of action of lead in the central nervous system are not well understood (Silbergeld, 1983). Various neuropsychological deficits and neuroanatomical changes have been associated with exposure to lead, and no single mechanism appears to be sufficient to account for all its diverse effects. It is more likely that lead acts at several cellular and subcellular sites, and affects neuronal functions in several different brain areas during development (Palmer et al., 1981; McCauley et al., 1982; Bjrrklund et al., 1983; Petit et al., 1983; Cooper and Manakis, 1983; Winder, 1984). It is also known that lead affects several biochemical processes in the brain (Shellenberg, 1984; Miller et al., 1990; Bresseler and Goldstein, 1991). Certain brain regions, however, appear to be particularly sensitive to lead administration at specific times of development. It has, for instance, been shown that postnatal exposure to lead leads to disturbances in hippocampal structure, e.g., loss of granular and pyramidal cells as well as changes in dendritic spines (Campbell et al., 1982; Kiraly and Jones, 1982; Slomianka et al., 1989; see also Petit et al., 1983). Other studies have demonstrated that lead affects cholinergic and serotonergic afferent connections to the hippocampus (see Alfano et al., 1983)
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and that changes in hippocampal afterdischarge occur after neonatal lead exposure (McCarren and Eccles, 1983; McCarren et al., 1984). Behavioral alterations following lead exposure have also been related to hippocampal dysfunction (Petit et al., 1983). In addition, following lead treatment during postnatal development in rats, alterations in the morphology of cerebellum have been observed, such as atrophy of the molecular layer and reduced granule cell density (Lorton and Anderson, 1986). We have previously demonstrated that postnatal treatment with lead acetate causes permanent impairment of spontaneous Purkinje cell discharge in cerebellum (Bjrrklund et al., 1983). The cerebellum appears to be involved in motor learning (Black et al., 1990) and lesions of the cerebellum have been shown to affect equilibrium (Zion et al., 1990). The hippocampal formation is known to be critical for several important functions related to cognition (see Walker and Olton, 1984) and may also be involved in exploratory behavior (see Morris, 1983). Furthermore, the dentate gyrus cells of the hippocampus and the cells derived from the external germinal layer in the cerebellum are produced largely postnatally in rodent, in contrast to neurons in other brain regions that are primarily produced prenatally (Rodier, 19807 Smart, 1991). It has also been shown that the cerebellum as well as the hippocampus seem to be highly sensitive to lead intoxication during development (Hoffer et al., 1987). Thus, although lead appears to be a generally nonspecific toxin, a selectivity can be expected if the exposure occurs during defined time periods when particularly sensitive regions mature. It is therefore important to study the effects of lead after various treatment paradigms (chronic vs acute exposure, specific time periods, etc.). In our study we observed behavioral effects that indicate effects on brain structures and transmitter systems that mature late, during the period of the lead exposure. It is therefore possible that some of the behavioral alterations observed may be related to impairment in maturation of interneurons in hippocampus and cerebellum. However, further studies are needed to define in detail the structural loci that may be involved in the functional deficits seen after postnatal lead exposure. The brain levels of lead determined in the present study were similar to the levels measured in a previous study after the same treatment protocol, when differences in measurements of weight are accounted for, i.e., wet weight versus dry weight (Bjrrklund et al., 1983). In this previous study, blood levels of approximately 225 ~g/liter were seen at Day 21 in the group receiving the lower dose of lead (PbAc l) and 3250 ~xg/liter in the group receiving the higher dose of lead; blood lead levels of 34 and 94 ixg/liter, respectively, were seen at Day 72. A blood lead level of concern of
Postnatal Lead Exposure Affects Motor Skills and Exploratory Behavior in Rats J O H A N L U T H M A N , * ' t A G N E T A OSKARSSON,~ LARS OLSON,* AND BARRY H O F F E R t
*Department of Histology and Neurobiology, Box 60400, Karolinska Institute, S-104 O1 Stockholm, Sweden; ?Departments of Pharmacology and Psychiatry, University of Colorado Health Sciences Center, Denver, Colorado; and SToxicology Laboratory, National Food Administration, Box 622, S-751 26 Uppsala, Sweden Received September 3, 1991 The present study was undertaken to investigate the behavioral effects of postnatal lead exposure. Newborn male Sprague-Dawley rats were given l or 8 mg/kg lead acetate intraperitoneally daily for 20 days. Control rats received 1 mg/kg sodium acetate, or 8 mg/kg sodium acetate in oversized litters. The high dose lead acetate group and the high dose, oversized sodium acetate group showed impaired weight and length increment during the end of the treatment. Rats treated with the higher dose of lead showed delayed eye opening. The time required to turn in a negative geotaxis test was transiently longer in rats treated with the higher dose of lead. A tendency of reduced forepaw grasping ability was seen in lead-treated rats during the end of the lead exposure. Ambulation and rearing in an open field were lower for the rats treated with the higher dose of lead acetate during certain periods of development. Impaired performance in a balancing rod test was also seen in the rats treated with the higher dose of lead at the adult stage, while no difference was seen in ambulation or gnawing activity during tail pinch-induced stress. Thus, lead intoxication in rats during the early postnataI period, with doses that approximate those in children, induced transient as well as persistent dysfunctions in exploratory behavior and motor skills. These observed actions of lead may be related to impaired maturation of sensitive brain regions which develop postnatally. © 1992AcademicPress, Inc.
INTRODUCTION
Exposure to lead is a well-known health problem, which may constitute an environmental pandemic (Grandjean, 1978; Needleman, 1980; Singhal and Thomas, 1980). It appears that some of the most profound effects of lead intoxication are found in the central nervous system (CNS), which seems to be especially vulnerable during development (Bornschein et al., 1980; Bjfrklund et al., 1980; Needleman, 1981, 1983; Bull et al., 1983; Winder, 1984). The effects of lead on the developing CNS may partly be due to the fact that immature organisms absorb lead to a larger extent than adults (Forbes and Reina, 1972; Ziegler et al., 1978; Mykkanen et al., 1979). However, the developing CNS is also more susceptible to the toxic effects of lead (Brown, 1975) and, even after lead exposure has ceased, functional effects of lead persist (Fox et al., 1977; Palmer et al., 1981; Bjfrklund et al., 1983; Munoz et al., 1986). Thus, intoxication with lead during critical periods of brain development appears to induce not only a developmental delay but also a permanent deficit (McMichael et al., 1988). 236 0013-9351/92 $5.00 Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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In humans, exposure to sufficiently high concentrations of lead during development has been reported to induce impairments in functions related to both cognition and motor performances (David et al., 1972; David, 1974; Needleman et al., 1980; Singhal and Thomas, 1980, Winneke et al., 1982). Human lead intoxication is known to occur as a consequence of various forms of exposure. Thus, the developing organism can be exposed to lead during gestation, during lactation, or later, due to a more general environmental distribution, e.g., air or drinking water (EPA, 1986). Another form of lead intoxication that may appear during development is derived from a more short term lead exposure from specific sources, e.g., ingestion of leaded-paint chips (ATSDR, 1988). The effects of lead exposure have been thoroughly studied in experimental animals in order to obtain a more comprehensive knowledge of behavioral, morphological, and biochemical actions. Combined pre- and postnatal exposure by administration of lead via the dam's drinking water has been the most commonly used model experimentally (Pentschew and Garro, 1966). However, this model is not devoid of drawbacks because of indirect actions on the pups due to effects of lead on the dams (Bornschein et al., 1979; Michaelson, 1980). It has also become clear that exposure to lead at later stages of development may induce detrimental effects, although these effects may differ from the actions of constant and early exposure during development (e.g., see Bull et al., 1983). In fact, postnatal exposure of lead has been shown to induce electrophysiological (Bjrrklund et al., 1983), neurochemical (see SheUenberg, 1984), and behavioral, i.e., food reinforcement (Cory-Slechta et al., 1985), changes that may last long after exposure has ceased. Thus, lead appears to induce a variety of neurotoxic actions at developmental stages later than those frequently regarded as being most susceptible. However, even though behavioral studies generally are sensitive indices of subtle damages (Riley and Vorhees, 1986), only a few studies have been performed on behavioral effects of postnatal lead exposure (see Cory-Slechta et al., 1985), and no comprehensive study has been done on the development of motor functions after postnatal lead exposure. In the present study we describe behavioral effects in rats during a limited postnatal period of direct exposure to lead. This treatment protocol, mimicking one form of more restricted lead exposure that may occur in humans during development, has previously been shown to induce alterations in the electrophysiological activity in adult cerebellum (Bjrrklund et al., 1983). The analysis was focused on lead-induced changes in physical development, sensorimotor skills, and exploratory activity during development. MATERIAL AND METHODS L e a d Treatments and A n i m a l Care
Twenty pregnant Sprague-Dawley rats were obtained at Day 18 of pregnancy from the breeder (Harlan). Immediately after delivery, the male offspring were randomly allocated to litters of 10 pups der dam, except for three litters that consisted of 15 pups/dam. The litters were divided into four different treatment
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groups: (1) two litters received I mg/kg sodium acetate (NaAc 1; 99.9% purity; Sigma) dissolved in distilled water (freshly boiled), given in daily intraperitoneal (ip) injections for 20 days; (2) three litters received 1 mg/kg lead acetate (PbAc 1) ip daily for 20 days; (3) three litters received 8 mg/kg lead acetate (PbAc 8) ip daily for 20 days; and (4) two oversized litters received 8 mg/kg sodium acetate (NaAc 8) ip daily for 20 days to control for nutritional changes. Treatments, physical examinations, and behavioral tests were performed at 12.00-16.00 hr in a small room with white artificial light and a fan producing a background noise of approximately 35 dB. The dams were removed to a separate cage before physical and behavioral measurements and injections of the pups were performed. The pups were kept in their home cage after handling and blood at the injection site was removed before the dam was taken back to the cage. Water and standard laboratory-quality food pellets (R3) were provided ad libitum. A small number of pups died (approximately 10% per litter) during the first 2-week period postnatally; thus the different groups consisted of 9-10 rats, or 13-15 rats (oversized group), per dam. Animals that died during the treatment period were briefly examined for general pathological changes, including an examination of the injection sites in the peritoneum. The rats were weaned at 35 days postpartum and kept at 4 rats per cage.
Physical Development Weight and length of the pups were measured daily during the first 20 days after birth. The weight was thereafter measured at 5-day intervals following behavioral tests. The day of complete fur development (fur at the back and the belly) was recorded as well as the day when both eyes were fully open.
Neurological Examination Righting ability was tested at Days 1-10 postnatally on a sandpaper-covered (extrafine grade) fiat testing surface. The rat was placed in a supine position and then released. The duration from the release in supine position to righting with all four legs stretched out was recorded. Negative geotaxis was tested on a sandpaper covered ramp with an 15° inclination. Each rat was placed in the middle of the ramp with the head facing down and the ability to successfully turn 180° was recorded. Time until complete and successful turning was measured for a maximum period of 30 sec. Lack of turning during the 30 sec period, or falling, terminated the test. The ability of rats to hang with the forelimbs was tested at Days 16, 19, and 20 postnatally. The rats were allowed to grasp a 6.5-mm-thick plastic rod, which was kept at 20 cm above a padded surface. Time between release and falling was recorded.
Open Field Activity The open field behavior was recorded at regular intervals at Postnatal Days 23-50. The open field consisted of a black box with an interior size of 100 x 100
B E H A V I O R A L E F F E C T S OF P O S T N A T A L L E A D
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cm and a height of 30 cm, and with the floor divided into 25 segments of 25 x 25 cm. The rats were placed in the corner to the right of the observer, with the head of the rat facing into the corner. Each rats was studied separately for a 2-min period and the box was cleaned with water and soap between tests. Ambulation was recorded every time the rat passed, with all four limbs, from one segment into another segment, while rearing was recorded when the rat raised without any of the forepaws in contact with the floor. Balancing R o d Balance rod performance was tested at Day 48 postnatally on a round beam as adapted from Wallace et al. (1980). The apparatus was a 60-cm-long wooden dowel rod of 5 cm diameter that was grooved and suspended between two 20 x 20-cm safety platforms 60 cm above a padded surface. Each rat was placed in the center of the horizontal rod and the time required to reach either of the safety platforms was recorded for a maximum of 60 sec. The testing was halted if the rat failed to reach the platform within the 60-sec time period or fell off the rod. Tail Pinch At Day 50, the activity of the rats during tail pinch was recorded. The rats were studied separately in their plastic home cages for 2 min. A wooden pin was placed in the cage. Tail pinch was induced by applying a paper clip on the lower third of the tail. The cage was divided into two halves and ambulations were recorded each time the rat passed from one side of the cage to the other. Gnawings were recorded when the rat started to chew on the wooden pin or on its tail. Lead Determinations At 21 and 51 days of age, seven animals per group were sacrificed for brain lead level determinations. The lead levels were determined with atomic absorption spectrophotometry utilizing an electrothermal flameless atomization unit (Oskarsson et al., 1986). Brains were analyzed after dry-ashing at 450°C and the ash was dissolved in 10 ml 0.1 M nitric acid (Jorhem et al., 1984). The lead concentration was determined by Zeeman-corrected ETA-AAS, using Livov's platform and the method of standard addition. The results were blank corrected. Bovine liver (Nist 1577a) was analyzed in duplicate three times in order to control the accuracy of the method. The result was 0.146 +- 0.012 mg/kg (mean - SD) compared to the certified level of 0.135 +-- 0.015 mg/kg (mean -+ 95%/95% tolerance limit). Statistical Analysis The results of the weight and length determinations, as well as the results from the behavioral tests, were compared using ANOVA followed by post hoc, comparisons by the Scheffe F test. The fur and eye development, as well as the percentage success in the negative geotaxis test and the percentage failure in the balancing rod test, was compared using the Kruskal-Wallis test, followed by post hoc comparisons using nonparametric Tukey-type multiple comparisons.
240
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RESULTS
Physical Development The mean weights of the rats in the various treatment groups did not differ significantly until 20 days postnatally. Thus, the high dose lead acetate group and the high dose, oversized sodium acetate group showed a slightly lower weight increment than the other two groups [F(3,92) = 9.99] (Fig. 1A). Thereafter, a gradual difference was seen in the mean weights, the high dose lead acetate group and the high dose, oversized sodium acetate group still showing a slightly lower weight increment than the other two groups. Thus, at Postnatal Day 30 the high dose lead acetate group and the high dose, oversized sodium acetate group were significantly lower in weight (59 --+ 3 and 62 -_+ 2 g) compared to the low lead acetate group and the control group (73 -+ 3 and 72 --+ 2 g). At Postnatal Day 40 the high dose lead acetate group and the high dose, oversized sodium acetate group weighed 105 + 4 and 108 --- 3 g, respectively, and the low lead acetate group and A 35
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BEHAVIORAL EFFECTS OF POSTNATAL LEAD
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the control group 123 _+ 4 and 126 + 4 g, respectively. At the end of the experiment, Day 50 postnatally, these differences were still evident; the high dose lead acetate group and the high dose, oversized sodium acetate group were significantly lower in weight (171 -+ 7 and 180 -+ 4 g) compared to the low lead acetate group and the control group (193 -+ 7 and 194 + 6 g). The mean lengths of the rats followed a similar pattern. Thus, at 18 and 20 days of age a significant difference was seen [F(3,91) = 6.768]. The rats in the groups treated with the higher dose of lead acetate and the higher dose of sodium acetate (oversized group) were slightly shorter than the rats treated with the lower sodium acetate and lead acetate doses (Fig. 1B). The lengths of the rats were not measured after 20 days postnatally. There were no detrimental effects on fur development produced by the lead acetate treatments. In fact, the fur development was slightly more rapid in the rats treated with the lower dose of lead acetate, compared to the other groups (data not shown). Fur development was completed for all rats by Day 12. On the other hand, a delay in eye opening was observed in the rats treated with the higher lead acetate dose compared to both control groups. The eye opening was completed in all rats by Day 19 (Fig. 2). The pups in the group treated with the higher dose of lead acetate that died during the treatment period showed minor deposits in the peritoneum, while no deposits could be found in pups that died in the other groups. No obvious inflammatory reaction was seen at the injection sites in the different groups.
Neurological Development No difference was seen between the different groups in the percentage of rats that were able to turn in the negative geotaxis test at Days 1 to 12 postnatalty (Fig. 3A). However, at Day 12, when all rats successfully completed this test, a significant group difference was seen in turning time [F(3,92) = 7.416]. In the group
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treated with the higher dose of lead acetate, the turning time was longer compared to the two control groups (Fig. 3B). At Day 14, however, no significant difference was seen between the different groups [F(3,90) = 0.409] (data not shown). The time required for the righting reflex was not different for the different treatment groups when tested at Days 1 to 10 postnatally. In fact, a faster righting was seen at Day 2 in the group treated with the lower dose of lead acetate (Fig. 4). The ability to hang by the forepaws was tested at Days 16, 19, and 20 postnatally. A tendency toward reduced ability was seen at both Day 19 [F(3,91) = 1.387] and Day 20 [F(3,91) = 1.596] in the lead acetate-treated groups compared to the control groups (Fig. 5).
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Open Field Activity The activity of the rats in the large open field arena was tested at regular intervals from Days 23 to 50 postnatally. A significant increase in the main effect was seen on ambulation [F(9,333) = 27.699]. The group treated with the higher dose of lead acetate demonstrated significantly lower ambulatory activity compared to the other groups at Day 26, while no other significant differences were seen between the different groups at the various time points (Fig. 6A). At Day 27, as well as at Day 35, the high lead acetate dose group also demonstrated less rearing activity compared to the other groups. A significant group interaction was seen on the rearing activity [F(3,342) = 5.892] (Fig. 6B). 18-
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L U T H M A N ET AL.
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FIG. 6. Ambulation (A) and rearing (B) activity in an open field at Postnatal Days 23 to 50. The data are expressed as mean (n = 8-13); SEM was less than 15% of means. (a) P < 0.05, as compared to the sodium acetate, 1 mg/kg group (NaAc 1). (b) P < 0.05, as compared to the sodium acetate, 8 mg/kg group (NaAc 8). (c) P < 0.05, as compared to the lead acetate, 1 mg/kg group (PbAc 1). Statistical comparisons performed using analysis of variance (ANOVA) with the Scheffe F test.
Balancing Rod At Day 48, the rats were tested at the balancing rod. The majority of the rats managed to successfully walk to the platforms located at either end of the rod. However, a significantly higher degree of failure was seen in the group of rats treated with the higher dose of lead acetate (Fig. 7). No difference was seen in the time to reach either platform in the rats that successfully completed the test (data not presented). Tail Pinch
Ambulation and gnawing behavior during tail pinch were tested in the home cage environment at Day 50. N o significant differences were seen in the ambulatory behavior during the 2-rain tail pinch between the different treatment groups
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[F(3,39) = 1.363]. Furthermore, gnawing behavior did not differ after tail pinch [F(3,38) = 0.425] (Fig. 8). Lead Levels The brain lead levels were measured at the end of exposure (Day 21) and at 51 days postnatally. Significant group effects were seen at both time points [F(3,27) = 121.18 and F(3,27) = 173.56]. At Day 21, the brain lead levels were more than 13 times higher in the group treated with 1 mg/kg lead acetate and almost 50 times higher in the group treated with 8 mg/kg lead acetate. At 51 days, the brain lead levels in the lead acetate-treated animals were approximately a third of the levels seen at the end of exposure. In the 1 mg/kg lead acetate group the lead levels were almost three times the levels of controls, while in the 8 mg/kg lead acetate group the lead levels were more than 10 times higher than those in the control group. No difference in brain lead levels were seen in the two groups receiving sodium acetate (Table 1).
DISCUSSION It is apparent that lead intoxication at later stages of brain development can induce functional deficits that are qualitatively different from chronic lead exposure started during pregnancy (see Bull et al., 1983). Since one form of lead intoxication that may appear during development is a more short term postnatal lead exposure from specific sources (see ATSDR, 1988), it is of importance to further characterize the effects of postnatal lead exposure in experimental animals. The present study demonstrates several behavioral effects of lead exposure during the early postnatal period. The major findings document deficiencies in negative geotaxis during early development after lead exposure, as well as altered activity in the open field. Furthermore, a less efficient performance in the horizontal rod
246
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balance test was seen at a later stage. All these changes were fairly modest, although clearly dose-dependent and consistant. It has been argued that lead-induced malnutrition may influence behavioral changes observed in lead-treated animals (Michaelson, 1980) and nutritional deficiency during development can affect locomotor activity (Loch et al., 1978). In many experiments lead exposure has been effected via the food or drinking water. This method could cause reduction in feeding of treated nursing mothers, which in turn could result in malnourished pups. To minimize nutritional deficiencies due to maternal intoxication, the rats were directly injected ip with lead acetate in the present study. Another possible route of direct administration of lead is per os (p.o.). However, this administration technique presents some technical difficulties in newborn rats. Thus, it is hard to give p.o. administrations consistantly in newborn rats. Also, the lead acetate has to be given in higher concentrations with p.o. administrations, which may lead to a higher degree of precipitation than that observed with the ip injections, and disturbed food intake. To further avoid nonspecific effects of the treatment on behavioral parameters
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TABLE 1 CONCENTRATIONS OF LEAD IN THE BRAINS OF RATS TREATED POSTNATALLY FOR 20 DAYS WITH LEAD ACETATE OR SODIUM ACETATE Brain lead level (~g/kg wet weight) At 21 days postnatally S o d i u m acetate, 1 mg/kg L e a d acetate, 1 mg/kg L e a d acetate, 8 mg/kg Sodium acetate, 8 mg/kg A t 51 days postnatally Sodium acetate, 1 mg/kg L e a d acetate, 1 mg/kg L e a d acetate, 8 mg/kg Sodium acetate, 8 mg/kg
35 351 1312 34
--- 4 -+ 17a - 97 a'b'c - 12
44 110 434 43
--- 7 + 5a +- 26 ~'b'C --- 4
Note. D a t a are e x p r e s s e d as m e a n -+ S E M (n = 7). Statistical c o m p a r i s o n performed using analysis of variance ( A N O V A ) with the Scheffe F test. a p < 0.05, as c o m p a r e d to the s o d i u m acetate, 1 mg/kggroup. b p < 0.05, as c o m p a r e d to the s o d i u m acetate, 8 mg/kg group. c p < 0.05, as c o m p a r e d to the lead acetate, lmg/kg group.
due to any lead-induced reduction in body weight, one control group was reared oversized. The rats in this group manifested weight, length, and fur development that were comparable to those of the group of rats treated with the higher dose of lead acetate. Even though no differences were seen in general body development in the group treated with the higher dose of lead acetate, compared to the oversized control group, a delay in one parameter of physical development studied was observed, i.e., eye opening, concomitant with various neurological and behavioral effects, discussed below. It is therefore evident that lead intoxication can affect specific aspects of somatic development. This finding may be due to the fact that certain processes occur under a limited time period, cf. eye opening to fur development, rendering them more sensitive to lead. Thus, by studying several different physical parameters, sensitive processes or structures can be detected. The observed impairment in some functional tests during development, but not in others, suggests that postnatal lead exposure affects specific subsets of motor skills. For instance, the success rate in the negative geotaxis test was not altered by the lead treatment, whereas the rats treated with the higher dose of lead needed more time to complete the turning. Furthermore, the success rate or the time needed for the righting reflex was not affected. Neither was the performance in the forepaw hanging test significantly affected. It is therefore tempting to speculate that these selective impairments may be related to lead-induced dysfunctions in specific neuronal regions, which may show up as transient dysfunctions. In the present study the rats were tested at regular intervals in a large open field arena for a short period (2 min). Although this test has been criticized because of the influence of emotionality factors (Renner, 1990), it provides at least a crude estimate of exploratory behavior. Rats treated with the higher dose of lead had a significant deficiency in the exploratory behavior during a specific period after the lead treatment, as evidenced by lower ambulation as well as rearing scores. It was
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originally suggested that lead exposure during development induces hyperactivity in rodents (Silbergeld and Goldberg, 1973). However, further experimentation has indicated a situation and measure specificity, as well as a dose dependency, of lead-induced effects on spontaneous motor activity (Driscoll and Stegner, 1978; Zimering et al., 1982). Indeed, hyperactivity is not a uniform consequence of lead intoxication and hyperactive behavior may indicate a failure to habituate rather than a true alteration in motor performance (Driscoll and Stegner, 1978). As mentioned above, the decreased activity of the lead-treated rats in the open field test may have been related to a higher degree of emotional stress. However, exploratory behavior in rats does not appear to be directly related to emotionality (Maier et al., 1988). Furthermore, stress induced by tail pinch (Antelman et al., 1975) did not induce any apparent differences. It has previously been shown that lead exposure during postnatal life leads to greater response to stress in rats (Barrett and Liversey, 1985). Differences in the experimental design may account for the lack of any specific stress-induced alterations in the lead-treated rats studied here, e.g., direct injection of lead (this study) or exposure via the dams. However, tail pinch is a rather nonspecific test for stress (cf. Barrett and Liversey, 1985). Thus, alterations in the sensory transmission may occur following lead exposure (see Winder, 1984), which could affect the interpretation of the tail pinch test. The rats treated with the higher dose of lead showed an impairment in the balancing rod test. This test appears to be an especially sensitive method to detect alterations in motor coordination skills (see Zion et al., 1990). In this test, impaired performance has been shown to occur in senescent rats, an effect that has been suggested to relate to dysfunction in the cerebellum (Bickford et aI., 1992; see also Friedeman and Gerhardt, 1992). The deficiency in equilibrium behavior as well as the longer time needed to complete the negative geotaxis task suggests that the postnatal lead exposure in fact induces an impairment in motor coordination skills. Furthermore, it appears that this deficiency persists after termination of the lead exposure. The sites of action of lead in the central nervous system are not well understood (Silbergeld, 1983). Various neuropsychological deficits and neuroanatomical changes have been associated with exposure to lead, and no single mechanism appears to be sufficient to account for all its diverse effects. It is more likely that lead acts at several cellular and subcellular sites, and affects neuronal functions in several different brain areas during development (Palmer et al., 1981; McCauley et al., 1982; Bjrrklund et al., 1983; Petit et al., 1983; Cooper and Manakis, 1983; Winder, 1984). It is also known that lead affects several biochemical processes in the brain (Shellenberg, 1984; Miller et al., 1990; Bresseler and Goldstein, 1991). Certain brain regions, however, appear to be particularly sensitive to lead administration at specific times of development. It has, for instance, been shown that postnatal exposure to lead leads to disturbances in hippocampal structure, e.g., loss of granular and pyramidal cells as well as changes in dendritic spines (Campbell et al., 1982; Kiraly and Jones, 1982; Slomianka et al., 1989; see also Petit et al., 1983). Other studies have demonstrated that lead affects cholinergic and serotonergic afferent connections to the hippocampus (see Alfano et al., 1983)
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and that changes in hippocampal afterdischarge occur after neonatal lead exposure (McCarren and Eccles, 1983; McCarren et al., 1984). Behavioral alterations following lead exposure have also been related to hippocampal dysfunction (Petit et al., 1983). In addition, following lead treatment during postnatal development in rats, alterations in the morphology of cerebellum have been observed, such as atrophy of the molecular layer and reduced granule cell density (Lorton and Anderson, 1986). We have previously demonstrated that postnatal treatment with lead acetate causes permanent impairment of spontaneous Purkinje cell discharge in cerebellum (Bjrrklund et al., 1983). The cerebellum appears to be involved in motor learning (Black et al., 1990) and lesions of the cerebellum have been shown to affect equilibrium (Zion et al., 1990). The hippocampal formation is known to be critical for several important functions related to cognition (see Walker and Olton, 1984) and may also be involved in exploratory behavior (see Morris, 1983). Furthermore, the dentate gyrus cells of the hippocampus and the cells derived from the external germinal layer in the cerebellum are produced largely postnatally in rodent, in contrast to neurons in other brain regions that are primarily produced prenatally (Rodier, 19807 Smart, 1991). It has also been shown that the cerebellum as well as the hippocampus seem to be highly sensitive to lead intoxication during development (Hoffer et al., 1987). Thus, although lead appears to be a generally nonspecific toxin, a selectivity can be expected if the exposure occurs during defined time periods when particularly sensitive regions mature. It is therefore important to study the effects of lead after various treatment paradigms (chronic vs acute exposure, specific time periods, etc.). In our study we observed behavioral effects that indicate effects on brain structures and transmitter systems that mature late, during the period of the lead exposure. It is therefore possible that some of the behavioral alterations observed may be related to impairment in maturation of interneurons in hippocampus and cerebellum. However, further studies are needed to define in detail the structural loci that may be involved in the functional deficits seen after postnatal lead exposure. The brain levels of lead determined in the present study were similar to the levels measured in a previous study after the same treatment protocol, when differences in measurements of weight are accounted for, i.e., wet weight versus dry weight (Bjrrklund et al., 1983). In this previous study, blood levels of approximately 225 ~g/liter were seen at Day 21 in the group receiving the lower dose of lead (PbAc l) and 3250 ~xg/liter in the group receiving the higher dose of lead; blood lead levels of 34 and 94 ixg/liter, respectively, were seen at Day 72. A blood lead level of concern of
