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The Effects of Inorganic Lead on Cholinergic Transmission
The Effects of Inorganic Lead on Cholinergic Transmission ALAN M. GOLDBERG Department of Environmental Health Sciences, The Johns Hopkins University, School ofHygiene and Public Health, Baltimore, MD 21205 (U.S.A.)
INTRODUCTION Our present understanding of the biologic effects of lead is largely based on the study of individuals suffering from overt lead poisoning and on the experience gained in the management of persons exposed to lead through the occupational setting. The exposure to lead in these groups is considerably greater than that in the normal population encountering lead via food sources and environmental pollution. About one of four young children who survive an attack of acute encephalopathy, as a result of lead poisoning, suffer severe and permanent neurological consequences. Byers and Lord (1 943) have delineated the nature and severity of central nervous system injury that follows lead poisoning in early childhood. The severe forms of acute lead encephalopathy, including convulsive episodes, are fortunately becoming less common; the subtle neurological deficits and mental impairment which might include sensory loss and a decrease in the I.Q. are more difficult to assess. The children appear to suffer from motor incoordination, lack of sensory perception, impaired learning, short attention spans and are generally easily distracted (National Academy of Science, 1972). These behavioral changes are difficult to distinguish from the changes seen after organic brain damage or environmental deprivation. ANIMAL MODELS OF LEAD EXPOSURE The administration of inorganic lead during critical periods of development can lead to behavioral dysfunction in experimental animals. Behavioral changes have been described in mice (Silbergel and Goldberg, 1973), rats (Saureoff and Michaelson, 1973; Shih et al., 1976; Overman, 1977) and monkeys (Allen et al., 1974). The major behavioral effects observed have been changes in spontaneous motor activity, agressiveness, arousal, patterning of behavior, and vocalization. One criticism raised against many experimental studies has been that the results on intact animals are largely based on experiments where high doses of lead, on an absolute scale have been administered. Actually, since the sensitiv-
466 ity of different animal species to lead is very different, the lead exposure necessary in experimental work also varies. It is apparent from the literature that one cannot use blood levels of lead in different species to compare the severity of exposure. Rodents with blood levels of lead exceeding 100 pg% do not present symptoms of overt intoxication, while man with the same blood level of lead is clearly intoxicated. Therefore, to suggest that exposure in any study is high or low can only be argued on the basis of species sensitivity. Various laboratories use different regiments of lead administration. Generally, rat or mouse pups are exposed to lead through the mother’s milk. While most investigators initiate lead exposure on the day pf parturition, others expose their animals on day two or three of life. In certain studies the mothers have been exposed to lead for their lifetime and the f2 generation, also exposed to lead, are the subjects of the investigation. As indicated above, data on behavioral changes produced by the exposure to lead are not consistent between laboratories. Reports from two laboratories describe either hyperactivity or overactivity in the mouse (Silbergeld and Goldberg, 1973; Maker et al., 1975). With the rat, hyperactivity has been reported from at least five different laboratories (Sauerhoff and Michaelson, 1973; Golter and Michaelson, 1975; Shih et al., 1976; Grant et al., 1975; Overmann, 1977). Some studies from the same laboratories, however, as well as from other laboratories have not revealed this effect (Sobotka et al., 1975; Modak et al., 1975; Grant et al., 1976; Krehbiel et al., 1978). To evaluate the apparent contradiction, several features of these studies should be taken into account. First, the route and level of exposure have in most cases been different. Second, the methods of measuring the behavioral changes have not been standardized. Third, the nutritional status of the animals has not been controlled as regards the intake of calories and nutritional factors such as other trace metals. Fourth, the litter size and thus the nutrition and the psychological factors have not been uniform. With this lack of uniformity it is difficult to compare directly the studies that have been reported. However, it may be noted that the attenuated or paradoxical pharmacological effects of amphetamines in lead exposed animals, originally observed in mice (Silbergeld and Goldberg, 1974) have been reported in the rat independently of the appearance of hyperactivity (Shih et al., 1976; Sobotka et al., 1975). The effects on the cholinergic system have also been consistent, independent of the species studied. In our recent studies, we have developed a model of lead exposure that does not retard growth or produce undernourishment. The protocol has worked well with rats and mice; other species have not been tested. On the day of parturition the mothers receive 5 mg/ml lead acetate in their drinking water. The mothers of control animals receive tap water. Litters are reduced to 3 pups each within 24 h of birth. Neonatal animals are thus exposed to lead through the milk of their mothers from postnatal days 1 through 21. They are weaned at 28 days and group-housed by sex. Animals exposed t o lead using this protocol show no growth retardation.
467 EFFECTS OF LEAD ON THE CHOLINERGIC SYSTEM
Peripheral system Kostial and Vouk (1957) demonstrated that lead added in vitro blocked ganglionic transmission in the superior cervical ganglion of the cat and produced a decrease in the release of acetylcholine (ACh); this effect of lead appeared to be related to the interaction of lead and calcium. Manalis and Cooper (1 973), using the frog sartorius muscle preparation, demonstrated that lead increases miniature end-plate potential frequency and decreases end-plate potential amplitude. Goldberg and van den Bercken (unpublished) confirmed these observations but observed that in the hemidiaphragm of the rat, the endplate potential amplitude was decreased but minature end-plate potential frequency was unchanged. Similar results in the rat hemidiaphragm have been reported by Bornstein and Pickett (1 977). Silbergeld et al., (1 974) demonstrated that in vitro lead can decrease the force of contraction of the diaphragm when the phrenic nerve is stimulated. Further, they demonstrated that the force of contraction was not impaired when the muscle was stimulated directly. The ability of lead to decrease the end-plate potential amplitude and the force of muscle contraction cannot be explained by a postsynaptic inhibition since lead dose not alter the postsynaptic sensitivity to ACh (Silbergeld et al., 1974). All of these results are consistent with the suggestion that lead added in vitro decreases the release of ACh.
Steady-state levels Steady-state levels of choline and ACh have been measured in mice and rats exposed to lead by Carroll et al. (1977) and Shih and Hanin (1978). These groups failed to observe any changes in either compound. On the other hand, Modak et al. ( 1975) did observe a small but significant decrease in the levels of ACh in the diencephalon of the rat.
Enzymes
No changes in the activity of choline acetyltransferase have been observed after exposure to lead but cholinesterase activity was depressed by approximately 20%in all studies (Sobotka et al., 1975; Modak et al., 1975; Carrol et al., 1977). In some of these studies the decrease reached statistical significance. Whether this is a biologically important phenomenon is difficult to determine. Release and turnover Carroll et al. (1977) studied the potassium induced release of ACh from brain tissue. The question that was being asked was whether inhibition of ACh release by lead in vitro was a general property of lead. ACh release was induced by exposing the brain minces to elevated levels of potassium (35 mM) for periods up to 1 h. As previously shown, the release of ACh was linear for at least 1 h and was calcium dependent. In control mice the release of ACh was in
468 the range of 300 nmol/g wet wt h. In Iead exposed animals the release was decreased by about 50%. These results demonstrate that chronic lead exposure inhibits the potassium induced release of ACh from brain tissue. Presumably, the impairment of ACh release is a direct effect of lead and not a secondary consequence of the lead exposure. Shih and Hanin (1978) have presented evidence that lead exposure in the rat results in a decrease in the turnover rate of ACh. They exposed rats t o lead acetate from birth. In their study there was a decrease in growth rate of the lead exposed animals. At approximately 4 5 days of age, control and lead treated rats were administered radiolabelled choline phosphate, a precursor of choline and ACh. Specific activities of ACh were not altered, but the specific activity of choline was significantly elevated in all brain areas studied as compared to age-matched controls. The in vivo ACh turnover rate in cortex, hippocampus, midbrain, and striatum was decreased by 33-45%. These findings provide evidence for an inhibitory effect of lead exposure on central cholinergic function in vivo. Pharmacology Many drugs produce unexpected pharmacological responses in lead exposed animals. Methylphenidate and amphetamine produced a paradoxical or attenuated response in lead treated animals. Although these drugs are generally thought of as aminergic stimulants they have many additional effects not related t o their catecholaminergic action. Amphetamines have been shown t o increase ACh release from brain. In attempting to see whether methylphenidate also had a cholinergic action we did the following experiment (Carroll e t al., 1977). Lead exposed animals were treated with 40 mg/kg methylphenidate and killed 2 h later. The brains from control, lead exposed, and lead exposed animals given methylphenidate were removed and the potassium induced release of ACh measured. As in previous experiments, lead exposed animals had a decreased release of ACh; this decrease was completely reversed in the methylphenidate-treated animals. These data are consistent with those of Shih e t al., (1 976 and 1978) who previously reported evidence suggesting a cholinergic effect of methylphenidate. Collectively, these data suggest a cholinergic link in the mechanisms of action of methylphenidate. SUMMARY
Low levels of inorganic lead chronically administered during critical periods of development result in behavioral alteration and neurochemical changes. Among the changes seen consistently are alterations in central and peripheral cholinergic function. Administration of several drugs that influence cholinergic metabolism influences the behavioral effects of lead. In the peripheral nervous system, lead in vitro has been shown to impair cholinergic function in several different experimental models. Lead inhibits the release of ACh from the superior cervical ganglion of the cat during stimulation of the preganglionic fibers, decreases the size of the end-plate potential in the frog and rat neuro-
469 muscular preparations, and reduces the force of muscle contraction during nerve stimulation. The pharmacological evidence obtained in studies on whole animals and direct measurements of ACh release and turnover indicate that the release of ACh in the central and peripheral nervous systems is impaired by both acute in vitro and by chronic in vivo exposure t o lead. ACKNOWLEDGEMENTS The studies reported from the author’s laboratory were supported by NIEHS grant 00454 and 00034. REFERENCES Allen, J.R., McWay, P.J. and Suomi, S.J. (1974) Pathological and behavioral changes in rhesus monkeys exposed t o lead. Environ. Health Perspect., 7,239-246. Bornstein, J.C. and Pickett, J.B. (1977) Some effects of lead ions o n transmitter release at rat neuromuscular junctions. Soc. Neurosci., 3, 370. Byers, R.K. and Lord, E.E. (1943) Late effects of lead poisoning o n mental development. Amer. J. Dis. Childh., 66, 471-494. Carroll, P.T., Silbergeld, E.K., and Goldberg, A.M. (1 977) Alterations of central cholinergic function in lead-induced hyperactivity. Biochem. Pharmacol., 26, 397-402. Colter, M. and Michaelson, I.A. (1 975) Growth, Behaviour and brain catecholamines in leadexposed neonatal rats: a reappraisal. Science, 178, 359-36 1. Grant, L.D., Howard, J.L., Alexander, S. and Krigman, M.R. (1975) Low level lead exposure: behavioral effects. Environ. Health Perspect., 10, 267. Grant, L.D., Breese, J.L., Howard, J.L., Krigman, M.R. and Mushak, P. (1976) Neurobiology of lead-intoxication in the developing rat. Fed. Proc., 35, 503. Kostial, K. and Vouk, V.B. (1957) Lead ions and synaptic transmission in the superior cervical ganglion of the cat. Brit. J. Pharrnacol., 12, 219-222. Krehbiel, D., David, G., LeRoy, L. and Bowman, R . (1978) Absence of hyperactivity in leadexposed developing rats. Environ. Health Perspect., 18, 147-157. Maker, H.S., Lehrer, G.M. and Silides. D.J. (1975) The effect of lead o n mouse brain development. Environ. Res., 1 0 , 79-91. Manalis, R.S. and Cooper, G.P. (1973) Presynaptic and postsynaptic effects of lead at the frog neuromuscular junction. Nature (Lond.), 243, 354-355. Modak, A.T., Weintraub, S.T. and Stavinoha, W.B. (1975) Effect of chronic ingestion o f lead on the central cholinergic system in rat brain regions. Toxicol. appl. Pharmacol., 3 4 , 340- 347. National Academy of Sciences (1 972) Lead, Airborn Lead in Perspecrive. Overmann, S.R. (1 977) Behavioral effects of asymptomatic lead exposure during neonatal development in rats. Toxicol. appl. Pharmacol., 41,459-472. Sauerhoff, M.W. and Michaelson, I.A. (1973) Effect of inorganic lead o n monoamines in brain of developing rat. Pharmacologist, 15, 165. Shih, T.M. and Hanin, I. (1978) Effects of chronic lead exposure on levels of acetylcholine and choline and o n acetylcholine turnover rate in rat brain areas in vivo. Psychopharmacol., in press. Shih, T.M., Khachaturina, Z.S., Barry 111, H. and Hanin, I. (1976) Cholinergic mediation of the inhibitory effect of methylphenidate o n neuronal activity in the reticular formation. Neuropharmacol., 15, 55-60. Shih, T.M., Khachaturina, Z. and Hanin, I. (1978) Involvement of both cholinergic and catecholaminergic pathways in t h e central action of methylphenidate: a study utilizing leadexposed rats. Psychopharrnacol., in press. Silbergeld, E.K. and Goldberg, A.M. (1973) A lead-induced behavioral disorder. Life Sci., 13,1275-1283.
470 Silbergeld, E.K. and Goldberg, A.M. ( 1974) Lead-induced behavioral dysfunction. An animal model of hyperactivity. Exp. Neurol., 42, 146- 157. Silbergeld, E.K., Fales, J.T. and Goldberg, A.M. (1974) The effect of inorganic lead o n the neuromuscular junction. Neuropharmocol., 13,795-801. Sobotka, T.J., Brodie, R.E. and Cook, M.P. (1975) Psychophysiologic effects of early lead exposure. Toxicol., 5, 175-191.
INTRODUCTION Our present understanding of the biologic effects of lead is largely based on the study of individuals suffering from overt lead poisoning and on the experience gained in the management of persons exposed to lead through the occupational setting. The exposure to lead in these groups is considerably greater than that in the normal population encountering lead via food sources and environmental pollution. About one of four young children who survive an attack of acute encephalopathy, as a result of lead poisoning, suffer severe and permanent neurological consequences. Byers and Lord (1 943) have delineated the nature and severity of central nervous system injury that follows lead poisoning in early childhood. The severe forms of acute lead encephalopathy, including convulsive episodes, are fortunately becoming less common; the subtle neurological deficits and mental impairment which might include sensory loss and a decrease in the I.Q. are more difficult to assess. The children appear to suffer from motor incoordination, lack of sensory perception, impaired learning, short attention spans and are generally easily distracted (National Academy of Science, 1972). These behavioral changes are difficult to distinguish from the changes seen after organic brain damage or environmental deprivation. ANIMAL MODELS OF LEAD EXPOSURE The administration of inorganic lead during critical periods of development can lead to behavioral dysfunction in experimental animals. Behavioral changes have been described in mice (Silbergel and Goldberg, 1973), rats (Saureoff and Michaelson, 1973; Shih et al., 1976; Overman, 1977) and monkeys (Allen et al., 1974). The major behavioral effects observed have been changes in spontaneous motor activity, agressiveness, arousal, patterning of behavior, and vocalization. One criticism raised against many experimental studies has been that the results on intact animals are largely based on experiments where high doses of lead, on an absolute scale have been administered. Actually, since the sensitiv-
466 ity of different animal species to lead is very different, the lead exposure necessary in experimental work also varies. It is apparent from the literature that one cannot use blood levels of lead in different species to compare the severity of exposure. Rodents with blood levels of lead exceeding 100 pg% do not present symptoms of overt intoxication, while man with the same blood level of lead is clearly intoxicated. Therefore, to suggest that exposure in any study is high or low can only be argued on the basis of species sensitivity. Various laboratories use different regiments of lead administration. Generally, rat or mouse pups are exposed to lead through the mother’s milk. While most investigators initiate lead exposure on the day pf parturition, others expose their animals on day two or three of life. In certain studies the mothers have been exposed to lead for their lifetime and the f2 generation, also exposed to lead, are the subjects of the investigation. As indicated above, data on behavioral changes produced by the exposure to lead are not consistent between laboratories. Reports from two laboratories describe either hyperactivity or overactivity in the mouse (Silbergeld and Goldberg, 1973; Maker et al., 1975). With the rat, hyperactivity has been reported from at least five different laboratories (Sauerhoff and Michaelson, 1973; Golter and Michaelson, 1975; Shih et al., 1976; Grant et al., 1975; Overmann, 1977). Some studies from the same laboratories, however, as well as from other laboratories have not revealed this effect (Sobotka et al., 1975; Modak et al., 1975; Grant et al., 1976; Krehbiel et al., 1978). To evaluate the apparent contradiction, several features of these studies should be taken into account. First, the route and level of exposure have in most cases been different. Second, the methods of measuring the behavioral changes have not been standardized. Third, the nutritional status of the animals has not been controlled as regards the intake of calories and nutritional factors such as other trace metals. Fourth, the litter size and thus the nutrition and the psychological factors have not been uniform. With this lack of uniformity it is difficult to compare directly the studies that have been reported. However, it may be noted that the attenuated or paradoxical pharmacological effects of amphetamines in lead exposed animals, originally observed in mice (Silbergeld and Goldberg, 1974) have been reported in the rat independently of the appearance of hyperactivity (Shih et al., 1976; Sobotka et al., 1975). The effects on the cholinergic system have also been consistent, independent of the species studied. In our recent studies, we have developed a model of lead exposure that does not retard growth or produce undernourishment. The protocol has worked well with rats and mice; other species have not been tested. On the day of parturition the mothers receive 5 mg/ml lead acetate in their drinking water. The mothers of control animals receive tap water. Litters are reduced to 3 pups each within 24 h of birth. Neonatal animals are thus exposed to lead through the milk of their mothers from postnatal days 1 through 21. They are weaned at 28 days and group-housed by sex. Animals exposed t o lead using this protocol show no growth retardation.
467 EFFECTS OF LEAD ON THE CHOLINERGIC SYSTEM
Peripheral system Kostial and Vouk (1957) demonstrated that lead added in vitro blocked ganglionic transmission in the superior cervical ganglion of the cat and produced a decrease in the release of acetylcholine (ACh); this effect of lead appeared to be related to the interaction of lead and calcium. Manalis and Cooper (1 973), using the frog sartorius muscle preparation, demonstrated that lead increases miniature end-plate potential frequency and decreases end-plate potential amplitude. Goldberg and van den Bercken (unpublished) confirmed these observations but observed that in the hemidiaphragm of the rat, the endplate potential amplitude was decreased but minature end-plate potential frequency was unchanged. Similar results in the rat hemidiaphragm have been reported by Bornstein and Pickett (1 977). Silbergeld et al., (1 974) demonstrated that in vitro lead can decrease the force of contraction of the diaphragm when the phrenic nerve is stimulated. Further, they demonstrated that the force of contraction was not impaired when the muscle was stimulated directly. The ability of lead to decrease the end-plate potential amplitude and the force of muscle contraction cannot be explained by a postsynaptic inhibition since lead dose not alter the postsynaptic sensitivity to ACh (Silbergeld et al., 1974). All of these results are consistent with the suggestion that lead added in vitro decreases the release of ACh.
Steady-state levels Steady-state levels of choline and ACh have been measured in mice and rats exposed to lead by Carroll et al. (1977) and Shih and Hanin (1978). These groups failed to observe any changes in either compound. On the other hand, Modak et al. ( 1975) did observe a small but significant decrease in the levels of ACh in the diencephalon of the rat.
Enzymes
No changes in the activity of choline acetyltransferase have been observed after exposure to lead but cholinesterase activity was depressed by approximately 20%in all studies (Sobotka et al., 1975; Modak et al., 1975; Carrol et al., 1977). In some of these studies the decrease reached statistical significance. Whether this is a biologically important phenomenon is difficult to determine. Release and turnover Carroll et al. (1977) studied the potassium induced release of ACh from brain tissue. The question that was being asked was whether inhibition of ACh release by lead in vitro was a general property of lead. ACh release was induced by exposing the brain minces to elevated levels of potassium (35 mM) for periods up to 1 h. As previously shown, the release of ACh was linear for at least 1 h and was calcium dependent. In control mice the release of ACh was in
468 the range of 300 nmol/g wet wt h. In Iead exposed animals the release was decreased by about 50%. These results demonstrate that chronic lead exposure inhibits the potassium induced release of ACh from brain tissue. Presumably, the impairment of ACh release is a direct effect of lead and not a secondary consequence of the lead exposure. Shih and Hanin (1978) have presented evidence that lead exposure in the rat results in a decrease in the turnover rate of ACh. They exposed rats t o lead acetate from birth. In their study there was a decrease in growth rate of the lead exposed animals. At approximately 4 5 days of age, control and lead treated rats were administered radiolabelled choline phosphate, a precursor of choline and ACh. Specific activities of ACh were not altered, but the specific activity of choline was significantly elevated in all brain areas studied as compared to age-matched controls. The in vivo ACh turnover rate in cortex, hippocampus, midbrain, and striatum was decreased by 33-45%. These findings provide evidence for an inhibitory effect of lead exposure on central cholinergic function in vivo. Pharmacology Many drugs produce unexpected pharmacological responses in lead exposed animals. Methylphenidate and amphetamine produced a paradoxical or attenuated response in lead treated animals. Although these drugs are generally thought of as aminergic stimulants they have many additional effects not related t o their catecholaminergic action. Amphetamines have been shown t o increase ACh release from brain. In attempting to see whether methylphenidate also had a cholinergic action we did the following experiment (Carroll e t al., 1977). Lead exposed animals were treated with 40 mg/kg methylphenidate and killed 2 h later. The brains from control, lead exposed, and lead exposed animals given methylphenidate were removed and the potassium induced release of ACh measured. As in previous experiments, lead exposed animals had a decreased release of ACh; this decrease was completely reversed in the methylphenidate-treated animals. These data are consistent with those of Shih e t al., (1 976 and 1978) who previously reported evidence suggesting a cholinergic effect of methylphenidate. Collectively, these data suggest a cholinergic link in the mechanisms of action of methylphenidate. SUMMARY
Low levels of inorganic lead chronically administered during critical periods of development result in behavioral alteration and neurochemical changes. Among the changes seen consistently are alterations in central and peripheral cholinergic function. Administration of several drugs that influence cholinergic metabolism influences the behavioral effects of lead. In the peripheral nervous system, lead in vitro has been shown to impair cholinergic function in several different experimental models. Lead inhibits the release of ACh from the superior cervical ganglion of the cat during stimulation of the preganglionic fibers, decreases the size of the end-plate potential in the frog and rat neuro-
469 muscular preparations, and reduces the force of muscle contraction during nerve stimulation. The pharmacological evidence obtained in studies on whole animals and direct measurements of ACh release and turnover indicate that the release of ACh in the central and peripheral nervous systems is impaired by both acute in vitro and by chronic in vivo exposure t o lead. ACKNOWLEDGEMENTS The studies reported from the author’s laboratory were supported by NIEHS grant 00454 and 00034. REFERENCES Allen, J.R., McWay, P.J. and Suomi, S.J. (1974) Pathological and behavioral changes in rhesus monkeys exposed t o lead. Environ. Health Perspect., 7,239-246. Bornstein, J.C. and Pickett, J.B. (1977) Some effects of lead ions o n transmitter release at rat neuromuscular junctions. Soc. Neurosci., 3, 370. Byers, R.K. and Lord, E.E. (1943) Late effects of lead poisoning o n mental development. Amer. J. Dis. Childh., 66, 471-494. Carroll, P.T., Silbergeld, E.K., and Goldberg, A.M. (1 977) Alterations of central cholinergic function in lead-induced hyperactivity. Biochem. Pharmacol., 26, 397-402. Colter, M. and Michaelson, I.A. (1 975) Growth, Behaviour and brain catecholamines in leadexposed neonatal rats: a reappraisal. Science, 178, 359-36 1. Grant, L.D., Howard, J.L., Alexander, S. and Krigman, M.R. (1975) Low level lead exposure: behavioral effects. Environ. Health Perspect., 10, 267. Grant, L.D., Breese, J.L., Howard, J.L., Krigman, M.R. and Mushak, P. (1976) Neurobiology of lead-intoxication in the developing rat. Fed. Proc., 35, 503. Kostial, K. and Vouk, V.B. (1957) Lead ions and synaptic transmission in the superior cervical ganglion of the cat. Brit. J. Pharrnacol., 12, 219-222. Krehbiel, D., David, G., LeRoy, L. and Bowman, R . (1978) Absence of hyperactivity in leadexposed developing rats. Environ. Health Perspect., 18, 147-157. Maker, H.S., Lehrer, G.M. and Silides. D.J. (1975) The effect of lead o n mouse brain development. Environ. Res., 1 0 , 79-91. Manalis, R.S. and Cooper, G.P. (1973) Presynaptic and postsynaptic effects of lead at the frog neuromuscular junction. Nature (Lond.), 243, 354-355. Modak, A.T., Weintraub, S.T. and Stavinoha, W.B. (1975) Effect of chronic ingestion o f lead on the central cholinergic system in rat brain regions. Toxicol. appl. Pharmacol., 3 4 , 340- 347. National Academy of Sciences (1 972) Lead, Airborn Lead in Perspecrive. Overmann, S.R. (1 977) Behavioral effects of asymptomatic lead exposure during neonatal development in rats. Toxicol. appl. Pharmacol., 41,459-472. Sauerhoff, M.W. and Michaelson, I.A. (1973) Effect of inorganic lead o n monoamines in brain of developing rat. Pharmacologist, 15, 165. Shih, T.M. and Hanin, I. (1978) Effects of chronic lead exposure on levels of acetylcholine and choline and o n acetylcholine turnover rate in rat brain areas in vivo. Psychopharmacol., in press. Shih, T.M., Khachaturina, Z.S., Barry 111, H. and Hanin, I. (1976) Cholinergic mediation of the inhibitory effect of methylphenidate o n neuronal activity in the reticular formation. Neuropharmacol., 15, 55-60. Shih, T.M., Khachaturina, Z. and Hanin, I. (1978) Involvement of both cholinergic and catecholaminergic pathways in t h e central action of methylphenidate: a study utilizing leadexposed rats. Psychopharrnacol., in press. Silbergeld, E.K. and Goldberg, A.M. (1973) A lead-induced behavioral disorder. Life Sci., 13,1275-1283.
470 Silbergeld, E.K. and Goldberg, A.M. ( 1974) Lead-induced behavioral dysfunction. An animal model of hyperactivity. Exp. Neurol., 42, 146- 157. Silbergeld, E.K., Fales, J.T. and Goldberg, A.M. (1974) The effect of inorganic lead o n the neuromuscular junction. Neuropharmocol., 13,795-801. Sobotka, T.J., Brodie, R.E. and Cook, M.P. (1975) Psychophysiologic effects of early lead exposure. Toxicol., 5, 175-191.