EXPERIMENTAL
NEUROLOGY
64,482-492
(1979)
Effects of Lead Exposure during Development on Neocortical Dendritic and Synaptic Structure TED L. PETIT Department
of Psychology,
AND JANELLE
University Received
of Toronto, October
C. LEBOUTILLIER’ West Hill.
Ontario
MlC
lA4,
Canada
17, 1978
The effect of lead (Pb) on neocortical dendritic and synaptic development was examined in rats. Newborn pups were indirectly exposed to Pb by placing 4% Pb carbonate in their mother’s diet from postnatal days 1 to 25. The mean brain weight of the Pb-treated animals was reduced 13.2%; neocortical thickness was reduced 13.9%. For analysis of dendritic development, layer V pyramidal cells from area 3 of the sensorimotor cerebral cortex were examined using the Scholl method to determine the number of dendritic branches at 20-pm intervals from the cell body. Although there were no differences in the number of processes leaving the cell body, reductions in dendritic branches were observed at distances greater than 40 pm, reaching significance at 80 and 100 pm. In addition the length of the primary apical dendrite was reduced 5.6% in Pb-treated animals. Synaptic parameters were examined in the molecular layer of the occipital cortex in ethanol phosphotungstic acid-stained tissue. There was a 22.7% reduction in the number of synapses per 15,000x field. No significant differences were observed in the following synaptic parameters measured at 300,000~: presynaptic length and thickness, postsynaptic length and thickness, and cleft width.
INTRODUCTION Low-level lead (Pb) exposure, which results in hyperactivity and learning disabilities in children, has received increasing attention in recent years (4,9,20). Of equal importance, however, is exposure to high levels of Pb, which frequently culminates in seizures and mental deficiency (1, 16, 17). Behavioral changes similar to those observed in humans were also reported in laboratory animals exposed to Pb during development (22,30, Abbreviations: PN-postnatal, EPTA-ethanol phosphotungstic acid. i This research was supported by grant A0292 from the National Research Council of Canada. The authors would like to thank Ms. Monika Idler for her expert technical assistance in electron microscopy. 482 0014-4886/79/060482-l 1$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
LEAD
AND
NEOCORTICAL
DEVELOPMENT
483
32). As such, Pb exposure during development is one of the few forms of mental deficiency that can be reproduced and studied in the laboratory. There are presently two major methods for inducing Pb exposure in developing experimental animals: direct intubation (or injection) of pups (22,23) and indirect exposure through the maternal milk supply (3,5,24). The major problem with the former method is the stress inflicted on the infant; stress itself is known to cause major changes in the development of the brain and behavior. The maternal route has the disadvantage of possible malnutrition through deficiencies in either quantity or quality of maternal milk (2). Reduction of the litter size to less than half normal could reasonably be expected to overcome the majority of the problems with the latter technique. Exposure to Pb in young experimental animals was reported to produce a number of morphologic changes in the brain. Those include reduction in brain weight (13)) hemorrhagic lesions in and reduction of the weight of the cerebellum (15), reduced thickness of cerebral cortex (12) and hippocampus (19), reduced synaptic density (12), and alterations in cortical vascularization (28). To date, however, there has not been a detailed morphological analysis of the effects of Pb at the neuronal level. The reduced thickness and synaptic density in the neocortex of Pb-treated animals could possibly indicate reductions in neural differentiation. Some forms of mental deficiency were reported to be associated with reductions or alterations in dendritic development (10). In addition, other forms of developmental intervention have been shown to alter the ultrastructure of the synaptic junction (11). Such aberrations in neural differentiation could conceivably be a major factor underlying observed intellectual deficiences. It is possible that Pb exposure during development may disrupt intellectual functioning through similar alterations or reductions in dendritic or synaptic structure. This study was undertaken to assess that possibility. MATERIALS AND METHODS Female Long-Evans hooded rats were placed with males for 1 week, after which they were moved to individual nesting cages and maintained on standard laboratory chow and tap water ad libitum . Approximately 1 week prior to delivery, ground laboratory chow was substituted for the food pellets. Animals were checked each morning and afternoon for litters, and the day on which a litter was discovered was taken as postnatal day 0 (PN 0) for that group. On PN 1 all litters were reduced to six male pups, cross-fostered with other litters within 424 h of age, randomly assigned to either the Pb or
484
PETIT
AND
LEBOUTILLIER
control group, and started on the appropriate diet. Diets consisted of ad access to either ground standard laboratory chow (controls) or chow containing 4% Pb carbonate (Pb group). Animals were fed their respective diets and tap water ad libitum until PN 25, when all animals were returned to a diet of standard laboratory chow. Dendritic Analysis. On PN 28, five control and eight Pb-treated animals were weighed, anesthetized with Nembutal, and perfused intracardially with physiological saline. The brains were removed, weighed, and stained with a tungstate modification of the Golgi-Cox method, embedded in celloidin, and sectioned at 90 pm. All slides examined for evidence of deficient dendritic development were first coded to prevent experimenter bias. Representative layer V (giant) pyramidal cells were chosen from area 3 of the sensorimotor cerebral isocortex lying at the most rostra1 level of the hippocampus. Only completely impregnated cells with apical dendrites extending to the molecular layer were included. Cells were drawn at 400x with the aid of a camera lucida. Dendritic growth was analyzed by the Scholl (29) method, i.e., by counting the number of intersections of dendrites with each of a series of concentric circles at 20-pm intervals from the cell body. The length of the apical dendrite was measured from the point where it leaves the cell body to its point of greatest length in layer I. Fifty control cells and 80 Pb-exposed cells were counted, i.e., 10 cells per animal. In addition, the total depth of the dorsal cerebral cortex, from the top of the molecular layer to the lateral ventricles, was measured in all animals. Synaptic Analysis. On PN 28, five Pb-exposed and four control animals were anesthetized with Nembutal, and their skulls were rapidly removed. Neocortical tissue from the right dorsal occipital cortex was excised and fixed by immersion in a 2% glutaraldehyde solution in Millonig’s buffer for 2 h, with at least three changes of solution, followed by 1% osmium tetroxide in Millonig’s buffer for 1 h at 4°C. After dehydration in a graded series of ethanol, the tissue was stained 1 h in a 1% solution of phosphotungstic acid in absolute alcohol (EPTA) and embedded in Epon-Araldite mixture, and sections were cut on a Reichert OMU 3 ultramicrotome. Subsequently, all tissue was viewed with a Siemens Elmiskop 102 electron microscope. Synaptic density and synaptic structure were examined in photographs systematically taken throughout the molecular layer to obtain a random sample of the synaptic population. Synaptic density was determined by counting the number of synapses per 15,000~ field; 15 photomicrographs were examined for each animal. Presynaptic and postsynaptic thickness and length, as well as synaptic cleft width, were measured in approximately 50 synapses from each libitum
LEADANDNEOCORTICALDEVELOPMENT
485
Frc. 1. Homologous sections through the sensorimotor cortex of 28-day-old rats treated with Pb (A and C) and a control rat (B). A and C represent the maximum and minimum reductions (respectively) in neocortical development caused by Pb. Magnification: A, x80; B, x80; C, x80.
FIG. 486
B.
FIG. 487
C.
488
PETIT
AND
LEBOUTILLIER
animal. The synapses were photographed at 100,000~ and measured at 300,000 x . The presynaptic and postsynaptic thickness measurements refer to the maximum dimensions of these densities; the maximum presynaptic thickness measures the height of dense projections in those junctions where they are present. Osmic acid treatment was not used. For greater detail on the way measurements were carried out see (11). RESULTS As litters were reduced to six infants, all animals appeared to be healthy and well nourished for the first 20 days. On PN 22 to 25 some Pb-treated animals developed symptoms of paraplegia and urinary incontinence. When withdrawn from the Pb-containing diet on PN 25 those symptoms quickly disappeared. The mean brain weight of the Pb-treated animals used for dendritic analysis was 1.326 g compared to 1.528 g for the controls, a 13.2% reduction. Neocortical thickness was also reduced 13.9% in the Pb-treated brains (see Fig. 1). Dendritic Development. The length of the primary apical dendrite of layer V pyramidal cells was reduced 5.6% in Pb-treated animals; this reduction was not statistically significant. Analysis of variance of the data gathered by the Scholl method revealed a reduction in dendritic development in Pb-treated animals. As seen in Fig. 2, Pb did not cause a reduction in the number of dendritic processes leaving the cell body, i.e., in
-.......CONTROL LEAD
20
40
60
80
100
120
140
160
180
DISTANCE FROM CELL BODY (MICRONS)
FIG. 2. Number of intersections between dendritic branches from the cell body of layer V pyramidal cells from sensorimotor control animals.
and rings at 20-Mm intervals cortex of Pb-exposed and
LEAD AND NEOCORTICAL TABLE
489
DEVELOPMENT 1
Comparison of Synaptic Parameters from Control and Pb-Treated Rats in Ethanol-Phosphotungstic Acid-Stained Tissue
No.
N
Presynaptic length (nm)
Presynaptic thickness (nm)
Postsynaptic length (nm)
Postsynaptic thickness (rim)
Cleft width
1 2 3 4 5 Pb mean
49 51 54 38 52 244
183.9” 178.5 183.2 190.2 168.0 180.2* 2 3.9
44.5 53.3 45.1 46.4 51.3 48.2 + 0.8
188.8 184.7 191.8 196.9 174.5 186.8 2 3.9
35.1 41.1 37.0 36.3 40.9 38.2 ” 0.7
20.5 22.1 21.3 21.5 20.9 21.2 k 0.1
43 48 44 50
193.9 174.4 161.7 160.7
45.6 50.1 45.9 44.2
201.3 183.7 168.8 167.9
38.5 37.2 35.5 35.1
21.9 20.7 20.8 21.0
185
172.2 + 4.7
46.4 t 0.6
180.0 f 4.7
36.5 f 0.8
21.1 f 0.1
(nm)
Pb
Control 1 2 3 4 Control mean
a Mean. Q Means 2 standard error.
the first 20 pm. A reduction of dendritic branches was observed at distances greater than 40 pm from the cell body, with significant reductions at80and lOO~m(80~m,P < 0.001; lOOpm,P = 0.019). Thechangesinthe brains of some Pb-treated animals were so striking as to be noticeable at first glance (Fig. lA), whereas in others no differences were apparent on visual inspection alone (Fig. 1C). Synaptic Development. Pb caused a significant reduction in neocortical synaptic density. There was a mean of 4.72 synapses per 15,000x field in the neocortex of control animals compared to only 3.65 synapses per field in the Pb-treated animals, a reduction of 22.7% (Z’ < 0.05, two-tailed t-test). Analysis of synaptic dimensions revealed no significant differences between the two groups on any of the measures used i.e., presynaptic length or width, postsynaptic length or width, or cleft width (Table 1). Although we (26) had previously suspected a possible thinning of the presynaptic terminal in some Pb-treated animals, the results of this study failed to confirm that possibility. DISCUSSION Pb exposure during development resulted in a 13.2% reduction in whole brain weight, and a 13.9% reduction in neocortical thickness. Associated
490
PETIT
AND
LEBOUTILLIER
with this was a reduction in development of the dendritic tree of layer V giant pyramidal cells, and a reduced number of neocortical synapses. There were, however, no apparent changes in synaptic dimensions as measured in EPTA-stained material. Using the same exposure paradigm in our laboratory we previously reported mean blood Pb concentrations of 1297 &lo0 ml in the Pb-treated animals and 2 pg/lOO ml in controls at PN 25 (25). There are several factors which suggest that these Pb-induced changes were not due to malnutrition. First, we previously showed that maternal weight was reduced only during the first 4 days of exposure to 4% Pb carbonate, after which time weights quickly returned to normal (25). Second, with the reduction of litters to six pups, prior to PN 20 the Pb-exposed pups looked healthy and well nourished. We previously showed, however, that Pb-exposed animals did not gain weight at the same rate as controls (25). The difference in brain and body weights of the Pb-exposed and control animals is more likely due to increased weight gain in control animals as a result of litter reduction to less than one-half normal. Another factor that argues against malnutrition is that Jones and Dyson (11) reported reductions in synaptic parameters in malnourished rats; such reductions were not observed in our Pb-treated animals. The reduced dendritic development in Pb-treated animals may be due to several factors. Lead was reported to disrupt the normal morphology of neurotubules (21). Neurotubular disruption by other agents was previously shown to result in reduced dendritic development (27). Another possibility is that Pb reduces neuronal firing rates by competing with calcium (Ca) and magnesium (Mg) which are essential for normal excitability and action potential formation. Research from several approaches has indicated that normal stimulation rates are essential for the normal development and maintenance of dendritic and synaptic structure (7, 14, 23). One interesting aspect of Pb toxicology is its developmentally dependent effects. Lead concentrations which can be easily tolerated by the adult (either rat or human) produce a severe encephalopathy in the developing animal. If untreated, lifelong impairment in intellectual functioning (i.e., mental retardation) frequently follows this developmental encephalopathy, but is not seen after Pb exposure during adulthood (6). This implies that the mental deficiency seen in children might be attributed to a long-term alteration in the morphological development of the brain combined with the direct effects of Pb on neural physiology. The changes in morphological development so far reported include transient hemorrhagic lesions of the cerebellum; reductions in the cerebral cortex, hippocampus, and cerebellum; hypomyelination and reduced synaptic density; and reduced dendritic differentiation. It seems likely that reduced dendritic differentiation and concomitant reductions in synaptic contacts would account for a
LEAD AND NEOCORTICAL
DEVELOPMENT
491
large portion of the reduced neocortical thickness, because it was shown that neuronal and glial cell number is not reduced in Pb-exposed animals (12). Similar to earlier, less detailed, synaptic analysis (12), we did not observe any differences in synaptic parameters in EPTA-stained material. The question arises as to how a reduced dendritic field and number of synapses might alter neural function to perhaps yield deficient intellectual functioning. First, those reductions would result in neurons with a deficient capacity to receive and consequently integrate information. In addition, from our recent knowledge of dendritic spikes, and the role of dendritic length and branching in the propagation of spikes, those neurons might be expected to have an altered physiology (18). Others have shown that Pb competes with Ca and Mg at the neuronal level (8). Although there have been no electrophysiological measurements of the effect of this, there have been reports of reduced peripheral acetylcholine release and central dopamine release (3 1). These changes in the nerve physiology together with the reductions in neural morphology reported here may be, in part, major factors responsible for reduced intellectual capacity of children suffering from increased Pb burdens. REFERENCES 1. BEATTIE, A. D., M. R. MOORE, A. GOLDBERG, M. J. W. FINLAYSON, J. F. GRAHAM, E. M. MACKIE, J. C. MAIN, R. M. MURDOCH,AND G. T. STEWARD. 1975. Role ofchronic low-level lead exposure in the aetiology of mental retardation. Lancer 2: 589-592. 2. BORNSCHEIN, R. L., I. A. MICHAELSON, D. A. Fox, AND R. LOCH. 1977. Evaluation of animal models used to study effects of lead on neurochemistry and behavior. Pages 441-456 in S. D. LEE, Ed., Biochemical Effects of Environmental Pollutanis, Ann Arbor Science Publishers, Ann Arbor, Mich. 3. BROWN, D. R. 1975. Neonatal lead exposure in the rat: decreased learning as a function of age and blood lead concentrations. Toxicol. Appl. Pharmacol. 32: 628-637. 4. DAVID, 0. J. 1974. Association between lower level lead concentrations and hyperactivity in children. Environ. Health Perspect. 7: 17-25. 5. DRISCOLL, J. W., AND S. E. STEGNER. 1976. Behavioral effects of chronic lead ingestion on laboratory rats. Pharmacol. Biochem. Behav. 4: 411-417. 6. FELDMAN, R. G., M. K. HAYES, R. YOUNES, AND F. D. ALDRICH. 1977. Lead neuropathy in adults and children. Arch. Neurol. 34: 481-488. 7. GLOBUS, A., AND A. B. SCHEIBEL. 1967. The effect of visual deprivation on cortical neurons: a Golgi study. Exp. Neurol. 19: 331-345. 8. GOLDSTEIN, G. W. 1977. Lead encephalopathy: the significance of lead inhibition of calcium uptake of brain mitochondria. Brain Res. 136: 185- 188. 9. GREENGARD, J., B. ADAMS, AND E. BERMAN. 1965. Acute lead encephalopathy in young children. J. Pediat. 66: 707-711. 10. HUTTENLOCHER, P. R. 1977. Dendritic development in neocortex of children with mental defect and infantile spasm. Neurology (Minneapolis) 24: 203-210. 1I. JONES, D. J., AND S. E. DYSON. 1976. Synapticjunctions inundernourished rat brain-an ultrastructural investigation. Exp Neurol. 51: 529-535. 12. KRICMAN. M. R., M. J. DRUSE, T. D. TRAYLOR, M. H. W.ISON, L. R. NEWELL, AND
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14. 15.
16. 17.
18.
19.
20. 21. 22. 23.
24.
25. 26. 27. 28. 29. 30. 31. 32. 33.
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E. L. HOGAN. 1974. Lead encephalopathy in the developing rat: effect of cortical ontogenesis. J. Neuropathol. Exp. Neurol. 5: 671-685. KRIGMAN, M. R., M. J. DRUSE, T. D. TRAYLOR, M. H. WILSON, L. R. NEWELL, AND E. L. HOGAN. 1974. Lead encephalopathy in the developing rat: effect on myelination. J. Neuropathol. Exp. Neural. 33: 58-73. KUENZLE, C. C., AND A. KNUSEL. 1974. Mass training in a superemiched environment. Physiol. Behav. 13: 205-210. LAMPERT, P., F. GARRO, AND A. PENTSCHEW. 1967. Lead encephalopathy in suckling rats. Pages 207-222 in I. KLATZO AND F. SEITELBERGER, Eds., Symposium on Brain Edema. Springer, Vienna. LIN-Fu, J. S. 1970. Lead Poisoning in Children. U.S. Public Health Service Publ. No. 2108. LIN-Fu, J. S. 1975. Undue lead adsorption and lead poisoning in children-an overview. In International Conference on Heavy Metals in the Environment. University of Toronto Press, Toronto. LLINAS, R. 1975. Electroresponsive properties of dendrites in central neurons. Pages l-13 in G. W. KREUTZBERG, Ed.,Advances in Neurology, Vol. 12. Raven Press, New York. LOUIS-FERDINAND, R. T., D. R. BROWN, S. F. FIDDLER, W. C. DAUGHTREY, AND A. W. KLEIN. 1978. Morphometric and enzymatic effects of neonatal lead exposure in the rat brain. Toxicol. Appl. Pharmacol. 43: 351-360. NEEDLEMAN, H. L. 1973. Lead poisoning in children: neurologic implications of widespread subclinical intoxication. Semin. Psychiat. 5: 47-54. NIKLOWITZ, W. J. 1975. Neurofibrillary changes after acute experimental lead poisoning. Neurology (Minneapolis) 25: 927-934. OVERMANN, S. R. 1977. Behavioral effects of asymptomatic lead exposure during neonatal development in rats. Toxicol. Appl. Pharmacol. 41: 459-471. PARNAVELAS, J. G., G. LYNCH, N. BRECHA, C. W. COTMAN, AND A. GLOBUS. 1974. Spine loss and regrowth in hippocampus following deaferentiation. Nature (London) 248: 70-71. PENTSCHEW, A., AND F. GARRO. 1966. Lead encephalo-myelopathy of the suckling rat and its implications on the porphyrinopathic nervous diseases. Acta. Neuropathol. 6: 266-278. PETIT, T. L., AND D. P. ALFANO. 1979. Differential rearing following developmental lead exposure: Effects on brain and behavior. Pharmacol. Biochem. Behav., (in press). PETIT, T. L., AND J. C. LEBOUTILLIER. 1978. Development of dendritic and synaptic structure following early lead exposure. Sot. Neurosci. Abstr. 4: 123. PETIT, T. L., AND R. L. ISAACSON, 1977. Deficient brain development following Colcemid treatment in postnatal rats. Brain Res. 132: 380-385. PRESS, M. F. 1977. Lead encephalopathy in neonatal Long-Evans rats: Morphologic studies. Neuropathology 7: 169- 193. SCHOLL, D. A. 1956. The Organization of the Cerebral cortex. Methuen, London. SILBERGELD, E K., AND A. M. GOLDBERG. 1975. Pharmacological and neurochemical investigations of lead-induced hyperactivity. Neuropharmacology 14: 431-444. SILBERGELD, E. K., AND H. S. ADLER. 1978. Subcellular mechanisms of lead neurotoxicity. Brain Res. 148: 451-467. SOBOTKA, T. J., AND M. P. COOK. 1974. Postnatal lead acetate exposure in rats: possible relationship to minimal brain dysfunction. Am. J. Menr. Dejic. 79: 5-9. SOBOTKA, T. J., R. E. BRODIE, AND M. P. COOK. 1975. Psychophysiologic effectsofearly lead exposure. Toxicology 5: 175- 191.
NEUROLOGY
64,482-492
(1979)
Effects of Lead Exposure during Development on Neocortical Dendritic and Synaptic Structure TED L. PETIT Department
of Psychology,
AND JANELLE
University Received
of Toronto, October
C. LEBOUTILLIER’ West Hill.
Ontario
MlC
lA4,
Canada
17, 1978
The effect of lead (Pb) on neocortical dendritic and synaptic development was examined in rats. Newborn pups were indirectly exposed to Pb by placing 4% Pb carbonate in their mother’s diet from postnatal days 1 to 25. The mean brain weight of the Pb-treated animals was reduced 13.2%; neocortical thickness was reduced 13.9%. For analysis of dendritic development, layer V pyramidal cells from area 3 of the sensorimotor cerebral cortex were examined using the Scholl method to determine the number of dendritic branches at 20-pm intervals from the cell body. Although there were no differences in the number of processes leaving the cell body, reductions in dendritic branches were observed at distances greater than 40 pm, reaching significance at 80 and 100 pm. In addition the length of the primary apical dendrite was reduced 5.6% in Pb-treated animals. Synaptic parameters were examined in the molecular layer of the occipital cortex in ethanol phosphotungstic acid-stained tissue. There was a 22.7% reduction in the number of synapses per 15,000x field. No significant differences were observed in the following synaptic parameters measured at 300,000~: presynaptic length and thickness, postsynaptic length and thickness, and cleft width.
INTRODUCTION Low-level lead (Pb) exposure, which results in hyperactivity and learning disabilities in children, has received increasing attention in recent years (4,9,20). Of equal importance, however, is exposure to high levels of Pb, which frequently culminates in seizures and mental deficiency (1, 16, 17). Behavioral changes similar to those observed in humans were also reported in laboratory animals exposed to Pb during development (22,30, Abbreviations: PN-postnatal, EPTA-ethanol phosphotungstic acid. i This research was supported by grant A0292 from the National Research Council of Canada. The authors would like to thank Ms. Monika Idler for her expert technical assistance in electron microscopy. 482 0014-4886/79/060482-l 1$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
LEAD
AND
NEOCORTICAL
DEVELOPMENT
483
32). As such, Pb exposure during development is one of the few forms of mental deficiency that can be reproduced and studied in the laboratory. There are presently two major methods for inducing Pb exposure in developing experimental animals: direct intubation (or injection) of pups (22,23) and indirect exposure through the maternal milk supply (3,5,24). The major problem with the former method is the stress inflicted on the infant; stress itself is known to cause major changes in the development of the brain and behavior. The maternal route has the disadvantage of possible malnutrition through deficiencies in either quantity or quality of maternal milk (2). Reduction of the litter size to less than half normal could reasonably be expected to overcome the majority of the problems with the latter technique. Exposure to Pb in young experimental animals was reported to produce a number of morphologic changes in the brain. Those include reduction in brain weight (13)) hemorrhagic lesions in and reduction of the weight of the cerebellum (15), reduced thickness of cerebral cortex (12) and hippocampus (19), reduced synaptic density (12), and alterations in cortical vascularization (28). To date, however, there has not been a detailed morphological analysis of the effects of Pb at the neuronal level. The reduced thickness and synaptic density in the neocortex of Pb-treated animals could possibly indicate reductions in neural differentiation. Some forms of mental deficiency were reported to be associated with reductions or alterations in dendritic development (10). In addition, other forms of developmental intervention have been shown to alter the ultrastructure of the synaptic junction (11). Such aberrations in neural differentiation could conceivably be a major factor underlying observed intellectual deficiences. It is possible that Pb exposure during development may disrupt intellectual functioning through similar alterations or reductions in dendritic or synaptic structure. This study was undertaken to assess that possibility. MATERIALS AND METHODS Female Long-Evans hooded rats were placed with males for 1 week, after which they were moved to individual nesting cages and maintained on standard laboratory chow and tap water ad libitum . Approximately 1 week prior to delivery, ground laboratory chow was substituted for the food pellets. Animals were checked each morning and afternoon for litters, and the day on which a litter was discovered was taken as postnatal day 0 (PN 0) for that group. On PN 1 all litters were reduced to six male pups, cross-fostered with other litters within 424 h of age, randomly assigned to either the Pb or
484
PETIT
AND
LEBOUTILLIER
control group, and started on the appropriate diet. Diets consisted of ad access to either ground standard laboratory chow (controls) or chow containing 4% Pb carbonate (Pb group). Animals were fed their respective diets and tap water ad libitum until PN 25, when all animals were returned to a diet of standard laboratory chow. Dendritic Analysis. On PN 28, five control and eight Pb-treated animals were weighed, anesthetized with Nembutal, and perfused intracardially with physiological saline. The brains were removed, weighed, and stained with a tungstate modification of the Golgi-Cox method, embedded in celloidin, and sectioned at 90 pm. All slides examined for evidence of deficient dendritic development were first coded to prevent experimenter bias. Representative layer V (giant) pyramidal cells were chosen from area 3 of the sensorimotor cerebral isocortex lying at the most rostra1 level of the hippocampus. Only completely impregnated cells with apical dendrites extending to the molecular layer were included. Cells were drawn at 400x with the aid of a camera lucida. Dendritic growth was analyzed by the Scholl (29) method, i.e., by counting the number of intersections of dendrites with each of a series of concentric circles at 20-pm intervals from the cell body. The length of the apical dendrite was measured from the point where it leaves the cell body to its point of greatest length in layer I. Fifty control cells and 80 Pb-exposed cells were counted, i.e., 10 cells per animal. In addition, the total depth of the dorsal cerebral cortex, from the top of the molecular layer to the lateral ventricles, was measured in all animals. Synaptic Analysis. On PN 28, five Pb-exposed and four control animals were anesthetized with Nembutal, and their skulls were rapidly removed. Neocortical tissue from the right dorsal occipital cortex was excised and fixed by immersion in a 2% glutaraldehyde solution in Millonig’s buffer for 2 h, with at least three changes of solution, followed by 1% osmium tetroxide in Millonig’s buffer for 1 h at 4°C. After dehydration in a graded series of ethanol, the tissue was stained 1 h in a 1% solution of phosphotungstic acid in absolute alcohol (EPTA) and embedded in Epon-Araldite mixture, and sections were cut on a Reichert OMU 3 ultramicrotome. Subsequently, all tissue was viewed with a Siemens Elmiskop 102 electron microscope. Synaptic density and synaptic structure were examined in photographs systematically taken throughout the molecular layer to obtain a random sample of the synaptic population. Synaptic density was determined by counting the number of synapses per 15,000~ field; 15 photomicrographs were examined for each animal. Presynaptic and postsynaptic thickness and length, as well as synaptic cleft width, were measured in approximately 50 synapses from each libitum
LEADANDNEOCORTICALDEVELOPMENT
485
Frc. 1. Homologous sections through the sensorimotor cortex of 28-day-old rats treated with Pb (A and C) and a control rat (B). A and C represent the maximum and minimum reductions (respectively) in neocortical development caused by Pb. Magnification: A, x80; B, x80; C, x80.
FIG. 486
B.
FIG. 487
C.
488
PETIT
AND
LEBOUTILLIER
animal. The synapses were photographed at 100,000~ and measured at 300,000 x . The presynaptic and postsynaptic thickness measurements refer to the maximum dimensions of these densities; the maximum presynaptic thickness measures the height of dense projections in those junctions where they are present. Osmic acid treatment was not used. For greater detail on the way measurements were carried out see (11). RESULTS As litters were reduced to six infants, all animals appeared to be healthy and well nourished for the first 20 days. On PN 22 to 25 some Pb-treated animals developed symptoms of paraplegia and urinary incontinence. When withdrawn from the Pb-containing diet on PN 25 those symptoms quickly disappeared. The mean brain weight of the Pb-treated animals used for dendritic analysis was 1.326 g compared to 1.528 g for the controls, a 13.2% reduction. Neocortical thickness was also reduced 13.9% in the Pb-treated brains (see Fig. 1). Dendritic Development. The length of the primary apical dendrite of layer V pyramidal cells was reduced 5.6% in Pb-treated animals; this reduction was not statistically significant. Analysis of variance of the data gathered by the Scholl method revealed a reduction in dendritic development in Pb-treated animals. As seen in Fig. 2, Pb did not cause a reduction in the number of dendritic processes leaving the cell body, i.e., in
-.......CONTROL LEAD
20
40
60
80
100
120
140
160
180
DISTANCE FROM CELL BODY (MICRONS)
FIG. 2. Number of intersections between dendritic branches from the cell body of layer V pyramidal cells from sensorimotor control animals.
and rings at 20-Mm intervals cortex of Pb-exposed and
LEAD AND NEOCORTICAL TABLE
489
DEVELOPMENT 1
Comparison of Synaptic Parameters from Control and Pb-Treated Rats in Ethanol-Phosphotungstic Acid-Stained Tissue
No.
N
Presynaptic length (nm)
Presynaptic thickness (nm)
Postsynaptic length (nm)
Postsynaptic thickness (rim)
Cleft width
1 2 3 4 5 Pb mean
49 51 54 38 52 244
183.9” 178.5 183.2 190.2 168.0 180.2* 2 3.9
44.5 53.3 45.1 46.4 51.3 48.2 + 0.8
188.8 184.7 191.8 196.9 174.5 186.8 2 3.9
35.1 41.1 37.0 36.3 40.9 38.2 ” 0.7
20.5 22.1 21.3 21.5 20.9 21.2 k 0.1
43 48 44 50
193.9 174.4 161.7 160.7
45.6 50.1 45.9 44.2
201.3 183.7 168.8 167.9
38.5 37.2 35.5 35.1
21.9 20.7 20.8 21.0
185
172.2 + 4.7
46.4 t 0.6
180.0 f 4.7
36.5 f 0.8
21.1 f 0.1
(nm)
Pb
Control 1 2 3 4 Control mean
a Mean. Q Means 2 standard error.
the first 20 pm. A reduction of dendritic branches was observed at distances greater than 40 pm from the cell body, with significant reductions at80and lOO~m(80~m,P < 0.001; lOOpm,P = 0.019). Thechangesinthe brains of some Pb-treated animals were so striking as to be noticeable at first glance (Fig. lA), whereas in others no differences were apparent on visual inspection alone (Fig. 1C). Synaptic Development. Pb caused a significant reduction in neocortical synaptic density. There was a mean of 4.72 synapses per 15,000x field in the neocortex of control animals compared to only 3.65 synapses per field in the Pb-treated animals, a reduction of 22.7% (Z’ < 0.05, two-tailed t-test). Analysis of synaptic dimensions revealed no significant differences between the two groups on any of the measures used i.e., presynaptic length or width, postsynaptic length or width, or cleft width (Table 1). Although we (26) had previously suspected a possible thinning of the presynaptic terminal in some Pb-treated animals, the results of this study failed to confirm that possibility. DISCUSSION Pb exposure during development resulted in a 13.2% reduction in whole brain weight, and a 13.9% reduction in neocortical thickness. Associated
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with this was a reduction in development of the dendritic tree of layer V giant pyramidal cells, and a reduced number of neocortical synapses. There were, however, no apparent changes in synaptic dimensions as measured in EPTA-stained material. Using the same exposure paradigm in our laboratory we previously reported mean blood Pb concentrations of 1297 &lo0 ml in the Pb-treated animals and 2 pg/lOO ml in controls at PN 25 (25). There are several factors which suggest that these Pb-induced changes were not due to malnutrition. First, we previously showed that maternal weight was reduced only during the first 4 days of exposure to 4% Pb carbonate, after which time weights quickly returned to normal (25). Second, with the reduction of litters to six pups, prior to PN 20 the Pb-exposed pups looked healthy and well nourished. We previously showed, however, that Pb-exposed animals did not gain weight at the same rate as controls (25). The difference in brain and body weights of the Pb-exposed and control animals is more likely due to increased weight gain in control animals as a result of litter reduction to less than one-half normal. Another factor that argues against malnutrition is that Jones and Dyson (11) reported reductions in synaptic parameters in malnourished rats; such reductions were not observed in our Pb-treated animals. The reduced dendritic development in Pb-treated animals may be due to several factors. Lead was reported to disrupt the normal morphology of neurotubules (21). Neurotubular disruption by other agents was previously shown to result in reduced dendritic development (27). Another possibility is that Pb reduces neuronal firing rates by competing with calcium (Ca) and magnesium (Mg) which are essential for normal excitability and action potential formation. Research from several approaches has indicated that normal stimulation rates are essential for the normal development and maintenance of dendritic and synaptic structure (7, 14, 23). One interesting aspect of Pb toxicology is its developmentally dependent effects. Lead concentrations which can be easily tolerated by the adult (either rat or human) produce a severe encephalopathy in the developing animal. If untreated, lifelong impairment in intellectual functioning (i.e., mental retardation) frequently follows this developmental encephalopathy, but is not seen after Pb exposure during adulthood (6). This implies that the mental deficiency seen in children might be attributed to a long-term alteration in the morphological development of the brain combined with the direct effects of Pb on neural physiology. The changes in morphological development so far reported include transient hemorrhagic lesions of the cerebellum; reductions in the cerebral cortex, hippocampus, and cerebellum; hypomyelination and reduced synaptic density; and reduced dendritic differentiation. It seems likely that reduced dendritic differentiation and concomitant reductions in synaptic contacts would account for a
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large portion of the reduced neocortical thickness, because it was shown that neuronal and glial cell number is not reduced in Pb-exposed animals (12). Similar to earlier, less detailed, synaptic analysis (12), we did not observe any differences in synaptic parameters in EPTA-stained material. The question arises as to how a reduced dendritic field and number of synapses might alter neural function to perhaps yield deficient intellectual functioning. First, those reductions would result in neurons with a deficient capacity to receive and consequently integrate information. In addition, from our recent knowledge of dendritic spikes, and the role of dendritic length and branching in the propagation of spikes, those neurons might be expected to have an altered physiology (18). Others have shown that Pb competes with Ca and Mg at the neuronal level (8). Although there have been no electrophysiological measurements of the effect of this, there have been reports of reduced peripheral acetylcholine release and central dopamine release (3 1). These changes in the nerve physiology together with the reductions in neural morphology reported here may be, in part, major factors responsible for reduced intellectual capacity of children suffering from increased Pb burdens. REFERENCES 1. BEATTIE, A. D., M. R. MOORE, A. GOLDBERG, M. J. W. FINLAYSON, J. F. GRAHAM, E. M. MACKIE, J. C. MAIN, R. M. MURDOCH,AND G. T. STEWARD. 1975. Role ofchronic low-level lead exposure in the aetiology of mental retardation. Lancer 2: 589-592. 2. BORNSCHEIN, R. L., I. A. MICHAELSON, D. A. Fox, AND R. LOCH. 1977. Evaluation of animal models used to study effects of lead on neurochemistry and behavior. Pages 441-456 in S. D. LEE, Ed., Biochemical Effects of Environmental Pollutanis, Ann Arbor Science Publishers, Ann Arbor, Mich. 3. BROWN, D. R. 1975. Neonatal lead exposure in the rat: decreased learning as a function of age and blood lead concentrations. Toxicol. Appl. Pharmacol. 32: 628-637. 4. DAVID, 0. J. 1974. Association between lower level lead concentrations and hyperactivity in children. Environ. Health Perspect. 7: 17-25. 5. DRISCOLL, J. W., AND S. E. STEGNER. 1976. Behavioral effects of chronic lead ingestion on laboratory rats. Pharmacol. Biochem. Behav. 4: 411-417. 6. FELDMAN, R. G., M. K. HAYES, R. YOUNES, AND F. D. ALDRICH. 1977. Lead neuropathy in adults and children. Arch. Neurol. 34: 481-488. 7. GLOBUS, A., AND A. B. SCHEIBEL. 1967. The effect of visual deprivation on cortical neurons: a Golgi study. Exp. Neurol. 19: 331-345. 8. GOLDSTEIN, G. W. 1977. Lead encephalopathy: the significance of lead inhibition of calcium uptake of brain mitochondria. Brain Res. 136: 185- 188. 9. GREENGARD, J., B. ADAMS, AND E. BERMAN. 1965. Acute lead encephalopathy in young children. J. Pediat. 66: 707-711. 10. HUTTENLOCHER, P. R. 1977. Dendritic development in neocortex of children with mental defect and infantile spasm. Neurology (Minneapolis) 24: 203-210. 1I. JONES, D. J., AND S. E. DYSON. 1976. Synapticjunctions inundernourished rat brain-an ultrastructural investigation. Exp Neurol. 51: 529-535. 12. KRICMAN. M. R., M. J. DRUSE, T. D. TRAYLOR, M. H. W.ISON, L. R. NEWELL, AND
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