Morphometric effects of postnatal lead exposure on hippocampal development of the 15-day-old rat

Developmental Brain Research, 3 (1982) 595-612

595

Elsevier Biomedical Press

MORPHOMETRIC EFFECTS OF POSTNATAL LEAD EXPOSURE ON HIPPOCAMPAL DEVELOPMENT OF THE 15-DAY-OLD RAT

J E R R O L Y N N B. CAMPBELL, DOROTHY E. WOOLLEY*, VIJAYA K. VIJAYAN and STEPHEN R. O V E R M A N N

Departments of Animal Physiology and Human Anatomy, University of California, Davis, CA 95616 (U.S.A.) (Accepted August 21st, 1981)

Key words: lead toxicity - - hippocampus - - neuroanatomy - - neural development - - mossy fibers - neuropathology

SUMMARY

Neurotoxic sequelae of developmental lead exposure suggest that the hippocampus may be affected. Therefore, rats received low-level lead exposure via the milk of dams drinking 0.2 ~o lead acetate beginning at parturition, and mid-dorsal sections of the hippocampus and dentate gyrus (DG) from 15-day-old pups were examined by light and electron microscopy. Lead exposure did not reduce body weight nor produce obviously abnormal vascularity or signs of cytotoxicity in the hippocampal formation, and total numbers per section of dentate granule cells or CA3 pyramidal cells were not reduced. On the other hand, lead exposure reduced neuropil development as evidenced both by reduced areas of the dentate hilus and dentate infrapyramidal stratum moleculare and by increased number of hilar CA3 pyramidal cells per unit area. Also, lead exposure reduced numbers of several types of synaptic profiles per unit area in the suprapyramidal mossy fiber zone. Complex invaginated (CI) profiles, assumed to be mature mossy fiber boutons, were characterized by multiple membrane densities and deep invaginations around dendritic spines of pyramidal cells. Complex noninvaginated (CN) boutons exhibited bag-like profiles with multiple membrane densities. Smaller, less numerous, simple (S) profiles contacted either dendritic trunks (ST) or spines (SS). Lead exposure reduced the numerical density of CN, CI and SS profiles in the deep (close to stratum pyramidale) part of the proximal (close to DG) region of the suprapyramidal mossy fiber zone, but did not alter the numerical density of any of the profiles in the superficial (distal to stratum pyramidale) parts of either proximal or distal (close to CA1) regions. Average size of CN profiles in the distal region was * To whom reprint requests should be addressed. 0165-3806/0000-0000/$02.75 © Elsevier Biomedical Press

596 increased by lead exposure. The pattern of effects suggests that low-level lead exposure during development preferentially affects later developing structures within the hippocampal formation, rather than affecting mature structures.

INTRODUCTION Environmental lead exposure presents a different set of hazards for infants than it dces for adults. Young animals absorb more ingested lead from the gut and excrete it more slowly than do adults ~1,33-35,43,4s,49,64. Also, lead is deposited in the brain to a greater extent in the neonate than in the adult z4,4s,ag. Sequelae of excess childhood lead exposure, including mental retardation and recurrent seizures54,~9, reflect the accumulation of lead in the developing brain and suggest involvement of the hippocampal formation in childhood lead encephalopathy5°,51. Exogenous agents are likely to have their greatest effects upon tissues which either selectively concentrate the agent or are undergoing rapid growth and differentiation at the time of exposure 73. Both factors may contribute to neonatal lead encephalopathy. The hippocampus, amygdala and cerebellum contain higher concentrations of lead than do other parts of the brain in rats 15,16,2°,46 and in man 2s,54. Furthermore, the hippocampal formation, cerebellum and neoeortex are still in periods of rapid growth and differentiation during early postnatal life in the rat3,7,a,lv,z2,a2,6s and in man 1~,17. The immaturity of these tissues at birth suggests that postnatal lead exposure could interfere with the morphologically observable processes of mitosis, cellular migration, differentiation of dendritic and axonal processes, synaptogenesis and myelin production in the neonate, as well as exerting biochemical or cytotoxic effects. In light of the reports that lead is found in high concentrations in the hippocampal formation, the late period of development of the hippocampus and the suspected role of this structure in lead-induced seizure activity, we examined the hippocampal formation of 15-day-old control and lead-exposed rats to determine the extent of lead-induced morphometric alterations. We were particularly interested in studying the hippocampal mossy fiber system because of its high content of zinOS,zg,31, which is believed to be important in the functioning of this system8°,72, and because of the possibility that lead might cause nonfunctional displacement of the zinc. Such a displacement could have deleterious effects on mossy fiber synaptogenesis. In contrast to many previous investigations, we used relatively low-level lead exposure in order to study subtle effects on hippocampal morphology without overt cytotoxicity or vascular damage. Our previous work with low-level lead exposure during development in the rat indicated that some aspects of behavior were altered, and that the severity of maximal electroshock seizures was increasedz8,56,57. Parameters examined in the present study included numbers per section and numbers per unit area of granule cells, pyramidal cells, blood vessels and mossy fiber synaptic profiles, as well as sizes of mossy fiber boutons, and areas of morphologically distinct regions of the hippocampal formation.

597 MATERIALSAND METHODS

Lead exposure Pregnant Long-Evans rats were purchased from Simonsen Laboratories (Gilroy, CA). The lead exposure protocol has been described previously and does not alter growth of pup body weights 23,56,57. Litters were reduced to 6 female pups at parturition (day 0) and were maintained at 4-6 pups. Control dams were maintained on Purina rat chow and tap water ad libitum, while treated dams received 0.2 ~ lead acetate solution (1090 /~g lead/g solution) in place of tap water beginning at parturition. The dose of lead ingested by the pups can be estimated by the method of Bornschein et al. 10 to range from approximately 0.2/~g lead/g body weight/day on day 1 to approximately 0.6-1.0/~g lead/g body weight/day on days 15-20. Tissue preparation At 15 days of age weight-matched control and lead-exposed pups were sacrificed. Mean -4- S.E. body weights were 34.1 q- 0.7 g and 33.2 4- 0.9 g for control and leadexposed groups, respectively. A single pup from each litter was perfused via an aortic cannula. Forty milliliters of a fixative containing 1 ~ paraformaldehyde, 1.25 glutaraldehyde and 0.1 ~ CaC12 in 0.1 M sodium cacodylate buffer (pH 7.5) were perfused via gravity flow at a pressure of approximately 75 mm Hg at a flow rate of 25-30 ml/min for 1 min, followed by 50 ml of a fixative containing 2 ~ paraformaldehyde, 2.5 ~ glutaraldehyde and 0.1 ~ CaC12 in the same buffer, at a reduced pressure with a flow rate of 5-8 ml/min. Two to four hours after perfusion the brain was removed from the cranium and immersed in the second fixative for 5-8 days. The hippocampus was dissected and sectioned as described in Fig. 1. A single mid-dorsal slice from the hippocampus of each animal was prepared for light and electron microscopy by postfixation 1-2 h in 1 ~ osmium tetroxide, 0.2 M sucrose in 0.2 M sodium cacodylate buffer prior to being stained en bloc with uranyl acetate, dehydrated in a graded series of acetones and embedded in Durcupan ACM epoxy (Polysciences, Warrington, PA). Light microscopic analysis A single semithin (0.75/~m) section of the hippocampal formation of each of 7 control and 7 lead-exposed pups was stained with toluidine blue (1 ~ in 1 ~ sodium borate) for light microscopic analysis. A line drawn from the tip of the infrapyramidal leaf of the dentate gyrus (DG) to the lateral tip of the suprapyramidal leaf divided hilar from non-hilar portions of the hippocampus (Fig. 1C). A second line drawn from the lateral tip of the suprapyramidal leaf of the DG to the boundary between CA3 and CA1 formed the superficial border of the area included in measurements of the nonhilar stratum pyramidale and its associated apical dendritic zone (Fig. 1C). The areas of neuronal and neuropil layers were measured with a Zeiss MOP-3 digital analyzer on drawings traced with a camera lucida at magnifications ranging from 100 to 400 × as described in Fig. 1C. Nucleated pyramidal cells in the hilar and non-hilar portions of CA3 stratum pyramidale and in CA4, and nucleated granule cells in the supra- and

-

~~omtm~

Fig. 1. Structures of areas investigated in the hippocampal formation. A: the intact hippocampal formation dissected from the right hemisphere of a 15-day-old rat was divided (dashed line) at the hippocampal flexure into dorsal and ventral portions. The dorsal portion was sliced into sequentially numbered 225 pm sections on a tissue chopper. The center section (arrows) was prepared for light and electron microscopic analysis: S, septal pole; T, temporal pole. B: frontal section of the hippoeampus and dentate gyrus (DG) illustrating some major neuronal connections of the hippocampal formation. A single granule cell in the suprapyramidal leaf of the D G gives rise to a mossy fiber axon which traverses the suprapyramidal mossy fiber zone (hatched area) to synapse on the base of the apical dendritic shaft of a CA3 pyramidal cell. The CA3 pyramidal cell, in turn, contacts a pyramidal cell of CA1 via a Schaeffer collateral. At this level of the dorsal hippocampus the infrapyramidal mossy fiber zone (stippled area) extends only a short distance beyond the lateral extent of the dentate gyrus. C: the following areas were measured by light microscopy: 1,2, supra- and infrapyramidal limbs, respectively, of the dentate stratum moleculare; 3, 4, supra- and infrapyramidal limbs of the dentate stratum granulesum; 5, the dentate hilus, excluding the hilar portion of CA3 stratum pyramidale; 6, hilar CA3 stratum pyramidale; 7, non-hilar CA3 stratum pyramidale; and 8, the apical dendritic zone, including strata radiatum and lacunosum-moleculare, of the non-hilar CA3 pyramidal cells. D: thin sections for electron microscopic studies of the hippocampus included thelateral tip of the suprapyramidal leaf of the D G and most of the hippocampal CA3 stratum pyramidale. The CA3 stratum pyramidale was subdivided into a distal region (also called CA3 a), which consists of the curved portion of regio inferior, and a proximal region, consisting of CA3 b which is the straight portion of regio inferior lateral to the infrapyramidal mossy fiber zone, and the non-hilar portion of CA3 c. CA3 c is the part of stratum pyramidale in contact with the infrapyramidal mossy fiber zone (stippled), including that portion enclosed by the two leaves of the dentate gyrus. Squares within the suprapyramidal mossy fiber zone (hatched area) depict the nonoverlapping micrographic fields analyzed for this study. Micrographs from each column were divided into 4 categories: (l) deep, or the closest to the stratum pyramidale; (2) superficial, or those most distant from stratum pyramidale, but still within the mossy fiber zone; (3) intermediate micrographs; and (4) single micrograph columns. Data from proximal and distal regions are presented (Figs. 5 and 6) as deep only, superficial only and entire; entire includes intermediate and single column micrographs, as welt as deep and superficial micrographs.

599 infrapyramidal leaves of the DG were counted. Blood vessels within the apical dendritic zone of CA3 were counted. To compensate for the fact that slight differences in the angle of sectioning might alter the numbers of cells or vessels counted and areas of regions analyzed, values were calculated as total numbers of structures or areas per section, as numbers per unit area, and as ratios of granule cells or vessels per pyramidal cell.

Electron microscopic analysis A single thin hippocampal section (80-100 nm) from each of 8 control and 8 lead-exposed pups was used for electron microscopic analysis. Sections were mounted on a thin plastic film (0.5 ~ Formvar in ethylene dichloride) over a 1 × 2mm hole in a single-hole slot grid. This allowed observation and analysis of the entire suprapyramidal mossy fiber zone from the lateral extent of the dentate gyrus to the end of the mossy fiber zone near CA1. Standard mesh grids were unsuitable for quantitative analysis as grid bars covered as much as 25 ~ of the mossy fiber zone, thus eliminating whole subfields from analysis. Columns of nonoverlapping electron micrographs were taken at an initial magnification of 4800 × from proximal (close to DG) and distal (close to CA1) parts of the suprapyramidal mossy fiber zone (Fig. 1D). Micrographs from deep (closest to stratum pyramidale) and superficial (most distant from stratum pyramidale) parts of the suprapyramidal mossy fiber zone were analyzed separately in both proximal and distal regions. Intermediate micrographs and those from singlemicrograph columns were included along with deep and superficial micrographs to determine values for entire proximal or entire distal regions. A total of 6000-9000/~m 2 of neuropil area from 50-60 electron micrographs was analyzed for each animal, with a minimum of 700/tmZ in each of the subfields of the suprapyramidal mossy fiber zone. Thus, approximately 880 electron micrographs were analyzed. All micrographs were enlarged to a magnification of 14,000 × for analysis. One or more dense regions of either the postsynaptic plasma membrane alone, or of both pre- and postsynaptic plasmalemmae and at least 5 synaptic vesicles were minimum criteria for inclusion of structures as synaptic profiles. Synaptic profiles wele divided into 4 morphologic types as described in Results. The number of each profile type was determined, and the area of each complex profile which did not extend beyond the edges of the micrograph was determined using a Zeiss MOP-3 digital analyzer. Vascular elements, nucleated cell bodies and dendritic trunks with a diameter greater than 2 /zm were subtracted from the area of each mierograph to obtain the neuropil area.

Statistical analysis The data were examined with Student's t-test with pooled variancenL Because previous studies have consistently shown that early postnatal lead exposure reduces the number of boutons per unit area6, 87,44,e0 and the area of neuropi137,45,60,63 in the cerebellum or cerebral cortex, one-tailed tests were considered appropriate for these measures.Two-tailed tests were used for all other data. Probability values less than 0.05 are reported for one-tailed tests and values less than 0.10 are reported for twotailed tests.

600 TABLE I

Quantitative light microscopic characteristics of hippocampal vasculature hl non-hilar CA3 apical dendritic zone of 15-day-old control and Pb-exposed rats Values are means ± S.E. for 7 control and 7 Pb-exposed, weight-matched rats. No significant differences were found.

Per section

Per 1000 l~m~

Perpyramidal cell

Number of capillaries Control Pb-exposed

56 ± 5 58 ~ 3

0.18 ± 0.02 0.19 ± 0.01

0.38 ± 0.09 0.40 ± 0.02

Number of total vessels Control Pb-exposed

72 ± 6 74 ± 2

0.22 ± 0.02 0.25 ± 0.02

0.48 ± 0.05 0.51 ± 0.02

RESULTS

Light microscopic analys& The brains of lead-exposed rats showed no macroscopic signs of lead neurotoxicity such as cerebellar hemorrhage. Also, no qualitative differences between control and lead-exposed brains were evident at the light microscopic level; vascular damage or obvious signs of cytotoxicity such as pyknosis, vacuolization, gliosis and neurofibrillary tangles were not observed. Quantitative analysis of hippocampal vasculature in the non-hilar CA3 apical dendritic zone did not reveal an effect of lead treatment. The number of capillaries and total blood vessels, their number per 1000/~m 2 and their number per pyramidal cell were the same in control and lead-exposed groups (Table I). Quantitative analysis did reveal selective effects of postnatal lead exposure on the hippocampal formation. Measurements of neuropil areas showed that lead

300~ 1CONTROL E::L i 17-nPb'EXPOSED O

o

200 <0.04

I00

I,,z,J

z

0

S I E DENTATESTR. MOLECULARE

DENTATE CA3 HILUS APICAL DENDRITES

Fig. 2. Effects of early postnatal low-level lead exposure on the areas of various neuropil regions in the hippocampal formation of the 15-day-old rat. A general trend toward reduction in neuropil development in lead-exposed pups was significant in the infrapyramidal limb (I) of the dentate stratum moleculare, which consists of granule cell dendrites and their afferent connections, and in the dentate hilus. The areas of the suprapyramidal limb ,(S) and of the entire (E) area of the dentate stratum moleculare were not significantly affected. Probabilities are based on one-tailed t-test and are shown above appropriate bars

601 exposure reduced the area of the intrapyramidal portion of the dentate stratum moleculare by 22 % and the area of the dentate hilus by 15 % (Fig. 2). However, the areas of the suprapyramidal portion of the dentate stratum moleculare and the nonhilar CA3 apical dendritic zone were not significantly altered by lead exposure (Fig. 2). Analysis of area and neuron number in pyramidal and granule cell layers indicated only one quantitative effect of lead exposure. The numerical density or number of cells per 1000 # m 2 in the hilar portion of CA3 stratum pyramidale was increased in lead-exposed pups (Table II). This resulted from a 12% increase in number of hilar pyramidal cells combined with an 8 % decrease in the area of the hilar portion of CA3 stratum pyramidale, changes which alone were not statistically significant. There were no lead-induced changes in numerical densities of pyramidal cells in non-hilar regions or in numerical densities of supra- and infrapyramidal granule cells. Numbers of granule and pyramidal cells, areas of stratum pyramidale and stratum granulosum (Table II), numbers of CA4 pyramidal cells, and ratios of granule to pyramidal cells (data not shown) were unaffected by lead exposure. Electron microscopic analysis

The ultrastructure of the stratum pyramidale and mossy fiber zone and the location and extent of the mossy fiber zone were qualitatively similar in lead-exposed and control pups. In both groups the intra- and infrapyramidal portions of the mossy fiber zone extended only 20-30 /.tm beyond the lateral limit of the DG. More laterally, only a few complex mossy fiber boutons were seen in these positions in either treatment group. Inspection of intra- and infiapyramidal regions revealed no shift of complex mossy fiber boutons to ectopic locations. The boutons were divided into 4 morphologic types (Figs. 3 and 4). Complex (C) boutons were characterized by multiple membrane densities and simple (S) boutons by TABLE II Quantitative light microscopic characteristics of hippocampal pyramidal and granule cells in 15-day-old control and lead-exposed rats.

Values are means 4- S.E. obtained from one mid-dorsal hippocampal section per animal for each of 7 Pb-exposed, weight-matched rats. Cell type

Area of cellular layer (1000 itm 2)

Number of cells per section

Number of cells per 1000 i~m2

C

Pb

C

Pb

C

31 4- 4 117 4. 4 148 i 8

49 4- 6 173 ± 4 223 4- 6

54 4- 4 175 4- 11 229 4-10

1.40 4. 0.10 1.80 4- 0.09* 1.47 4. 0.05 1.504- 0.07 1.46 4- 0.03 1.56-t- 0.07

64 4- 5 35 ± 4 99 4- 8

473 4-17 319 :~ 20 791 4- 34

443 4- 29 304 4. 24 747 + 50

6.99 i 0.27 7.01 ± 0.38 8.08 4- 0.33 8.86 4- 0.42 7.39 ± 0.27 7.64 4. 0.33

CA3 pyramidal cells Hilar 34 4- 3 Non-hilar 118 ± 4 Entire 152 4- 6 Dentate granule cells Suprapyramidal 68 4- 4 Infrapyramidal 40 4- 3 Entire 108 + 6

* P ~< 0.01 by two-tailed t-test for significance of difference from control group.

Pb

602

J7 Pyr

o

Pyr

Fig. 3. Four morphologic types of synaptic profiles in the suprapyramidal mossy fiber zone. All synaptic profiles counted exhibited one or more dense regions on the plasma membrane and 5 or more synaptic vesicles. A: complex invaginated (CI) profiles were characterized by multiple membrane densities (often asymmetric, Gray type I contacts), and deep invaginations around the small dendritic processes and spines (s) upon which they synapsed. Some dendritic spines were completely surrounded by the bouton (*). Regions of CI boutons which contacted large dendritic trunks or perikarya of pyramidal cells (Pyr) generally exhibited symmetric membrane contacts. Clusters of mitochondria often paralleled the surface of dendrites or perikarya. Most CI boutons contained dense populations of 40 nm round, clear-cored synaptic vesicles. B: complex noninvaginated (CN) profiles lacked the deep invaginations of CI profiles. The multiple membrane densities were often symmetric, similar to those observed in regions of CI boutons which contacted dendritic trunks (den). The symmetric membrane densities may represent either Gray type II synapses or nonsynaptic punctata adherenstype adhesions. CN boutons had variable numbers of 40 nm clear-cored vesicles and some contained a few larger clear- or dense-cored vesicles. C: simple boutons contacting dendritic trunks (ST) exhibited a single membrane density (either symmetric or asymmetric) in contact with a large dendritic trunk or pyramidal cell perikaryon. Vesicle populations of ST profiles varied, but were often sparse. D: simple profiles contacting dendritic spines (SS) exhibited a single membrane density (generally asymmetric) in contact with small dendritic spines (s) having a diameter of less than 1 ~m. These boutons usually contained few synaptic vesicles, but denser populations of vesicles were occasionally observed. Some elongated SS profiles which exhibited many neurotubules appeared to be slight expansions of mossy fiber axons synapsing en passage.

603 a single membrane density. Complex boutons were either deeply invaginated (CI) or were noninvaginated (CN). Simple profiles contacted either dendritic trunks (ST) or spines (SS). CI profiles, assumed to be mature mossy fiber boutons, were deeply invaginated by the small dendritic processes upon which they synapsed and often exhibited dense membrane contacts on dendritic trunks as well (Figs. 3A, 4A). They usually contained relatively dense populations of round, clear-cored 40 nm synoptic vesicles, and sometimes exhibited mitochondria, neurotubules, and a few 80-120 nm clear-cored or dense-cored vesicles. CN profiles were bag-like and lacked the deep invaginations characteristic of CI (Figs. 3B, 4A, B), usually contacted large dendritic trunks and sometimes synapsed on dendritic spines. CN boutons contained more variable (often sparse) populations of 40 nm clear-cored vesicles. ST plofiles (Figs. 3C, 4B) and SS profiles (Figs. 3D, 4C) were generally smaller thart either types CI or CN, but contained vesicles similar to those in complex profiles and some contained mitochendria and neurotubules as well. It was not possible to characterize all membrane densities as symmetric or asymmetric. However, dense contacts on dendritic spines were often clearly asymmetric, while dense contacts on dendritic trunks were often clearly symmetric and some may have been punctata adherens. Thus, CI profiles often displayed many asymmetric dense contacts, CN profiles exhibited predominantly symmetric contacts, SS profiles usually exhibited asymmetric contacts, while the symmetry of ST contacts was often indistinct. The numerical density and size of CI profiles were approximately the same in all subfields of the mossy fiber zone studied, except in the superficial part of the distal region, in which CI profiles were both slightly smaller and less numerous (Figs. 5 and 6). Although CI and CN profiles did not differ strikingly in numerical density, CN profiles were significantly smaller than were CI profiles (Figs. 5 and 6). Further, CN profiles were distinctly smaller in the distal than in the proximal subfields (Fig. 6). ST boutons occurred most frequently in distal subfields and SS profiles tended to occur most often in superficial parts of the mossy fiber zone (Fig. 5). Postnatal lead exposure decreased by 15 ~ the numerical density of both CI and CN boutons in the deep part of the proximal region of the suprapyramidal mossy fiber zone (Fig. 5). The numerical density of CI boutons was reduced 12 ~ over the entire proximal portion of the mossy fiber zone in lead-exposed pups. Lead exposure also reduced the number of SS profiles per unit area by 31 ~ in the deep-proximal region. The number of ST profiles per unit area, however, was not affected in this region. Thus, the numerical density of 3 of the 4 bouton types was reduced by lead exposure in the deep subfield of the proximal mossy fiber zone, whereas lead exposure did not significantly alter the numerical density of any of the profiles in the superficial field of either proximal or distal regions of the mossy fiber zone (Fig. 5). The average cross-sectional area of CI profiles was not altered by lead exposure, but the mean size of CN profiles was increased in both the superficial subfield of the distal mossy fiber zone and in the entire distal mossy fiber zone (Fig. 6). In the proximal mossy fiber zone, CN profiles of lead-exposed rats tended to be larger than those of controls, but this did not reach statistical significance. Areas of SS and ST profiles were not measured.

604

605 DISCUSSION Neonatal exposure to low levels o f lead resulted in morphologic alterations in specific iegions o f the hippocampal formation in the absence o f reduced b o d y weight, encephalopathy, or overt signs o f cytotoxicity. The effects observed included: (1) reduced area of the stratum moleculare o f the infrapyramidal limb o f the dentate gyrus; (2) reduced area of the dentate hilus; (3) reduced numbers o f types CI, CN, and SS profiles per unit area in the deep part o f the proximal region o f the mossy fiber zone; and (4) increased size o f C N profiles in the superficial part o f the distal region o f the mossy fiber zone.

Regional variation and identification of synaptic profiles in the suprapyramidal mossy fiber zone The current report is the first electrort micrographic study to describe qualitatively and analyze quantitatively synaptic profiles t h r o u g h o u t the entire lateral extent o f the suprapyramidal mossy fiber zone at mid-dorsal level o f the developing rat hippocampus. Similar previous studies have examined only areas CA3 b and c 4 or area CA3 b a,6a and therefore did not present data on regional variation in type, n u m b e r or size o f profiles. A m a r a l and Dent 4 f o u n d the area o f large invaginated boutons to be 4.8 42.2/~m z (mean q- S.E.) in CA3 b and c in 14-day-old rats, which is larger than our 3.5 4- 0.2/~m 2 for the area o f CI profiles in the proximal region. This difference is likely due to the selection o f only the most mature-appearing b o u t o n s for measurement by these investigators. O u r data on numerical density o f CI profiles in the deep-proximal region (25 -q- 1/1000/~m 2) is in approximate agreement with Stirling and Bliss' report o f 30 4- 3/1000 p m 2 mossy fiber profiles in this region in their 15-day-old rats 6a. Some o f the CN, SS and ST profiles observed in the 15-day-old mossy fiber zone m a y be immature CI profiles. A m a r a l and Dent's illustration and description o f mossy fiber boutons at 3 days o f age resemble our SS and ST profiles, and our C N profiles are Fig. 4. Profile types in the mossy fiber zone of a 15-day-old rat. A: complex invaginated (CI) profiles exhibited several dense regions of the plasmalemma in contact with a large dendritic process (den) and smaller dendritic processes and spines which protruded into the bouton. One dendritic spine (s) appears to be surrounded by the CI profile. Another spine displays a spine apparatus (sa) which appears as a series of flattened membranous sacs. Complex noninvaginated profiles (CN) displayed multiple dense membrane regions, but were not deeply indented by postsynaptic elements. Both CI and CN profiles contained many small 40 nm clear-cored vesicles, and sometimes displayed a few larger clear-cored (arrows) or dense-cored (arrowheads) vesicles. Clusters of mitochondria (m) were often observed in complex bouton types near their contacts with large dendritic processes. M, myelinated axon. × 21,200. B: CN profiles in this micrograph displayed a wide range of vesicle populations. One of these profiles only showed clumps of vesicles over the dense membrane contacts (arrowheads). Simple profiles (ST) showed only a single dense membrane contact with large dendritic trunks (Tr). Small (0.3 #m in diameter) multivesieular bodies (MVB) occurred in dendritic processes of various sizes. × 19,700. C: a simple profile (SS) at higher magnification displayed a distinctly asymmetric membrane contact (arrow) on a small dendritic spine. SS profiles often showed a clump of vesicles over the membrane contact, as seen here, with few vesicles in the remaining cytoplasm of the profile. This provides striking contrast with the large number of vesicles observed in a portion of a nearby CI profile which can be seen at the lower left. Another small profile (p) contained vesicles, but displayed no dense region of either pre- or postsynaptic membranes, and therefore was not counted as a synaptic profile in this study, × 32,400.

606 Complex Invaginated (CI)

Complex Noninvaginoted (CN) I CONTROL

3 0 F~O.025

F-

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<0025

"~20

|~005

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,

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to

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Simple on Dendritic Trunks (ST) Simple on Dendritic Spines (SS)

o

.i"

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~T T~ ~

LZ

~,0, ~

~/~TT

I0

0

D S E PROXIMAL

D S E DISTAL

D S E D S E P R O X I M A L DISTAL

Fig. 5. Effect of low-level postnatal lead exposure on the numerical density of synaptic profiles, i.e. number of profiles per unit of neuropil area, of the suprapyramidal region of the mossy fiber zone. The mossy fiber zone was divided into proximal and distal regions which in turn were divided into deep (D) and superficial (S) subfields, or studied in their entirety (E), as described in Fig. 1. Lead exposure reduced the numerical density of complex invaginated (CI) profiles, complex noninvaginated (CN) profiles, and simple profiles contacting dendritic spines (SS) in the deep subfield of the proximal part of the mossy fiber zone. The numerical density of CI profiles was also significantly reduced in the entire proximal region. These same profile types were not affected by lead exposure in the superficial subfield of the proximal zone or in any subfield of the distal zone. Lead exposure had no effect on numerical density of simple profiles which synapsed on dendritic trunks (ST) in any region. Probabilities are based on one-tailed t-tests.

Complex Invoginoted (CI) 4.0

F

I

~n

ComplexNoninvoginated(CN) m CONTROL r7"71Pb-EXPOSED

5.0 20

~

I.O

O

D

S

E

PROXIMAL

D

S

E

DISTAL

D

S

E

PROXIMAL

D

S

E

DISTAL

Fig. 6. Effect of low-level postnatal lead exposure on mean size of complex synaptic profiles in the suprapyramidal mossy fiber zone in the 15-day-old rat. As in Fig. 5, the proximal and distal portions of the suprapyramidal mossy fiber zone were divided into deep (D) and superficial (S) zones or studied in their entirety (E). Lead exposure had no effect on the size of the CI profiles, but tended to increase the mean cross-sectional area of CN profiles. This increase in size of CN profiles was significant in the superficial subfield of the distal part and throughout the entire distal region of the mossy fiber zone. Probabilities are based on 2-tailed t-tests.

607 similar to the bag-like boutons that Amaral and Dent found in 10-day-old rats. Also, our CN and ST profiles resemble mossy fiber boutons observed by Stirling and Bliss69 in the 7-day-old rat. Alternatively, some CI, SS and ST profiles may be fragments of CI profiles which were not sectioned through an invaginated region. Further, some simple profiles may be synapses en passage or they may be of non-dentate origin. Other sources of afferents to the region of the mossy fiber zone include septal and commissural fibers, basket cells and other interneurons which synapse near the suprapyramidal mossy fiber zone with boutons similar to our SS and ST profiles T M .

Effects of neonatal lead exposure on brain development Previous investigations have shown that exposure of neonatal rodents to high levels of lead resulted in reduced body weightl,25,3n,37,4°,45,58,nl,n2,7°, reduced brain weighteS,86-ss,42,6°,vascular damage including hypervascularization and hemorrhages, especially in the cerebelluml,26,40,ss,nl,62,65,70,71, spontaneous seizuresaT,61,62 and paraplegia zn-3s,S8,61,62,7°. High level lead exposure also reduced dendritic growth and branching in the cerebellum45,nz and neocortex87, no, and reduced or delayed synaptogenesis in the neocortexZ7,60. Low-level lead exposure, which did not impair somatic growth or cause paraplegia, overt encephalopathy or grossly observable brain hemorrhages, also delayed synaptogenesis in the neocortex6,4a. Exposure to high levels of lead also resulted in reduced hippocampal weight, reduced dimensions of the hippocampus and dentate gyrus and reduced dimensions of the mossy fiber zone as seen by sulfide-silver staining39. Both high and low level lead exposure resulted in alteled development of the dendrites of the infrapyramidal granule cellsz, and low-level lead exposure reduced the thickness of the stratum pyramidale and stratum granulosum41. The low-level lead exposure employed in our study has previously been shown to allow normal somatic growth, although some but not all aspects of behavior were altered and the severity of maximal electroshock seizures was inereased23,sn,57. Nevertheless, the low-level lead exposure resulted in impairments of neuropil development and synaptogenesis which were qualitatively similar to those seen with neonatal exposure to higher lead levels, while paraplegia, vascular damage and other signs of overt encephalopathy or cytotoxicity were absent. Our results demonstrate that the effects of neonatal lead exposure were restricted to particular subfields of the developing hippocampal formation; hippocampal differentiation and growth were not uniformly impaired. Exposure of neonatal rats to lead may cause developmental impairment by both direct and indirect mechanisms. Direct effects of lead on brain tissues may involve nonfunctional displacement within neurons of essential divalent ions such as zinc, copper, iron or calcium by lead. The hippocampal formation is known to concentrate zinc and lead and to contain high levels of copper and iron la, 15,18-20,2s. Lead has been located in high concentration in the granular-molecular portion of the DG, in the dentate hilus and in CA3 of normal rats 15. Niklowitz and Yeager 52 suggested that lead may interfere with brain deposition of essential metals such as zinc, copper and iron. Lead has been shown to reduce the levels of these metals in some brain regions4n,52,

608 but the effect of lead exposure on metal levels in the developing hippocampal formation has not been examined. Microdissection of the hippocampal formation followed by atomic absorption spectroscopy has revealed very high concentrations of zinc in the molecular-granular and hilar portions of the DG, and in the CA3 region of the normal rat 15. In the hippocampal CA3 region, zinc has been localized to the giant mossy fiber synaptic boutons29,a1, 55. Although the precise function of zinc in the mossy fiber zone is unknown, chelation of this metal or its reduction by a zinc deficient diet resulted in functional deficits of the hippocampal formationa0,72. Furthermore, postnatal zinc deficiency has been demonstrated to impair growth of the hippocampus in rats 11. Copper is also concentrated in the granular-molecular and hilar portions of the DG and CA315, but the precise location of iron within the hippocampus has not been determined. Niklowitz and Yeager 5z showed that exposure of adult rabbits to tetraethyllead reduced iron concentration in the hippocampal formation and other brain tissues, and Michaelson and Sauerhoff46 demonstrated that exposure of rats to inorganic lead reduced copper levels in the cerebellum. While neither of these studies examined the metal levels in the developing hippocampal formation, the morphological effects of lead exposure reported here are localized to regions rich in copper and iron, as well as zinc, implying that these metals could be involved in lead-induced alterations of hippocampal morphology. Previous studies associated delayed neocortical synaptogenesis during lead exposure with neurochemical alterations 12,44. Bull et al. 12 demonstrated that exposure of rat pups to subencephalopathic levels of lead prenatally and postnatally via dams drinking lead-adulterated water produced significant dose-related transitory delays in the normal increase in cytochrome content of cerebral cortex, which was especially pronounced between 10 and 15 days of age, and delayed neocortical synaptogenesis44. It was concluded that these effects may have been secondary to direct effects of lead on immature brain mitochondrial metabolism. Whether similar effects of lead on brain metabolism may be related to the effects on development of the hippocampal formation noted here is not certain but must be considered an important possibility.

Interrelationship of affected hippocampal regions and significance of findings Most of the effects of lead exposure which are reported here can be related to impaired development of a single region - - the infrapylamidal leaf of the dentate gyrus. The reduction of the area of the infrapyramidal stratum moleculare and the decrease in the hilar area, which is traversed by the mossy fiber axons of dentate granule cells indicate reduced development, respectively, of the granule cell dendrites and mossy fiber axons and/or their collaterals. Lesion studies by Gaarskjaer z4 elucidated the stratified nature of the proximal part of the suprapyramidal mossy fiber zone, with the most superficial fibers arising from the suprapyramidal leaf of the DG, intermediate fibers arising from the crest of the DG, and the deepest fibers originating from the lateral portion of the infrapyramidal leaf. This implies that the area of the mossy fiber zone in which we find the greatest effect of lead on the numbers of boutons per unit area arises from the infrapyramidal leaf of the stratum granulosum.

609 Previous autoradiographic investigations have shown that neurogenesis in the extrahilar portion of stratum pyramidale precedes that in the hilar portion 7, and that neurogenesis in the suprapyramidal leaf of the DG precedes that in the infrapyramidal leafT,6s. Sulfide-silver staining studies4,69,~4 also indicate that the mossy fiber area most severely affected by lead in our study, i.e. the deep-proximal region, normally develops somewhat later than more superficial areas. Therefore, the pattern of effects of lead exposure in our study suggests that of the areas studied within the hippocampal formation, lead most severely affected those which develop latest. Apparently, later developing structures are susceptible during clitical stages of their development to levels of lead exposure which do not affect structures that are formed earlier. The probability of obtaining a profile of a bouton is directly proportional to both the numbers of the bouton per unit volume and to its size. The reduction in numbers of complex boutons per unit area in the mossy fiber zone noted here probably represents an actual reduction in numbers per unit volume, rather than a reduction in size, however, as our data indicate a trend towards increased size of the boutons concomitant with a reduction of bouton density. In our electron microscopic analysis no gross differences in dimensions of the mossy fiber zone were observed between control and lead-exposed rats. Furthermore, LeBoutillier et al. a9 demonstrated that neonatal exposure to lead reduced the dimensions of the mossy fiber zone and reduced hippocampal weight. Thus, it is unlikely that the reduced number of boutons per unit area which we have observed in lead-exposed animals is compensated by a concomitant increase in total area of the mossy fiber zone. Studies which show altered dendritic branching patterns in lead-exposed brain 2,37,45,6°,6a suggest that further studies employing Golgi-staining methods are needed to determine whether the number and length of dendritic branches and the density of dendritic spines are normal in areas which appeared unaffected by the low level of lead exposure in this study. Similarly, a lack of effect of lead on vascular density and the lack of gross vascular damage does not conclusively demonstrate the integrity of the blood-brain barrier in lead-exposed animals. Previous studies4,9,69,74 have indicated that at 15 days of age the rat hippocampal mossy fiber zone is nearing the end of a rapid phase of growth and maturation during which the numbers of boutons per unit area, size of boutons, complexity of profile shape, number of synaptic contacts per bouton, number of vesicles per profile and intensity of sulfide-silver staining have all increased dramatically. The increased size of CN mossy fiber profiles which we observed in lead-exposed rats might be explained by retardation of the process of invagination as CN profiles mature to the larger and more complex CI configuration. It is possible that our study of the hippocampus of the 15-day-old rat has highlighted a temporary delay in development rather than a permanent reduction in anatomical substrate for neurologic function. Light and electron microscopic analyses of the hippocampal formation in 90- and 600-day-old rats which have been exposed to lead from parturition until weaning are in progress to determine the time-course of the effects of lead observed in the 15-day-old rat.

610 ACKNOWLEDGEMENTS This research was supported by N I H G r a n t ES-01503. We gratefully acknowledge the secretarial assistance of Ms. M a r y Lou Rodriguez a n d t h a n k Dr. Walter S. Tyler of the California P r i m a t e Research Center for use of the Zeiss M O P digitizer.

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