* Program in Human Health and the Environment and
Department of Epidemiology and Preventive Medicine, University of Maryland, Baltimore, Maryland 21201
Received December 1, 2000; accepted May 3, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: methylmercury; neurodevelopment; cytokine; migration; cerebellum; cell signaling.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vitro, nanomolar and micromolar levels of methylmercury (0.510 µM) inhibit the movement of murine cerebellar neurons in culture systems. In these studies, movement was characterized by reaggregation of dissociated cells (Jacobs et al.1986), or migration within organotypic cultures (Kunimoto and Suzuki, 1997
).
Neuronal movement is regulated in part by precise temporal and spatial expression of neural cell adhesion molecules (NCAMs), which are cell-surface glycoproteins that guide neuronal migration, neurite elongation, and synaptogenesis through the regulation of cellcell recognition and adhesion (Regan, 1991, 1993
; Regan and Fox, 1995
). The NCAMs have disulfide-bonded immunoglobulin-like segments in the extracellular domain, providing a potential target site for disruption by mercury. In vivo exposure of rat pups to MeHgCl administered subcutaneously on alternate postnatal days 313 has been shown to alter the sialylated state of NCAM within the neuronal cell layers of the developing cerebellar cortex (Dey et al.1999
).
Cytokines, produced by glial cells, are among the signaling molecules that are reported to influence cell adhesion molecules in the developing nervous system. Interleukin 6 (IL-6) is produced by astroglial cells and at physiological concentrations has been shown to induce production and release of nerve growth factor (NGF) by astroglia (Frei et al.1989). IL-6 has also been shown to induce neurite extension in a neuronal cell line (Satoh et al.1988
) and has been proposed to function as a developmental neurotrophic factor (Gadient and Otten, 1994a
, b
; Otten et al.1994
). Two other cytokines, interleukin-1ß (IL-1ß) and tumor necrosis factor-
(TNF-
) are also produced in the brain by glial cells, and at physiological concentrations are capable of co-inducing each other and of inducing high levels of IL-6 (Giulian et al.1988
; Merrill, 1992
; Munoz-Fernandez et al.1994
). In tissue culture systems, glial-derived IL1-ß, TNF-
, and interferon-
(IFN-
) are all capable of inducing expression of cell surface adhesion molecules in glial cells of both rodent and human origin (Lee and Benveniste, 1999
; Merrill and Jonakait, 1995
; Satoh et al.1991
; Zhao and Schwartz, 1998
).
The proposed role of cytokines in the regulation of neuronal movement suggests a mechanism for the effects of MeHg on neural migration. Both mercury and methylmercury are immunotoxic, with well described effects on cytokine production and impaired ability to respond to cytokines related to both autoimmunity and immune suppression (Bagenstose et al.1999; El-Fawal et al.1999
; Hultman and Hansson-Georgiadis, 1999
; Kono et al.1998
; Mehler and Kessler, 1997
; Moszczynski, 1997
). However, no studies have been conducted to date on the potential role of altered cytokine-dependent cell signaling in mercury neurotoxicity.
In these studies we introduce a novel in vitro method by which the movement of individual granule cells can be observed and quantitated. We utilize this method to investigate the effects of altered cytokine production on neural movement and to quantitate the effects of MeHg on neural migration in vitro. These studies were also undertaken to test the hypothesis that cytokines may modulate the effects of MeHg on neuronal movement. Previous studies examining cytokine actions have been limited to evaluating the in vitro effects of cytokines on the production of CAMs and other molecules, mainly in transformed cell lines and monocultures. This study is the first attempt to examine the interactive effects of cytokine stimulation and mercury exposure on neuronal movement.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All steps were carried out under aseptic conditions within a laminar flow hood. Mouse pups were sacrificed by CO2 overexposure and cerebella removed into cold HBSS buffer (Gibco-BRL), at pH 7.4. The cerebella were minced and trypsinized for 15 min at 37°C. The digested tissue was triturated in modified HBSS containing 10 U/ml DNase Type I (Gibco-BRL) and 10% heat-inactivated fetal bovine serum (HI-FBS). The supernatant was centrifuged at 100 x g for 10 min and the cell pellet resuspended in modified minimal essential medium (MEM, Gibco-BRL) supplemented with 10% HI-FBS and 50 U/ml penicillin-50 µg/ml streptomycin-glutamine (Gibco-BRL). Cell number and viability were determined using a hemocytometer and trypan blue dye exclusion. Eight9 million viable cells were routinely obtained from each cerebellum. The cell suspension was adjusted to 8 x 105 viable cells/ml and plated on poly-D-lysine coated 6-well multiwell plates (Costar/Corning) at 2 ml (1.6 million cells) per well. Wells contained a CELLocate coverslip, etched with a 55-µm grid (Eppendorf Scientific, Inc), and coated with poly-D-lysine. Cultures were maintained at 37°C, 5% CO2/95% air, without further medium changes for the duration of the experiment.
Escherichia coli O55:B5 lipopolysaccharide (LPS) (Sigma) was made as a stock solution in PBS and diluted to 50 µg/ml. Just prior to using, the stock solution was vortexed vigorously (1015 min.), and added directly to cell cultures at a final concentration of 50 ng/ml. Methylmercuric chloride (MeHgCl; ICN Biomedicals, Inc) was added to the cultures at a final concentration of 0.1 µM. This concentration has been used in several in vitro studies in our laboratory, and produced effects on cytokine production without observable cell damage or cell death (Silbergeld et al., 2000). It is within the range utilized by Sager (1988) and others (Graff et al., 1997
; Kunimoto and Suzuki 1997
) in studying in vitro effects of MeHg.
Determination of neuronal migration.
The cerebellum is comprised of only 5 neuronal cell types: basket and stellate, Golgi, granule, and Purkinje cells. All cells selected for tracking were granule cell neurons, identified by the small round phase-bright soma characteristic of these cells (Bulleit and Cui, 1998; Cui and Bulleit, 1998
; Hertz et al.1989
). By 48 h in culture, the granule cells were distributed evenly. On day 4, cultures were dense enough to support cellcell contacts, and migration, but individual cells were still distinguishable. By 56 days in culture the granule cells had extended long processes, and most had contacted each other. Granule cells that did not extend processes by the time of observation were excluded from tracking, and were presumed to be either unhealthy or premigratory germinative neuroblasts (Mares et al.1970
).
Preliminary studies were undertaken to determine optimal conditions for studying individual neuron movement. It was observed that granule cells with extended processes and contacts with neighboring cells were most likely to move. Migration of these cells was in a relatively direct trajectory towards each other, and towards larger aggregates, often moving in a small group or line. Astroglia, which move rapidly compared with granule cells, often moved through the observation area during the observation period (10 h). Occasionally, granule cells moved with the glial cells, as if "hitching a ride."
For all the studies reported here, treatments were initiated on day 4 of culture. At 48 h after treatment (day 6 in culture), tracking of neuronal movement began and continued for 10 h, as described below.
Tracking of neurons was accomplished by photographing each coverslip, using a Nikon Diaphot 300 microscope with phase-contrast optics, magnification x20, at 0, 3, 6, and 10 h (48, 51, 54, and 58 h after treatment). Images were digitally recorded using an AlphaImager (Alpha Innotech Corp.) linked to a black and white video camera (Alpha Innotech Corp.) Individual cells could be identified from recorded images by their location on the CELLocate grid. Since the grid is labeled at regular intersections with an alphanumerical system, the same location can be identified and the same cells recorded repeatedly (Fig. 1). The distance of movement was measured for each neuron, from the photo images, for each observation interval over the 10-h period of observation (Fig. 2
). For each experiment, data was collected for 10 cells/well, from 3 wells/treatment group, over a 10-h period from 48 to 58 h posttreatment. These experiments were repeated 4 times to determine replicability of the methods.
|
|
Detection of cytokine mRNAs by RT-PCR.
Cytokine mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR) analysis using cells pooled from 3 wells in each treatment group. As a positive control, murine splenocyte cultures were established according to standard methods (Coligan et al.1998), and treated with Concanavalin A (Sigma) at 2 µg/ml for 24 h. RNA for analysis was isolated from cell cultures with Trizol reagent (Gibco-BRL) according to manufacturer's instructions. RNA (3.5 µg/reaction) was used as a template in first strand cDNA synthesis with random hexamers, using the Superscript Preamplification kit (Gibco-BRL) according to the manufacturer's instructions. cDNA was amplified using the following murine primer pairs: ß-actin, a 348-bp product; IL-1ß, a 515-bp product; IL-6, a 600-bp product, and TNF-
, a 446-bp product (Bohn et al.1994
). All RT-PCR reactions were set up in a final volume of 24 µl of master mix [1x PCR buffer/ 15 mM MgCl2, 500 µM dNTPs, 0.025 U Taq Gold (Perkin Elmer)], 0.5 µl of cDNA template, and 500 nM 5' and 3' primers. PCR was performed in a PTC-200 Peltier thermal cycler (MJ Research) with 30 cycles: 30 s of 95°C denaturation, 90 s of 60°C annealing, and 2.5 min of 72°C extension. PCR products were visualized by electrophoresis of the total product on 1% agarose gel in 0.5x Tris-boric acid-EDTA buffer. A 123-bp marker set (Boehringer-Mannheim) was run in parallel with the samples.
Detection of cytokine protein levels.
Cytokine release was measured from 3 pooled wells in each group in the same cultures on which migration was tracked, 18 h after treatment with LPS. This has been determined to be the time when LPS-stimulated cytokine release in neural cultures is at peak levels (Chao et al.1995; Lee et al.1993
). Cytokine levels were measured by HS enzyme-linked immunosorbent assay (ELISA), (Biosource International, Inc.) according to the manufacturer's instructions. The data were read using a Spectramax250 plate reader (Molecular Devices) and analyzed with a standard curve, using Softmax Pro software (Molecular Devices). Levels of cytokine as low as 50 pg/ml could be detected.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Combined Effects of LPS and Methylmercury
LPS and MeHg, added together to the cultures, resulted in significant reductions in the proportion of neurons that moved at all over the observation period, compared with all other groups, including controls (Tables 1, 2), and those treated with MeHg alone (Table 3
). Of the LPS + MeHg neurons that moved at all, the mean distance moved was 12.1 µm, significantly less than the LPS-treated cultures (Tables 1, 3
). LPS had no effect on movement in neurons treated with MeHg. Analysis by Wilcoxon rank sum test demonstrates that these LPS + MeHg neurons moved significantly less as a group than either LPS-treated neurons (Table 3
) or control neurons (Tables 2 and 3
).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of this study indicate that it is important to analyze data from these experiments in terms of the number of cells that move, distribution of movement, and mean movement within groups of neurons, because there is substantial variability in movement among neurons. In the early stages of neural development in the vertebrate cerebellum, the progenitor cells of the external granule layer of the cerebellum are in a highly proliferative state. Following the proliferative stage, the immature neurons migrate inwards to form the future internal granule cell layer. The "decision" by these cells to initiate migration is not well understood and may vary among cells, as reflected by the failure of all cells to move under any experimental condition. Moreover, within the culture system described here, of those neurons that move at all over the observation period their total distance traveled varies considerably, which can be detected by assessment of the distribution of movement within and between groups. Our data suggest that the 2 events of initiation of movement and distance traveled are differentially affected by the treatment regimes.
Effects of LPS on Neural Movement and Cytokine Release
Our data provide additional support for the hypothesis that cytokines may play a role in neural migration. LPS increased neuronal movement and enhanced release of IL-6 and TNF-. LPS stimulated the transcription of IL-1ß but did not increase the release of this cytokine into culture media. This result is consistent with other reports, that LPS increases the intracellular levels of IL-1ß without affecting its release (Lee et al.1993
).
LPS did not affect the proportion of neurons that moved but did influence the distance traveled by moving neurons, such that a larger proportion of neurons move a farther distance, compared with control cultures (Fig. 2). While not all LPS-exposed neurons were fast-moving, there was a shift in the distribution curve toward the faster-moving end compared with control cultures, such that an increased proportion of neurons were fast-moving compared with controls (Fig. 2
). These results are consistent with a role for cytokines in stimulating neuronal movement.
Effects of Methylmercury
In this study, we confirmed findings of others that MeHg, in vitro, can inhibit neuronal movement, measured for the first time at the level of individual cells. MeHg had 2 effects on neuronal migration: inhibition of the initiation of movement, and reduction in the mean distance of movement. These results are consistent with previously reported observations of the effects of MeHg to inhibit the movement of neurons in vitro (Jacobs et al.1986; Kunimoto and Suzuki, 1997
). Our findings are also consistent with preliminary in vivo observations by our laboratory of significantly decreased granule cell migration in cerebella at 10 days postnatal age in mouse pups exposed in utero to 100 or 200 µg/kg HgCl2 administered to pregnant dams every other day (J.B. Sass and I.A. Silva, unpublished observations).
Role of Cytokines in the Effects of MeHg
We investigated the effects of LPS on MeHg-induced changes in neuronal movement, in order to explore the potential role of cytokines in the neurotoxic effects of MeHg. The results are complex.. There was an apparent interaction between these 2 agents. First, in the presence of LPS a significantly smaller proportion of MeHg-exposed neurons moved at all as compared with cultures exposed to either MeHg-only or to LPS-only. LPS did not affect the mean movement of neurons that moved in cultures exposed to both LPS and MeHg. These data suggest that LPS may be able to partially overcome the inhibitory effects of methylmercury on initiation of movement, but not on the distance moved by neurons.
We did not observe a clear role for cytokines in these effects of MeHg. We found no effect in vitro on release of IL-6 or TNF- under conditions in which neuronal movement was affected. It is possible that MeHg interfered with receptors or signal transduction mechanisms used by IL-6 or TNF-
. Preliminary data indicate that exposure of mice to mercury in vivo blocks the ability of liver cells (Kupffer cells) to respond to IL-6 and IL-1ß (Silbergeld et al., in preparation). Alternately, it may be that mercury induces an altered kinetic profile of cytokine appearance, resulting in a shift in the time of peak production of cytokines, as has been shown for IL-6 appearance in the brains of lead-exposed mice (Kishikawa and Lawrence, 1998
). MeHg may affect other cytokines involved in neural migration, which were not measured in these experiments. Recent work by our laboratory has demonstrated that exposure of human monocytes or mouse splenocytes in vitro to low doses of mercury (mercuric chloride) results in inhibition of IFN
production (Silbergeld et al.2000
).). These data support further investigation of the role of these and other cytokines in mercury-induced developmental neurotoxicity.
The effects of MeHg in these experiments also involve mechanisms other than cytokines, as hypothesized here. MeHg could have affected the functional status of NCAMs secreted in response to cytokine stimulation, by binding to SH- groups, as proposed by Dey et al. (1999), although substantially higher concentrations of MeHg (at least 1 µM) were needed to affect NCAM status in vitro. One of the more sensitive targets of mercury is thought to be disruption of microtubule formation, resulting in alterations in the cytoskeleton (Sager, 1988). Disruption of microtubules has been reported in cultured cells (Sager et al., 1983
). However, Graff et al. (1997) reported that in vitro sensitivity of microtubules to methylmercury decreased significantly after 6 days in culture, the time at which our measurements were initiated. In other studies in our laboratory on MeHg developmental toxicity in preimplantation stage mouse embryos, exposure to MeHg at levels below 500 nM, five times higher than the concentration in these neural studies, did not observably affect microtubule integrity (Devine and Silbergeld, 1998
). Sager (1988) reported that a concentration of 500 nM appeared to be needed to reduce the number of assembled microtubules. Graff et al (1997) utilized concentrations of 12 µM MeHg to affect microtubules in carcinoma-derived neurons. Thus, it is possible that in the developing CNS, cytokine-related mechanisms may precede the effects of MeHg on microtubules; however, more work is needed to confirm this.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
2 Present address: Natural Resource Defense Council, 1200 New York Ave. NW, Washington, DC 20005.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beiswanger, C. M., Diegmann, M. H., Novak, R. F., Philbert, M. A., Graessle, T. L., Reuhl, K. R., and Lowndes, H. E. (1995). Developmental changes in the cellular distribution of glutathione and glutathione S-transferases in the murine nervous system. Neurotoxicology 16, 425440.[ISI][Medline]
Bohn, E., Heesemann, J., Ehlers, S., and Autenrieth, I. B. (1994). Early gamma interferon mRNA expression is associated with resistance of mice against Yersinia enterocolitica. Infect. Immun. 62, 30273032.[Abstract]
Brown, D. L., Reuhl, K. R., Bormann, S., and Little, J. E. (1988). Effects of methyl mercury on the microtubule system of mouse lymphocytes. Toxicol Appl Pharmacol. 94, 6675.[ISI][Medline]
Bulleit, R. F., and Cui, H. (1998). Methylmercury antagonizes the survival-promoting activity of insulin-like growth factor on developing cerebellar granule neurons. Toxicol. Appl. Pharmacol.. 153, 161168.[ISI][Medline]
Burbacher, T. M., Rodier, P. M., and Weiss, B. (1990). Methylmercury developmental neurotoxicity: A comparison of effects in humans and animals. Neurotoxicol Teratol. 12, 191202.[ISI][Medline]
Chang, L. W., and Hartmann, H. A. (1972). Ultrastructural studies of the nervous system after mercury intoxication: I. Pathological changes in the nerve cell bodies. Acta Neuropathol. 20, 122138.[ISI][Medline]
Chao, C. C., Hu, S., Sheng, W. S., and Peterson, P. K. (1995). Tumor necrosis factor-alpha production by human fetal microglial cells: Regulation by other cytokines. Dev. Neurosci. 17, 97105.[ISI][Medline]
Charleston, J. S., Body, R. L., Bolender, R. P., Mottet, N. K., Vahter, M. E., and Burbacher, T. M. (1996). Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term sub-clinical methylmercury exposure. Neurotoxicology 17, 127138.[ISI][Medline]
Choi, B. H. (1986). Methylmercury poisoning of the developing nervous system: I. Pattern of neuronal migration in the cerebral cortex. Neurotoxicology 7, 591600.[ISI][Medline]
Choi, B. H., Lapham, L. W., Amin-Zaki, L., and Saleem, T. (1978). Abnormal neuronal migration, deranged cerebral cortical organization, and diffuse white matter astrocytosis of human fetal brain: A major effect of methylmercury poisoning in utero. J. Neuropathol. Exp. Neurol. 37, 719733.[ISI][Medline]
Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W. (1998). Current Protocols in Immunology, Vol. 1. John Wiley and Sons, New York.
Cui, H., and Bulleit, R. F. (1998). Potassium chloride inhibits proliferation of cerebellar granule neuron progenitors. Brain Res. Dev. Brain Res. 106, 129135.[ISI][Medline]
Devine, P. J., and Silbergeld, E. K. (1998). Effects of methyl mercury on preimplantation mouse embryos. Toxicologist 37, 261.
Dey, P. M., Gochfeld, M., and Reuhl, K. R. (1999). Developmental methylmercury administration alters cerebellar PSA-NCAM expression and Golgi sialyltransferase activity. Brain Res. 845, 139151.[ISI][Medline]
Dolbec, J., Mergler, D., Sousa-Passos, C. J., Sousa de Morais, S., and Lebel, J. (2000). Methylmercury exposure affects motor performance of a riverine population of the Tapajos river, Brazilian Amazon. Int. Arch. Occup. Environ. Health. 73, 195203.[ISI][Medline]
El-Fawal, H. A., Waterman, S. J., De Feo, A., and Shamy, M. Y. (1999). Neuroimmunotoxicology: Humoral assessment of neurotoxicity and autoimmune mechanisms. Environ. Health Perspect. 107, 767775.[ISI][Medline]
Frei, K., Malipiero, U. V., Leist, T. P., Zinkernagel, R. M., Schwab, M. E., and Fontana, A. (1989). On the cellular source and function of interleukin 6 produced in the central nervous system in viral diseases. Eur. J. Immunol. 19, 689694.[ISI][Medline]
Gadient, R. A., and Otten, U. (1994a). Expression of interleukin-6 (IL-6) and interleukin-6 receptor (IL-6R) mRNAs in rat brain during postnatal development. Brain Res. 637, 1014.[ISI][Medline]
Gadient, R. A., and Otten, U. (1994b). Identification of interleukin-6 (IL-6)-expressing neurons in the cerebellum and hippocampus of normal adult rats. Neurosci Lett. 182, 243246.[ISI][Medline]
Giulian, D., Young, D. G., Woodward, J., Brown, D. C., and Lachman, L. B. (1988). Interleukin-1 is an astroglial growth factor in the developing brain. J. Neurosci. 8, 709714.[Abstract]
Goldberg, W. J., Levine, K. V., Tadvalkar, G., Laws, E. R., Jr., and Bernstein, J. J. (1992). Mechanisms of C6 glioma cell and fetal astrocyte migration into hydrated collagen-I gels. Brain Res. 581, 8190.[ISI][Medline]
Graff, R. D., Falconer, M. M., Brown, D. L., and Reuhl, K. R. (1997). Altered sensitivity of posttranslationally modified microtubules to methylmercury in differentiating embryonal carcinoma-derived neurons. Toxicol. Appl. Pharmacol. 144, 215224.[ISI][Medline]
Grandjean, P., Weihe, P., and White, R. F. (1995). Milestone development in infants exposed to methylmercury from human milk. Neurotoxicology 16, 2733.[ISI][Medline]
Grandjean, P., Weihe, P., White, R. F., Debes, F., Araki, S., Yokoyama, K., Murata, K., Sorensen, N., Dahl, R., and Jorgensen, P. J. (1997). Cognitive deficit in 7-year-old children with prenatal exposure to methylmercury. Neurotoxicol. Teratol. 19, 417428.[ISI][Medline]
Hertz, L., Peng, L., Hertz, E., Juurlink, B. H., and Yu, P. H. (1989). Development of monoamine oxidase activity and monoamine effects on glutamate release in cerebellar neurons and astrocytes. Neurochem. Res. 14, 10391046.[ISI][Medline]
Hultman, P., and Hansson-Georgiadis, H. (1999). Methyl mercury-induced autoimmunity in mice. Toxicol. Appl. Pharmacol. 154, 203211.[ISI][Medline]
Jacobs, A. J., Maniscalco, W. M., and Finkelstein, J. N. (1986). Effects of methylmercuric chloride, cycloheximide, and colchicine on the re-aggregation of dissociated mouse cerebellar cells. Toxicol. Appl. Pharmacol. 86, 362371.[ISI][Medline]
Kishikawa, H., and Lawrence, D. A. (1998). Differential production of interleukin-6 in the brain and spleen of mice treated with lipopolysaccharide in the presence and absence of lead. J. Toxicol. Environ. Health 53, 357373.[ISI]
Kono, D. H., Balomenos, D., Pearson, D. L., Park, M. S., Hildebrandt, B., Hultman, P., and Pollard, K. M. (1998). The prototypic Th2 autoimmunity induced by mercury is dependent on IFN-gamma and not Th1/Th2 imbalance. J. Immunol. 161, 234240.
Kunimoto, M., and Suzuki, T. (1997). Migration of granule neurons in cerebellar organotypic cultures is impaired by methylmercury. Neurosci. Lett. 226, 183186.[ISI][Medline]
Lee, S. J., and Benveniste, E. N. (1999). Adhesion molecule expression and regulation on cells of the central nervous system. J. Neuroimmunol. 98, 7788.[ISI][Medline]
Lee, S. C., Liu, W., Dickson, D. W., Brosnan, C. F., and Berman, J. W. (1993). Cytokine production by human fetal microglia and astrocytes. Differential induction by lipopolysaccharide and IL-1 beta. J. Immunol. 150, 26592667.
Maggi, R., Pimpinelli, F., Molteni, L., Milani, M., Martini, L., and Piva, F. (2000). Immortalized luteinizing hormone-releasing hormone neurons show a different migratory activity in vitro. Endocrinology 141, 21052112.
Mares, V., Lodin, Z., and Srajer, J. (1970). The cellular kinetics of the developing mouse cerebellum: I. The generation cycle, growth fraction, and rate of proliferation of the external granular layer. Brain Res. 23, 323342.[ISI][Medline]
Matsumoto, H., Koya, G., and Takeuchi, T. (1965). Fetal Minamota disease. A neuropathological study of two cases of intrauterine intoxication by methylmercury compound. J. Neuropathol. Exp. Neurol. 24, 563574.[ISI][Medline]
Mehler, M. F., and Kessler, J. A. (1997). Hematolymphopoietic and inflammatory cytokines in neural development. Trends Neurosci. 20, 357365.[ISI][Medline]
Merrill, J. E. (1992). Tumor necrosis factor alpha, interleukin 1, and related cytokines in brain development: Normal and pathological. Dev. Neurosci. 14, 110.[ISI][Medline]
Merrill, J. E., and Jonakait, G. M. (1995). Interactions of the nervous and immune systems in development, normal brain homeostasis, and disease. FASEB J. 9, 611618.
Moszczynski, P. (1997). Mercury compounds and the immune system: A review. Int. J. Occup. Med. Environ. Health. 10, 247258.[Medline]
Munoz-Fernandez, M. A., Cano, E., O'Donnell, C. A., Doyle, J., Liew, F. Y., and Fresno, M. (1994). Tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, and interleukin-6 but not TNF-beta induce differentiation of neuroblastoma cells: The role of nitric oxide. J. Neurochem. 62, 13301336.[ISI][Medline]
NRC (2000). Toxicological Effects of Methylmercury. National Academy Press, Washington, DC.
Otten, U., Scully, J. L., Ehrhard, P. B., and Gadient, R. A. (1994). Neurotrophins: Signals between the nervous and immune systems. Prog. Brain Res. 103, 293305.[ISI][Medline]
Regan, C. M. (1991). Regulation of neural cell adhesion molecule sialylation state. Int. J. Biochem. 23, 513523.[ISI][Medline]
Regan, C. M. (1993). Neural cell adhesion molecules, neuronal development and lead toxicity. Neurotoxicology 14, 6974.[ISI][Medline]
Regan, C. M., and Fox, G. B. (1995). Polysialylation as a regulator of neural plasticity in rodent learning and aging. Neurochem Res. 20, 593598.[ISI][Medline]
Reuhl, K. R., and Chang, L. W. (1979). Effects of methylmercury on the development of the nervous system: A review. Neurotoxicology 1, 2155.[ISI]
Reuhl, K. R., Chang, L. W., and Townsend, J. W. (1981a). Pathological effects of in utero methylmercury exposure on the cerebellum of the golden hamster: I. Early effects upon the neonatal cerebellar cortex. Environ. Res. 26, 281306.[ISI][Medline]
Reuhl, K. R., Chang, L. W., and Townsend, J. W. (1981b). Pathological effects of in utero methylmercury exposure on the cerebellum of the golden hamster: II. Residual effects on the adult cerebellum. Environ. Res. 26, 307327.[ISI][Medline]
Reuhl, K. R., and Pounds, J. G. (1981). Absorption and disposition of [203]Hg in the pregnant and nonpregnant hamster following oral administration of [203]Hg methylmercuric chloride. Environ. Res. 24, 131139.[ISI][Medline]
Sager, P. M. (1988). Selectivity of methyl mercury effects on cytoskeleton and mitotic progression in cultured cells. Toxicol. Appl. Pharmacol. 94, 473486.[ISI][Medline]
Sager, P. R., Aschner, M., and Rodier, P. M. (1984). Persistent, differential alterations in developing cerebellar cortex of male and female mice after methylmercury exposure. Brain Res. 314, 111.[Medline]
Sager, P. R., Doherty, R. A., and Olmsted, J. R. (1983). Interaction of methylmercury with microtubules in cultured cells and in vitro. Exp. Cell Res. 146, 127137.[ISI][Medline]
Sager, P. R., Doherty, R. A., and Rodier, P. M. (1982). Effects of methylmercury on developing mouse cerebellar cortex. Exp. Neurol 77, 179193.[ISI][Medline]
Satoh, J., Kastrukoff, L. F., and Kim, S. U. (1991). Cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1) in cultured human oligodendrocytes and astrocytes. J. Neuropathol. Exp. Neurol. 50, 215226.[ISI][Medline]
Satoh, T., Nakamura, S., Taga, T., Matsuda, T., Hirano, T., Kishimoto, T., and Kaziro, Y. (1988). Induction of neuronal differentiation in PC12 cells by B-cell stimulatory factor 2/interleukin 6. Mol. Cell. Biol. 8, 35463549.[ISI][Medline]
Schousboe, A., Meier, E., Drejer, J., and Hertz, L. (1989). Preparation of Primary Cultures of Mouse (Rat) Cerebellar Granule Cells. Alan R. Liss, New York.
Silbergeld, E. K., Sacci, J. B. J., and Azad, A. F. (2000). Mercury exposure and murine response to Plasmodium yoelli infection and immunization. Immunopharmacol. Immunotoxicol 22, 685696.[ISI][Medline]
Takeuchi, T. (1977). Pathology of fetal Minamata disease: The effects of methylmercury on human interuterine life. Pediatrician 6, 6987.
Tanaka, S., Sekino, Y., and Shirao, T. (2000). The effects of neurotrophin-3 and brain-derived neurotrophic factor on cerebellar granule cell movement and neurite extension in vitro. Neuroscience 97, 727734.[ISI][Medline]
Tessier-Lavigne, M., Placzek, M., Lumsden, A. G., Dodd, J., and Jessell, T. M. (1988). Chemotropic guidance of developing axons in the mammalian central nervous system. Nature 336, 775778.[ISI][Medline]
Zhao, B., and Schwartz, J. P. (1998). Involvement of cytokines in normal CNS development and neurological diseases: Recent progress and perspectives. J. Neurosci. Res. 52, 716.[ISI][Medline]