Methylmercury-Induced Decrement in Neuronal Migration May Involve Cytokine-Dependent Mechanisms: A Novel Method to Assess Neuronal Movement in Vitro

J. B. Sass*,1,2, D. T. Haselow{dagger} and E. K. Silbergeld*

* Program in Human Health and the Environment and {dagger} Department of Epidemiology and Preventive Medicine, University of Maryland, Baltimore, Maryland 21201

Received December 1, 2000; accepted May 3, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major toxic effect associated with methylmercury (MeHg) exposure in developing humans is damage to the nervous system, which involves inhibition of cell migration, particularly in the cerebellum. The mechanisms by which MeHg impairs neural migration are not fully known, especially at low doses. In this paper we report on a novel method for observing and quantitating the movement of individual cells in primary cultures of murine neonatal cerebellar cells, which offers an opportunity to assess the role of endogenous and exogenous factors on neural migration. We have used this system to test the hypothesis that treatment with methylmercury would inhibit movement of granule cell neurons, possibly via a cytokine-mediated mechanism. We demonstrate that LPS (50 ng/ml) increases movement of neurons, concomitant with increased levels of TNF-{alpha} and IL-6 secreted protein, and IL-1{alpha} mRNA. Treatment with LPS did not increase the number of neurons that moved, but, of the cells that did move, exposure to LPS significantly increased the total distances moved. Treatment with methylmercury (0.1 µM) decreased the number of moving cells and inhibited overall distance traveled by granule cells.

Key Words: methylmercury; neurodevelopment; cytokine; migration; cerebellum; cell signaling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neurotoxic effects of exposure to methylmercury (MeHg) in children are well documented and include both neurocognitive and neuromotor effects (Dolbec et al.2000Go; Grandjean et al.1995Go, 1997Go; NRC, 2000Go; Reuhl and Chang, 1979Go). Since MeHg readily crosses the placental barrier, its potential developmental toxicity is of extreme concern. The effects of in utero or perinatal exposures are particularly damaging, and result in morphological and behavioral disturbances; the extent of damage is dependent on dose, route of exposure, duration, and developmental timing. Consequences of exposure include alterations in neuronal and glial proliferation, neuronal migration, and final cytoarchitecture of affected brain regions (Beiswanger et al.1995Go; Sager et al.1984Go; Brown et al.1988Go; Charleston et al.1996Go; Choi et al.1978Go). The neuropathology following high level in utero exposure of humans and animals to MeHg is proposed to partly result from inhibition of neuronal migration (Burbacher et al.1990Go; Choi, 1986Go; Choi, et al.1978Go; Takeuchi, 1977Go). In particular, the cytoarchitecture and functioning of the cerebellar cortex is prominently affected (Chang and Hartmann, 1972Go; Choi et al.1978Go; Matsumoto et al.1965Go; Reuhl et al.1981bGo; Reuhl and Pounds, 1981Go). When hamster embryos were treated in utero with high levels of methylmercury (daily doses of 2 mg/kg on gestational days (GD) 10–15, or a single dose of 10 mg/kg on GD 10), they had an abnormally thickened external granule cell layer in the cerebellum, which suggested an inhibition of the migration of these neurons to deeper layers (Reuhl et al., 1981aGo) as a result of the MeHg treatment. There have been several studies demonstrating that low doses of mercury inhibit neuronal movement, particularly in the developing cerebellum of primates (Burbacher et al.1990Go) or rodents exposed in utero (Reuhl et al.1981aGo, bGo), as well as in cultured neuronal cells treated in vitro (Jacobs et al.1986Go; Kunimoto and Suzuki, 1997Go). This effect on cerebellar granule cell movement seen at low dose, in contrast with high dose exposures, such as the severe mercury toxicity disaster in Japan (Minamata disease), in which postmortem examination revealed atrophy of the cerebellar hemispheres, with a thinning of the granular neuron layer and widespread granule cell loss (Reuhl and Chang, 1979Go). Decreased mitotic activity of granular neurons, and thinning of the granule cell layer was also seen in newborn mice treated with large doses of methylmercury (4–8 mg Hg/kg body weight; Sager et al.1982).

In vitro, nanomolar and micromolar levels of methylmercury (0.5–10 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.1986Go), or migration within organotypic cultures (Kunimoto and Suzuki, 1997Go).

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 cell–cell recognition and adhesion (Regan, 1991Go, 1993Go; Regan and Fox, 1995Go). 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 3–13 has been shown to alter the sialylated state of NCAM within the neuronal cell layers of the developing cerebellar cortex (Dey et al.1999Go).

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.1989Go). IL-6 has also been shown to induce neurite extension in a neuronal cell line (Satoh et al.1988Go) and has been proposed to function as a developmental neurotrophic factor (Gadient and Otten, 1994aGo, bGo; Otten et al.1994Go). Two other cytokines, interleukin-1ß (IL-1ß) and tumor necrosis factor-{alpha} (TNF-{alpha}) 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.1988Go; Merrill, 1992Go; Munoz-Fernandez et al.1994Go). In tissue culture systems, glial-derived IL1-ß, TNF-{alpha}, and interferon-{gamma} (IFN-{gamma}) are all capable of inducing expression of cell surface adhesion molecules in glial cells of both rodent and human origin (Lee and Benveniste, 1999Go; Merrill and Jonakait, 1995Go; Satoh et al.1991Go; Zhao and Schwartz, 1998Go).

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.1999Go; El-Fawal et al.1999Go; Hultman and Hansson-Georgiadis, 1999Go; Kono et al.1998Go; Mehler and Kessler, 1997Go; Moszczynski, 1997Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Primary cultures of mouse cerebellum.
Cerebellar cultures were established from newborn mouse pups, to include both neuronal and glial elements, according to established methods (Jacobs et al.1986Go; Schousboe et al.1989Go), with modifications as described here. Cells were isolated from neonatal CD-1 mouse pups (Harlan Sprague-Dawley) at GD 25, dated from date of plug, a time when cerebellar granule cell neurons are still within the proliferative period of neurogenesis, just prior to the first wave of neural migration (Mares et al.1970Go; Schousboe et al.1989Go).

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. Eight–9 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 (10–15 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., 2000Go). It is within the range utilized by Sager (1988) and others (Graff et al., 1997Go; Kunimoto and Suzuki 1997Go) 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, 1998Go; Cui and Bulleit, 1998Go; Hertz et al.1989Go). By 48 h in culture, the granule cells were distributed evenly. On day 4, cultures were dense enough to support cell–cell contacts, and migration, but individual cells were still distinguishable. By 5–6 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.1970Go).

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. 1Go). 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. 2Go). 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.



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FIG. 1. Murine cerebellar cultures at 6 days in vitro. Cells are plated on a CELLocate grid coverslip. The distance between grid lines is 55 µm. A depicts control (untreated) cultures at 6 days in vitro. B depicts sister cultures exposed to 50 ng/ml LPS for 2 days. C depicts cultures exposed to 0.1 µM MeHgCl for 2 days. D depicts cultures treated with both 50 ng/ml LPS and 0.1 µM MeHgCl for 2 days. All panels depict sister cultures taken from the same experiment and photographed at the same time; bar = 55 µm.

 


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FIG. 2. Frequency distributions of movement of granule cell neurons. Frequency distributions are shown of movement of granule cell neurons in culture over a 10-h observation period, beginning at 48 h after treatment. Histogram shows the distance moved by neurons in untreated cultures and cultures treated with 50 ng/ml LPS, 0.1 µM MeHgCl, or both LPS and MeHgCl.

 
Statistical analysis.
Neuronal movement was analyzed by study group and experiment (i.e., control group and each of the 3 treatment groups), stratified by the date of the experiment, and described using simple statistics. Means and standard deviations were calculated for each group with and without nonmoving cells. For each group, a frequency distribution was created to visually depict the differences in patterns of movement between the study groups. Statistical tests were performed to compare (1) each treatment group versus the control group, and (2) each treatment group versus each other treatment group. For all group versus group comparisons, differences in the proportion of neurons that moved in each group were examined with a Chi-square test. To examine whether mean movements differed by group, an analysis of variance was performed with and without Duncan's Multiple Range test (which accounts for the potential problem of finding significant results by chance alone when performing multiple comparisons). This multiple comparison method was chosen, rather than one of those designed by Bonferroni, Tukey or Dunnett, because it is not overly conservative, allows for different numbers and different variances in each of the groups being compared, and accounts for treatment group versus treatment group comparisons, respectively. Because the data were significantly skewed, movement between groups was also compared using a nonparametric Wilcoxon rank sum test with a normal approximation. All tests were two-sided. Results were considered significantly different if the calculated p value was less than or equal to 0.05. All analyses were performed in SAS version 8.01 (SAS institute Inc., Cary, NC).

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.1998Go), 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-{alpha}, a 446-bp product (Bohn et al.1994Go). 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.1995Go; Lee et al.1993Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuronal Movement in Control Cultures
Under the culture conditions described here, glia proliferated and neurons extended processes by day 4 in culture, the time selected for treatment and subsequent observation, These conditions elicited maximal neuronal movement. The results are relatively consistent across 4 separate experiments, as shown in Table 1Go. However, within a culture there was considerable variability in the distance moved by individual neurons. As shown in the frequency distribution in Figure 2Go, in all cultures, a number of cells did not move at all over the 10-h observation period. In controls, 70% of the observed neurons moved during 10 h (Table 1Go). Overall mean movement, including nonmoving neurons, was 8.9 µm. Of only neurons that moved at all, mean movement was 12.8 µm.


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TABLE 1 Summary of Descriptive Statistics of Neuron Movement Compared across All Four Experiments
 
Effects of LPS
LPS did not increase the proportion of neurons that moved (Table 1Go). However, of those neurons that moved at all, LPS-treated neurons moved significantly farther than those in the control group (Table 2Go). Analysis by Wilcoxon rank-sum test showed no significant differences between the distribution of cell movement in controls and LPS treated cultures (p = 0.17; Table 2Go), when both moving and nonmoving neurons are included in the analysis.


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TABLE 2 Summary of Neuron Movement Compared across All Four Experiments: Control Group vs. Treatment Groups
 
Effects of Methylmercury
Methylmercury (0.1 µM) significantly decreased the proportion of neurons that moved. In MeHg cultures, only 58% of observed neurons moved at all, as compared to 70% of control neurons (Table 1Go). Of the cells that moved, MeHg-exposed neurons moved a mean distance of 9.3 µm, not significantly different from untreated controls (Table 1Go). Analysis by a Wilcoxon rank sum test demonstrated that as a group, neurons exposed to MeHg moved significantly less than controls (p = 0.001) (Table 2Go), when all neurons are included in the analysis.

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, 2GoGo), and those treated with MeHg alone (Table 3Go). 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, 3GoGo). 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 3Go) or control neurons (Tables 2 and 3GoGo).


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TABLE 3 Summary of Neuron Movement Compared across All Four Experiments: Treatment Groups vs. Each Other
 
Effects of LPS and MeHg on Resting and Stimulated Release of IL-6, IL-1ß, and TNF-{alpha}
In the absence of LPS, both control and MeHg cultures produced very low amounts of IL-1ß, IL-6 and TNF-{alpha} (Table 4Go, averaged data from 2 experiments). Levels of IL-6 and TNF-{alpha} rose dramatically in response to LPS,. This was consistent with RT-PCR analysis, which demonstrated that IL-6 and TNF-{alpha} transcripts were detectable only in cell cultures stimulated with LPS (Fig. 3Go). While LPS had no effect on secreted protein levels of IL-1ß by ELISA at 18 or 30 h after stimulation, RT-PCR demonstrates that IL-1ß transcripts were elevated in response to LPS (Fig. 3Go). Treatment with MeHg did not affect LPS-stimulated cytokine production or release. (Fig. 3Go).


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TABLE 4 Levels of IL-6 and TNF-{alpha}, Determined by ELISA, from the Supernatants of Cerebellar Cultures Collected at 18 h after Treatment
 


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FIG. 3. mRNA expression of TNF-{alpha}, IL-6, and IL-1ß. Transcript isolated from dissociated cell cultures derived from the spleen and cerebellum of CD-1 mice. Cell cultures were unstimulated, or stimulated with either Con A (spleen) or LPS (cerebellum). All RT-PCR products were generated under identical reaction conditions, and compared with ß-actin. Lanes 1, 5, 10, and 14 depict reactions that used cDNA from ConA-stimulated spleen cultures as template. Lanes 2, 6, 11, and 15 depict reactions that used cDNA from unstimulated cerebellar cultures as template. Lanes 3, 7, 12, and 16 depict reactions that used cDNA from LPS-treated cerebellar cultures as template. Lanes 4, 8, 13, and 17 depict reactions using cDNA isolated from cerebellar cultures treated with LPS and methylmercury as template. Lanes 1–4 depict ß-actin (348 bp). Lanes 5–8 depict TNF-{alpha} (446 bp), Lanes 10–13 depict IL-6 (600 bp). Lanes 14–17 depict IL-1ß (515 bp). Lanes 9 and 18 show a 123-bp molecular weight marker, with each fragment being 123 bp different in length from the adjacent fragment (i.e., 123 bp, 246 bp, 369 bp, etc.).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Method
We have demonstrated that neuronal migration can be studied in vitro at the individual cell level, in contrast to methods which assess movement by the reaggregation of dissociated cells (Jacobs et al.1986Go; Tanaka et al.2000Go) or the ability of cells to move through a collagen gel (Goldberg et al.1992Go; Maggi et al.2000Go; Tessier-Lavigne et al.1988Go). The methods described here allow the quantitative assessment of velocity and direction of cell movement, in addition to qualitative assessments of individual cell states. Agents can be added directly to the cell culture system, as demonstrated in this paper, or animals can be treated in utero and effects can be assessed ex vivo.

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-{alpha}. 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.1993Go).

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. 2Go). 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. 2Go). 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.1986Go; Kunimoto and Suzuki, 1997Go). 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-{alpha} 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-{alpha}. 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, 1998Go). 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{gamma} production (Silbergeld et al.2000Go).). 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, 1988Go). Disruption of microtubules has been reported in cultured cells (Sager et al., 1983Go). 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, 1998Go). 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 1–2 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
 
The authors wish to acknowledge the generous help of J. L. Powell for reagents used in RT-PCR, B. K. Krueger and L. L. Bambrick for help with culture techniques and analysis; and L. A. Vergato for performing ELISAs. Drs. J. L. Powell and B. K. Krueger made many helpful comments throughout these studies. Research in this publication was supported by Grant Number 5T32ES07263 from the National Institute of Environmental Health Sciences (NIEHS) and by a University of Maryland School of Medicine Bressler Research Fund Award.


    NOTES
 
1 To whom correspondence should be addressed at Program in Human Health and the Environment, University of Maryland, Baltimore, 10 S. Pine St., 9-34 MSTF, Baltimore, MD 21201. Fax: (410) 706-0727. E-mail: esilbergeld{at}epi.umaryland.edu. Back

2 Present address: Natural Resource Defense Council, 1200 New York Ave. NW, Washington, DC 20005. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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