Continuous exposure to low concentrations of methylmercury impairs cerebellar granule cell migration in organotypic slice culture

doi:10.1016/j.neuro.2008.12.010

NeuroToxicology

Volume 30, Issue 2, March 2009, Pages 203-208

https://doi.org/10.1016/j.neuro.2008.12.010 Get rights and content

Abstract

Chronic, low-level perinatal exposure to methylmercury (MeHg) is associated with neurological and motor deficits that appear to result from cerebellar dysfunction. Neuropathological studies suggest that these deficits are due to impaired cerebellar granule cell (CGC) migration. Although neuronal migration in vivo and in vitro has been shown to be impaired during acute and/or high level exposure to MeHg, the cellular effects of chronic exposure to submicromolar and micromolar levels of MeHg during development are not clear. The majority of CGC migration in rats occurs between postnatal days 8 and 14 (P8 and 14); migration peaks on P10 and 11. Organotypic cultures of parasagittal slices of cerebellum from P8 rats were exposed to low levels of MeHg (0.2–5.0 μM) for 3 or 7 days, and CGC viability and migration were assessed. MeHg-induced cell death was time- and concentration-dependent. After 3 days of exposure CGC viability decreased in 3 μM MeHg and declined to 42.7% in 5 μM MeHg. Cultures treated with MeHg for 7 days showed decreased CGC viability in 1 μM MeHg, which declined to 62.8% in 3 μM MeHg. CGC migration was assessed by BrdU pulse-chase labeling. Migration into the internal granule cell layer (IGL) was impaired in cultures exposed to ≥1 μM MeHg for 3 days or ≥0.5 μM for 7 days. CGCs failed to initiate migration from the external germinal cell layer at the same level of exposure. For those cells which initiated migration, MeHg reduced the number that migrated into the IGL. This implied a slowing of migration once it had begun. These effects occurred with no overall change in cerebellar cortical structure, or loss of granule cell viability. Thus, chronic exposure to low micromolar concentrations of MeHg impairs development of the cerebellar cortex in a slice culture model.

Introduction

Immature cerebellar granule cells (CGCs) migrate from the external germinal cell layer (EGL) through the molecular layer (ML) to form the internal granule cell layer (IGL) postnatally. The specific mechanisms orchestrating CGC migration are not yet clearly understood. CGCs that do not migrate to their appropriate destination typically fail to mature properly. Deficits in neuronal migration are involved in numerous pathological conditions as indicated by heterotrophic, or misplaced, neurons in the cerebellum; examples include Zelweger's Syndrome, Cornelia de Lange Syndrome, autosomal recessive cerebellar hypoplasia, paroxysmal ataxia, and Fryns Syndrome. Clinical manifestations of malformed cerebellar cortex include deficits in psychomotor development, ataxia, and epilepsy (Gressens, 2000).

The cerebellum is particularly sensitive to damage by methylmercury (MeHg). Gross/overt clinical manifestations of this toxicity include cerebellar-based ataxia (Berlin, 1976, Chang et al., 1977, Takeuchi, 1982). The effect in exposed children appears to be directly due to CGC loss, even though higher concentrations of MeHg are found in the Purkinje cells (Hunter and Russell, 1954). MeHg causes a characteristic lack of CGC migration and subsequently, laminar cortical organization in the developing cerebellum in vivo (Rustam and Hamdi, 1974, Reuhl and Chang, 1979, Choi, 1989).

Significant impairment of CGC migration by MeHg has been shown in vitro (Sass et al., 2001) and in organotypic slice culture (Kunimoto and Suzuki, 1997) under acute and/or high level, but not chronic low-level conditions of exposure to MeHg. Sass et al. (2001) described a decrease in migration of murine neonatal CGC in primary culture following acute application of 0.1 μM MeHg. However, an ideal migration culture system would maintain in vivo-like cortical structure allowing for interplay between the CGCs and the Bergmann glia on which they migrate. One such model system is organotypic slice culture. Kunimoto and Suzuki (1997) show that treating organotypic cultures of postnatal rat cerebellar slices for 3 days with 3 μM MeHg selectively impaired CGC migration by approximately 20% without impairing or killing surrounding neurons. Only 70.3% of cells had migrated by 4 days in vitro (DIV) in the control samples. However, CGC migration in rat occurs over more than 7 days (Altman, 1972). If organotypic cultures are not treated with MeHg for the entire duration of the developmental window, affected CGCs may recover and migrate, or more CGCs may be generated and migrate to the IGL. Therefore, MeHg treatment over the entire migratory period may cause more pronounced deficits than those caused by less chronic treatment paradigms. Experimental models using chronic, low-level MeHg treatment would also better mimic the current patterns of MeHg intoxication.

The purpose of this study was to examine the effects of chronic submicromolar levels of MeHg on immature CGC viability and migration. Whereas a number of studies have examined possible mechanisms of MeHg neurotoxicity within individual neurons in primary culture, cell migration is guided and influenced by surrounding cells. Organotypic slice cultures provide cortical structure and cell–cell interactions that are similar to or the same as those in vivo. This makes this an ideal model system for studies of migration, and especially the role of ligand-gated receptor responses and Ca²⁺ regulation during migration.

An organotypic slice culture system of P8–9 rat pups cerebellar vermis was used. CGC mitosis is prevalent in P8–9 pups, and CGC migration peaks at P10–11 (Altman, 1972, Kunimoto and Suzuki, 1997). Four clearly recognizable layers (EGL, molecular layer, Purkinje cell layer (PCL), and IGL) can be seen at P9–12 (Komuro et al., 2001, Davids et al., 2002). At that time, CGCs are in all stages of development. Purkinje cells are aligned in a monolayer with apical cones, primary dendrites, and a few short secondary branches. In as much as MeHg can cause granule cell death, as well as impaired migration, we examined cytotoxicity initially, to ascertain MeHg concentrations for which cell death would occur, and hence obscure determinations of impaired migration.

Section snippets

Organotypic slice culture

All experiments were conducted in accordance with NIH guidelines on use of experimental animals and were approved by the Michigan State University Institutional Animal Use and Care Committee. The organotypic culture system was modified from that of previous reports (Komuro and Rakic, 1993, Haydar et al., 1999). Sagittal slices of cerebellum were prepared from P8 to 9 Sprague-Dawley rat pups. Slices were cut approximately 400 μm thick, and maintained viability for over 8 days. Sagittal slices

Viability

Cell viability following 3 and 7 days of MeHg treatment (4 and DIV8, respectively) is shown in Fig. 1. There were no significant changes in viability in slice cultures (p > 0.01) over time in the absence of MeHg, however, exposure to MeHg induced cytotoxicity in both a concentration- and time-dependent manner. After 3 days at 5 μM, MeHg viability was reduced significantly and at 10 μM MeHg, virtually all cells were dead. Increasing exposure length resulted in cytotoxicity at lower concentrations of

Discussion

Results of the present study are consistent with the following conclusions: (1) at micromolar concentrations MeHg is cytotoxic to organotypic cerebellar cultures. This effect occurs in both a time- and concentration-dependent manner. (2) Lower concentrations of MeHg impair the normal migration of CGCs from the EGL and again do so in a concentration-dependent manner. Over longer periods of exposure, some migration occurs in the MeHg-treated cultures, but it is less than in MeHg-free cultures,

Conflict of interest

None declared.

Acknowledgments

This work was supported in part by funds from the Center for Alternatives to Animal Testing, Johns Hopkins University and intramural support from the Michigan State University College of Osteopathic Medicine. The Leica confocal microscope was purchased off a grant from the State of Michigan Life Sciences Corridor Initiative of the Michigan Economic Development Corp. The word processing and graphical assistance of Erin E. Koglin, Julie VanRaemdonck and Tara S. Oeschger, respectively is

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Continuous exposure to low concentrations of methylmercury impairs cerebellar granule cell migration in organotypic slice culture

Abstract

Introduction

Section snippets

Organotypic slice culture

Viability

Discussion

Conflict of interest

Acknowledgments

Prog Neurobiol

Eur Neuropsychopharmacol

Brain Res Brain Res Protoc

Cell Calcium

Neurosci Lett

Neurotoxicology