A novel system applying the 2-deoxyglucose method to fish for characterization of environmental neurotoxins.

Methods of identifying and preventing ecotoxicity related to environmental stressors on wildlife species are underdeveloped. To detect sublethal effects, we have devised a neurochemical method of evaluating environmental neurotoxins by a measuring changes in regional neural activity in the central nervous system of fish. Our system is a unique adaptation of the 2-deoxyglucose (2-DG) method originally developed by L. Sokoloff in 1977, which is based on the direct relationship between glucose metabolism and neural functioning at the regional level. We applied these concepts to test the assumption that changes in neural activity as a result of chemical exposure would produce measurable effects on the amount of [(14)C]2-DG accumulated regionally in the brain of Tilapia nilatica. For purposes of this study, we utilized the excitotoxin N-methyl-D-aspartate (NMDA) to characterize the response of the central nervous system. Regional accumulation of [(14)C]2-DG was visualized by autoradiography and digital image processing. Observable increases in regional [(14) C] 2-DG uptake were evident in all NMDA-treated groups as compared to controls. Specific areas of increased [(14)C] 2-DG uptake included the telencephalon, optic tectum, and regions of the cerebellum, all areas in which high concentrations of NMDA-subtype glutamate receptors have been found in Tilapia monsanbica. These results are consistent with the known neural excitatory action of NMDA.

Methylmercury-induced decrement in neuronal migration may involve cytokine-dependent mechanisms: a novel method to assess neuronal movement in vitro.

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-1alpha 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 microM) decreased the number of moving cells and inhibited overall distance traveled by granule cells.

Restricted expression of the zebrafish hsp90alpha gene in slow and fast muscle fiber lineages.

Members of the heat shock protein 90 (Hsp90) family of molecular chaperones play important roles in allowing a select group of intracellular signaling molecules reach and maintain functionally active conformations. We have previously shown that hsp90alpha gene expression in early zebrafish embryos is restricted to a subgroup of paraxial-mesoderm derived somitic cells prior to muscle formation and that the gene is downregulated in mature trunk and tail muscle fibers. Here we have compared the expression of the hsp90alpha gene to muscle regulatory genes during development of slow and fast muscle fibers in normal embryos and in embryos carrying mutations which affect somitic muscle formation. We show that hsp90alpha is first expressed early during the development of slow somitic muscle progenitors shortly following myoD activation and at a point prior to or co-incident with the expression of other known muscle regulatory genes. Expression of hsp90alpha is also activated in the midline of flh mutants when these cells switch from a notochord to a muscle fate. Conversely, expression is not detectable in cells of the paraxial mesoderm lineage which fail to converge in spt mutants and which do not activate expression of other muscle specific marker genes. Finally, expression of hsp90alpha is downregulated in slow muscle fibers by 24 h of age but becomes detectable in the later developing fast fibers at this time. Thus, hsp90alpha is expressed in developing muscle progenitors during short temporal and spatial windows of both slow and fast fiber lineages in the zebrafish somite.

HSP90alpha gene expression may be a conserved feature of vertebrate somitogenesis.

We have previously demonstrated that the hsp90alpha and hsp90beta genes in zebrafish are expressed in dramatically different spatial and temporal patterns in early embryos. In the case of hsp90alpha, expression is spatially restricted within the somites to putative myogenic cells which also express mRNA encoding the myogenic bHLH transcription factor myoD and is downregulated along with myoD following myogenesis. In the present study, we have examined hsp90alpha gene expression in developing chicken embryos using a gene-specific probe. We show that hsp90alpha gene expression is also localized to a subset of cells within the somites of chicken embryos and that the expression pattern correlates closely to that observed for myoD. Furthermore, expression of the hsp90alpha gene is strongly upregulated throughout the embryo following heat shock in a manner similar to that observed in heat-shocked zebrafish embryos. The data suggest that the hsp90alpha gene may play an evolutionarily conserved role during somitogenesis in vertebrates in addition to providing protection to all cells of the embryo following stress.

Heat shock protein gene expression during embryonic development of the zebrafish.

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development of a wide range of organisms. Our laboratory has been involved in an analysis of heat shock gene expression in the zebrafish, a model system which is now utilized extensively for the examination of early embryonic development of vertebrates. Members of the zebrafish hsp47, hsp70 and hsp90 gene families have been cloned and shown to be closely related to their counterparts in higher vertebrates. Expression of these genes has been examined using Northern blot and whole mount in situ hybridization analyses. Both the hsp47 and hsp90 genes are expressed in a highly tissue-restricted manner during normal development. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes during early zebrafish development.

Heat shock genes and the heat shock response in zebrafish embryos.

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development in a wide range of organisms. Our laboratory has initiated an analysis of heat shock protein gene expression in the zebrafish, a model system that is now utilized extensively for the examination of early embryonic development of vertebrates. We have cloned members of the zebrafish hsp47, hsp70, and hsp90 gene families and shown them to be closely related to their counterparts in higher vertebrates. Whole mount in situ hybridization and Northern blot analyses have revealed that these genes are regulated in distinct spatial, temporal, and stress-specific manners. Furthermore, the tissue-specific expression patterns of the hsp47 and hsp90 alpha genes correlate closely with the expression of genes encoding known chaperone targets of Hsp47 and Hsp90 in other systems. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes in zebrafish embryos during normal development and following exposure to environmental stress.

Specific localization of zebrafish hsp90 alpha mRNA to myoD-expressing cells suggests a role for hsp90 alpha during normal muscle development.

Members of the eukaryotic hsp90 family function as important molecular chaperones in the assembly, folding and activation of a select group of cellular signalling molecules and transcription factors. Several of the molecules with which hsp90 interacts, such as the bHLH transcription factor myoD, are known to be important regulators of developmental events in vertebrates. However, little information is available in support of any specific role for hsp90 in developing embryos in vivo. In this study, we provide the first in vivo evidence that the hsp90 alpha gene may play a role in the process of myogenesis. We show that constitutive hsp90 alpha mRNA in zebrafish embryos is restricted primarily to a subset of cells within the somites and pectoral fin buds which also express myoD. Furthermore, expression of the hsp90 alpha gene is down-regulated along with myoD in differentiated muscles of the trunk at a time when levels of mRNA encoding the muscle structural protein alpha-tropomyosin remain high. No hsp90 alpha mRNA is detectable within the CNS at control temperatures. In contrast, heat shock-induced expression of the hsp90 alpha gene occurs throughout the embryo at all stages of development examined. The expression patterns strongly suggest that the hsp90 alpha gene plays a specific role in the normal process of myogenesis in addition to providing protection to all cells of the embryo during periods of environmental stress.

HSP 90 alpha and HSP 90 beta genes are present in the zebrafish and are differentially regulated in developing embryos.

We have employed a polymerase chain reaction-based cloning strategy to demonstrate that both hsp 90 alpha and hsp 90 beta genes are present in the zebrafish. The fact that zebrafish represents the most primitive vertebrate in which hsp 90 genes have been isolated to date has allowed us to determine that the duplication event which generated the hsp 90 alpha and hsp 90 beta genes occurred shortly before the emergence of the teleosts from the rest of the vertebrate lineage. In expression studies using Northern blot analysis, hsp 90 beta mRNA was found to be present at control temperatures throughout normal embryonic development whereas hsp 90 alpha mRNA was barely detectable. Upon heat shock, hsp 90 alpha mRNA levels increased dramatically in all developmental stages examined. The levels of hsp 90 beta mRNA increased 2-3 fold during heat shock of early stage embryos. Thus, the hsp 90 alpha gene is strongly upregulated during heat shock in zebrafish embryos whereas expression of the hsp 90 beta gene appears to be weakly induced.

Aluminum pretreatment impairs the ability of astrocytes to protect neurons from glutamate mediated toxicity.

A number of laboratories have shown that astrocytes protect neurons from glutamate excitotoxicity. The experiments described in this paper were designed to address the question whether prior exposure of astrocytes to aluminum (in the form of aluminum citrate) interfered with the ability of astrocytes to protect neurons from glutamate excitotoxicity. Our culture paradigm consisted of highly enriched cultures of neurons and astrocytes grown on separate coverslips; this design enables one to subject either the neurons or the astrocytes to specific treatments and recombine the cells into the same petri dish simply by moving coverslips from dish to dish. We have confirmed findings of other laboratories that astrocytes could protect neurons from glutamate-induced death when glutamate (100 microM) is added to the culture medium. We have also demonstrated that prior treatment of astrocytes with 100 microM aluminum citrate impairs this ability of astrocytes to promote neuronal survival. No differences, however, were observed in the ability of control and aluminum-treated astrocytes to take up glutamate. These findings suggest that aluminum may cause astrocytes to: (i) secrete a factor that makes neurons more susceptible to glutamate-induced toxicity; (ii) secrete a neuronotoxic factor in the presence of glutamate; or (iii) reduce secretion of a factor that protects neurons from glutamate excitotoxicity.

A simple, yet versatile, co-culture method for examining neuron-glia interactions.

There is increasing interest in examining neuron-glial interactions in the developing and the mature central nervous system. Here, we describe a system that allows examination of interactions mediated by diffusible molecules even when such interactions involve more than 2 cell populations. Our procedure involves culturing highly enriched populations of neurons and glial cells on separate coverslips. Such coverslips can be readily moved from one petri dish to another, thus enabling the investigator to subject the neurons and the glial cells to different experimental manipulations. The coverslips can then be placed in any desired combination within a petri dish. This gives one great flexibility in examining neuron-glial interactions. Using this approach, we unequivocally demonstrate that astrocytes protect neurons from glutamate excitotoxicity.