Effects of Trace Metal Profiles Characteristic for Autism on Synapses in Cultured Neurons

Figure 1

Toxicity profiles of trace elements in primary hippocampal neuronal cell culture. ((a), (b)) The amount of cell death was calculated using various concentrations for each trace metal by assessing the number of neuronal apoptotic nuclei (identified by MAP2 and DAPI staining) per optic field (from 5 fields of view and cultures) normalized against the total number of neurons per optic field. The results show no changes in cell health over a wide spectrum of concentrations for Al, Fe, and Pb, and a linear correlation between metal level and cell death for Cd, Cu, and Zn. A linear correlation might also exist for Hg and Se although an exponential increase in cell death beyond a certain concentration is more likely. Addition of Mg increased cell survival in a low concentration range and with higher concentrations leading to cell death (a). (b) Using -tests, values were calculated to evaluate, whether changes between different concentrations compared to untreated control cells are significant. (c) As an alternative read-out, the number of primary, secondary, and tertiary dendrites was investigated. Cells were stained with MAP2 antibody. As signs of cell death, neurons show fragmentation (pinching off) dendrites, starting with branches more distal from the soma. Dendrites showing signs of fragmentation were not counted. High concentrations of metals that were shown to lead to cell death ((a), (b)) lead to a reduction in dendritic branching starting from tertiary dendrites and also affecting secondary and primary in the most toxic cases. Although, for some metal, no significant cell death in a certain concentration was seen, and already a fragmentation of dendrites is visible (e.g., 5 μM Hg). Mg and Zn show a significant increase in dendritic branching restricted to tertiary dendrites in a lower concentration range. (d) To assess whether some trace elements display a synaptogenic effect, synapses were labeled using Bassoon fluorescence and the number of Bassoon positive puncta was measured per 10 μm dendrite length on primary dendrites (3 dendrites per cell, 10 cells in total per metal and concentration). While synapse number is decreased correlating with toxic effects for most metal ions, such as Cd, Cu, and Se, an increase in synapse number can be seen for Fe and lower concentrations of Mg and Zn (seen as a trend). ((a)–(d)) All cells were treated with metals between DIV 10 and DIV 14 before analysis.