Synaptic Structure Quantification in Cultured Neurons

Abstract Behavioral problems (e.g., learning and memory) following developmental exposure to toxicants suggests that dysregulation of the process of synapse formation and function may occur. The ability to assess these changes is thus of value. This unit describes a method to investigate toxicant‐induced changes to synaptic structure formation in primary hippocampal neurons using immunocytochemical labeling of the pre‐ and post‐synaptic markers synaptophysin and PSD‐95, confocal imaging, and three‐dimensional object analysis. Protocols for the long‐term culturing of primary hippocampal neurons and of primary cortical astrocytes, as well as their co‐culture, are included. While the described methods focus on how astrocytes influence synapse formation and how toxicants may interfere in this process, modifications to the experimental plan can easily be implemented. This would allow for the investigation of the effects of toxicants after treating neurons alone, or both astrocytes and neurons in co‐culture. With the common endpoint of synapse structure formation, differences between varying treatment paradigms can expand the understanding of the influence of particular toxicants on these diverse cell types and provide insight into potential mechanisms of effect and the contributions of each to synapse formation. Curr. Protoc. Toxicol . 60:12.22.1‐12.22.32. © 2014 by John Wiley & Sons, Inc.