Abstract
The ability to quantify changes of synaptic structure, whether associated with the formation of synapse in early development or the degeneration of synapses in adult life in an in vitro culture system, is important for understanding the underlying mechanisms. Astrocytes play a vital role in neuronal development and functioning, including synapse formation and stabilization. The method described in this chapter allows for the determination of the modulation by astrocytes of synaptic structure formation in hippocampal neurons. Using a sandwich coculture system, highly pure, hippocampal neurons are grown in culture for 14 days on glass coverslips, after which they are inverted, without contact, over separately cultured astrocytes or pretreated astrocytes for 24 h. Neuronal immunocytochemical staining of the presynaptic marker, synaptophysin, and the postsynaptic marker, PSD-95, is used to assess the localization of synaptic proteins into pre and postsynaptic structures. Deconvolved, confocal images are used to determine a mean puncta intensity threshold for use in rendering the surface of the synaptic structures using three-dimensional object analysis software. Once rendered in three-dimensional space, automatic quantification of the number of pre- and postsynaptic specializations and the number of those structures that overlap is obtained, allowing the ability to compare how different treatments may modulate the formation of synapses. Because synapses not only consist of distinct pre- and postsynaptic specializations, but are also defined by their apposition, the determination and study of synapse formation can only benefit by methods that use all of the available data to assess the actual structure.
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Acknowledgments
The Digital Microscopy Center of the Cellular Morphology Core of the Center on Human Development and Disability (CHDD) at the University of Washington, Seattle, WA, for access to the equipment and particularly Glen MacDonald for his technical expertise in the use of confocal microscope, deconvolution and object analysis, and theory.
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Roqué, P.J., Guizzetti, M., Giordano, G., Costa, L.G. (2011). Quantification of Synaptic Structure Formation in Cocultures of Astrocytes and Hippocampal Neurons. In: Costa, L., Giordano, G., Guizzetti, M. (eds) In Vitro Neurotoxicology. Methods in Molecular Biology, vol 758. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-170-3_25
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DOI: https://doi.org/10.1007/978-1-61779-170-3_25
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