Quantification of synaptic structure formation in cocultures of astrocytes and hippocampal neurons

Pamela J. Roqué, Marina Guizzetti, Gennaro Giordano, Lucio G. Costa

Research output: Chapter in Book/Report/Conference proceedingChapter

8 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationIn Vitro Neurotoxicology
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages361-390
Number of pages30
ISBN (Print)9781617791697
DOIs
StatePublished - Jan 1 2011

Publication series

NameMethods in Molecular Biology
Volume758
ISSN (Print)1064-3745

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Keywords

  • Astrocytes
  • Confocal microscopy
  • Deconvolution
  • Hippocampal neurons
  • ImmunoÂ-cytochemistry
  • Postsynaptic density protein-95
  • Synaptophysin
  • Three-dimensional analysis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Roqué, P. J., Guizzetti, M., Giordano, G., & Costa, L. G. (2011). Quantification of synaptic structure formation in cocultures of astrocytes and hippocampal neurons. In In Vitro Neurotoxicology: Methods and Protocols (pp. 361-390). (Methods in Molecular Biology; Vol. 758). Humana Press Inc.. https://doi.org/10.1007/978-1-61779-170-3_25