Abstract
Labeling fixed brain tissue with fluorescent synaptic and cellular markers can help assess circuit connectivity. Despite the diffraction-limited resolution of light microscopy there are several approaches to identify synaptic contacts onto a cell-of-interest. Understanding which image quantification methods can be applied to estimate cellular and synaptic connectivity at the light microscope level is beneficial to answer a range of questions, from mapping appositions between cellular structures or synaptic proteins to assessing synaptic contact density onto a cell-of-interest. This chapter provides the reader with details of the image analysis methods that can be applied to quantify in situ connectivity patterns at the level of cellular contacts and synaptic appositions.
Correspondence may be addressed to either author.
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Acknowledgments
The dot-finding routine was developed by Josh Morgan and further refined by the collaborative efforts of past and present Rachel Wong Lab members. Credit for the dot-finding routine goes to the concerted efforts of Josh Morgan, Daniel Kerschensteiner, Adam Bleckert, and Haruhisa Okawa. Daniel Kerschensteiner, Florentina Soto, Josh Morgan, and Adam Bleckert together developed the correlation analysis. We are grateful to Rachel Wong and Adam Bleckert for critical feedback and suggestions during preparation of this chapter. This work is supported by NIH grants (EY10699, EY17101, and EY14358 to R. Wong and a Vision Core grant EY01730); a Human Frontier Science Program grant (RGP0035 to L. Lagnado, F. Schmitz, and R. Wong); a Human Frontier Science Program Long term fellowship (R. Sinha); and a Knights Templar Eye Foundation career starter grant (M. Hoon). The immunolabeling for Ribeye and GABAA receptors shown in the Figures has been possible due to antibodies generously provided by Frank Schmitz and Jean-Marc Fritschy.
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Hoon, M., Sinha, R., Okawa, H. (2017). Using Fluorescent Markers to Estimate Synaptic Connectivity In Situ. In: Poulopoulos, A. (eds) Synapse Development. Methods in Molecular Biology, vol 1538. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6688-2_20
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DOI: https://doi.org/10.1007/978-1-4939-6688-2_20
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