Quantifying synapses: an immunocytochemistry-based assay to quantify synapse number. Ippolito DM, Eroglu C (2010). JoVE., doi: 10.3791/2270.
This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (available upon request from firstname.lastname@example.org) under the ImageJ* analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that is already utilized in a number of studies on synaptic connectivity. (See the studies that used this method)
*Puncta Analyzer does not function with new versions of Image J. Please download and use ImageJ 1.29 here.
Rapid Golgi analysis method for efficient and unbiased classification of dendritic spines. Risher W.C., Ustunkaya T., Singh J.A., and Eroglu C. (2014) PLoS ONE, Sep 10;9(9):e107591.
Dendritic spines are the primary recipients of excitatory synaptic input in the brain. Spine morphology provides important information on the functional state of ongoing synaptic transmission. One of the most commonly used methods to visualize spines is Golgi-Cox staining, which is appealing both due to ease of sample preparation and wide applicability to multiple species including humans. However, the classification of spines is a time-consuming and often expensive task that yields widely varying results between individuals. Here, we present a novel approach to this analysis technique that uses the unique geometry of different spine shapes to categorize spines on a purely objective basis. This rapid Golgi spine analysis method successfully conveyed the maturational shift in spine types during development in the mouse primary visual cortex. This approach, built upon freely available software, can be utilized by researchers studying a broad range of synaptic connectivity phenotypes in both development and disease.(See the studies that used this method)