Biology subjects

Savage, J. T.

Publications and source records attributed to Savage, J. T..

2 recordsLinked to original sources

bioRxiv2023

SynBot: An open-source image analysis software for automated quantification of synapses

The formation of precise numbers of neuronal connections, known as synapses, is crucial for brain function. Therefore, synaptogenesis mechanisms have been one of the main focuses of neuroscience. Immunohistochemistry is a common tool for visualizing synapses. Thus, quantifying the numbers of synapses from light microscopy images enables screening the impacts of experimental manipulations on synapse development. Despite its utility, this approach is paired with low throughput analysis methods that are challenging to learn and results are variable between experimenters, especially when analyzing noisy images of brain tissue. We developed an open-source ImageJ-based software, SynBot, to address these technical bottlenecks by automating the analysis. SynBot incorporates the advanced algorithms ilastik and SynQuant for accurate thresholding for synaptic puncta identification, and the code can easily be modified by users. The use of this software will allow for rapid and reproducible screening of synaptic phenotypes in healthy and diseased nervous systems. MotivationLight microscopy imaging of pre- and post-synaptic proteins from neurons in tissue or in vitro allows for the effective identification of synaptic structures. Previous methods for quantitative analysis of these images were time-consuming, required extensive user training, and the source code could not be easily modified. Here, we describe SynBot, a new open-source tool that automates the synapse quantification process, decreases the requirement for user training, and allows for easy modifications to the code.

neuroscience↗

bioRxiv2023

GEARBOCS: An Adeno Associated Virus Tool for In Vivo Gene Editing in Astrocytes

CRISPR/Cas9-based genome engineering enables rapid and precise gene manipulations in the CNS. Here, we developed a non-invasive astrocyte-specific method utilizing a single AAV vector, which we named GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/Cas9 System). We verified GEARBOCS specificity to mouse cortical astrocytes and demonstrated its utility for three types of gene manipulations: knockout (KO); tagging (TagIn); and reporter knock-in (GeneTrap) strategies. Next, we deployed GEARBOCS in two test cases. First, we determined that astrocytes are a necessary source of the synaptogenic factor Sparcl1 for thalamocortical synapse maintenance in the mouse primary visual cortex. Second, we determined that cortical astrocytes express the synaptic vesicle associated Vamp2 protein and found that it is required for maintaining excitatory and inhibitory synapse numbers in the visual cortex. These results show that the GEARBOCS strategy provides a fast and efficient means to study astrocyte biology in vivo. MotivationAstrocytes are indispensable for brain development, function, and health. However, molecular tools to study astrocyte biology and function in vivo have been largely limited to genetically modified mice. Here, we developed a CRISPR/Cas9-based gene editing strategy within a single AAV vector that enables efficient genome manipulations in astrocytes. We designed and optimized this easy-to-use viral tool to understand gene expression, protein localization and function in astrocytes in vivo.

neuroscience↗