Optimization of Whole Brain Clearing Techniques for the Molecular Investigation of Arc

• Main Author: Nguyen, Minhkhoi
• Other Authors: Barnes, Carol A.
• Language: en_US
• Published: The University of Arizona. 2017
• Online Access: http://hdl.handle.net/10150/625693; http://arizona.openrepository.com/arizona/handle/10150/625693

The immediate early gene Arc has been implicated in synaptic plasticity and has also been shown to be unique in its behavior-driven expression and localization. Thus, it has been used as a molecular marker for behavior-driven neuronal activity, especially in hippocampal and cortical neurons. Fluorescence in situ hybridization (FISH) for Arc mRNA reveal distinct compartmentalization of the transcript: Arc is found in the nucleus of neurons activated within the last few minutes, moves to the cytoplasm staining in neurons activated over ~25 minutes ago, and both intranuclear foci and cytoplasmic Arc mRNA is observed in neurons activated at both time points if the behavior at the two time points is identical. With the development of several tissue clearing techniques like CUBIC, CLARITY, and EDC-CLARITY, it is now possible to use Arc FISH to label whole brains in order to map out intact neural networks in response to behavior. Conventional Arc FISH utilized full length antisense Arc riboprobes (~3.1 kb) in 20 micron thin sections, however in cleared whole brains, the full length riboprobes may not fully penetrate the tissue. We sought to resolve the issue of tissue penetration with hybridization chain reaction (HCR), which uses DNA probes less than 150 bases in length. The DNA probes can also be amplified by using HCR fluorescence hairpins thus providing better tissue penetration and signal amplification. Male Fischer 344 rats were given maximal electroconvulsive shock (MECS) to induce Arc transcription in a high percentage of hippocampal and cortical neurons. The animals were sacrificed and the brains were extracted to be cleared by CUBIC or EDC-CLARITY. Utilizing HCR amplified FISH, Arc-positive cells were found in the dentate gyrus of the hippocampus and in the cerebral cortex.