OPTIMIZATION OF WHOLE BRAIN CLEARING TECHNIQUES AND MOLECULAR INVESTIGATION OF IEGS INVOLVED IN SPATIAL MEMORY PROCESSES

摘要

Fluorescence in situ hybridization (FISH) has been used to track the localization of Arc transcripts as a molecular method for quantifying behavior-driven activity in the hippocampus. But with the development of several tissue clearing techniques like CUBIC, CLARITY, and passive-CLARITY, the opportunity to use Arc FISH on transparent brains to map out intact neural networks is now feasible. Convention has been to use the full length Arc riboprobe (~3.1 kb) for FISH in 20 micron thick sections. However, in whole cleared brains, the full length Arc cRNA probe cannot easily penetrate the brain due to its thickness. We sought a solution to the issue of penetration with hybridization chain reaction (HCR). HCR is a technique that utilizes DNA probes instead of RNA riboprobes. Since DNA probes are inherently more stable, and HCR probes are less than 150 bases in length, this should drastically improve penetration. In addition, HCR fluorescence hairpins self-polymerize off of the DNA probe and thus, amplify the fluorescence signal. Fischer 344 (F344) rats were given maximal electroconvulsive shock (MECS) to induce transcription, were anesthetized, and then decapitated with a rodent guillotine. The brains were extracted, fixed in paraformaldehyde, and cleared using the CUBIC reagent (containing aminoalcohols) for 6-7 weeks. Using HCR amplified fluorescence in situ hybridization, we expect to visualize Arc-positive cells in rat hippocampus.