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

Persistent Link:
http://hdl.handle.net/10150/613373
Title:
OPTIMIZATION OF WHOLE BRAIN CLEARING TECHNIQUES AND MOLECULAR INVESTIGATION OF IEGS INVOLVED IN SPATIAL MEMORY PROCESSES
Author:
NGUYEN, MINHKHOI
Issue Date:
2016
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
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.
Type:
text; Electronic Thesis
Degree Name:
B.S.
Degree Level:
Bachelors
Degree Program:
Honors College; Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Barnes, Carol A.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleOPTIMIZATION OF WHOLE BRAIN CLEARING TECHNIQUES AND MOLECULAR INVESTIGATION OF IEGS INVOLVED IN SPATIAL MEMORY PROCESSESen_US
dc.creatorNGUYEN, MINHKHOIen
dc.contributor.authorNGUYEN, MINHKHOIen
dc.date.issued2016-
dc.publisherThe University of Arizona.en
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en
dc.description.abstractFluorescence 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.en
dc.typetexten
dc.typeElectronic Thesisen
thesis.degree.nameB.S.en
thesis.degree.levelBachelorsen
thesis.degree.disciplineHonors Collegeen
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorUniversity of Arizonaen
dc.contributor.advisorBarnes, Carol A.en
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