Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.
Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.
ORCID
N/A
Access Type
Open Access Thesis
Document Type
thesis
Degree Program
Neuroscience & Behavior
Degree Type
Master of Science (M.S.)
Year Degree Awarded
2016
Month Degree Awarded
May
Abstract
The purpose of this research is to advance development of a photochemical tool designed to probe the role of ionotropic glutamate receptor signaling in neurodegenerative processes, and to delve more deeply into the biological processes underlying the role of these receptors in signaling and memory formation. This ligand-targeted nanoprobe was designed and developed in our lab to label endogenous calcium-permeable AMPARs (CP-AMPARs) in live cells with minimal disruption to native receptor activity. Nanoprobe is designed to use naphthyl acetyl spermine (NASPM) as a photocleavable ligand to target and covalently label native CP-AMPARs with a non-perturbing, fluorescent marker that then allows observation of these receptors using standard epifluorescence microscopy. My contribution to this work, outlined in the aims below, is the characterization of nanoprobe using electrophysiology and fluorescent imaging to evaluate its effectiveness as an endogenous CP-AMPAR label on live neurons.
Aim 1: To use whole cell patch clamp electrophysiology to test the labeling of CP-AMPARs with nanoprobe by recording changes in glutamate-evoked current through heterologously expressed GluA1-L497Y homomultimers during, pre- and post- nanoprobe labeling.
Aim 2: To use fluorescent imaging to evaluate nanoprobe labeling of glutamate receptors endogenously expressed in hippocampal neurons by co-labeling nanoprobe-treated neurons with traditional antibodies to AMPAR and synaptic targets.
Aim 3: To use nanoprobe to detect endogenously expressed CP-AMPARs on live neurons during the course of neuron development. Live neuronal cultures will be imaged before and after labeling with nanoprobe in young dissociated cultures (DIV 1-2) and in maturing cultures (DIV 14-17).
Conclusions: Whole cell patch clamp electrophysiology results provide evidence that nanoprobe will label CP-AMPARs in a minimally-perturbing fashion that allows the receptors to resume normal activity after photolytic-release of ligand as designed. Fixed cell imaging of CP-AMPAR nanoprobe labeling was largely ineffective, and live cell imaging was not conclusive, but provided supporting evidence that nanoprobe targets and labels NASPM-sensitive endogenous glutamate receptors on live dissociated hippocampal neurons
DOI
https://doi.org/10.7275/8748533
First Advisor
James J Chambers
Second Advisor
Gerald B Downes
Third Advisor
Jerrold S Meyer
Movie2 (1).mov (3294 kB)
Movie3 (1).avi (2675 kB)
Movie4.avi (38131 kB)
Movie5.avi (71124 kB)
Recommended Citation
Combs-Bachmann, Rosamund Elizabeth, "Evaluating a Novel Photochemical Tool for Labeling and Tracking Live, Endogenous Calcium-Permeable AMPARs" (2016). Masters Theses. 343.
https://doi.org/10.7275/8748533
https://scholarworks.umass.edu/masters_theses_2/343
Included in
Biology Commons, Cell and Developmental Biology Commons, Molecular and Cellular Neuroscience Commons, Neurosciences Commons, Pharmacology, Toxicology and Environmental Health Commons