{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/122134"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/122134","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Studying synaptic organization and dynamics of AMPA receptors in primary neuronal cultures and brain tissue using super-resolution fluorescence microscopy","abstract":"Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-12-01","abstract_html":"Submission published under a 24 month embargo labeled &#x27;U of I Access&#x27;, the embargo will last until 2025-12-01","abstract_has_math":false,"creators":["Vaidya, Rohit Mohan"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics & Quant Biology","degree_department":null,"school":null,"contributors":["Selvin, Paul R","Chung, Hee Jung","Gruebele, Martin","Grosman, Claudio"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-12","date_published":"2023-12","updated_at":"2026-07-22T22:25:00Z","subjects":["Super-resolution Imaging","Super-resolution Fluorescence Microscopy","Ampa Receptor"],"languages":["en","eng"],"rights":["Copyright 2023 Rohit Vaidya"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/122134","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Selvin, Paul R","Chung, Hee Jung","Gruebele, Martin","Grosman, Claudio"]},{"key":"dc:creator","label":"Author","values":["Vaidya, Rohit Mohan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2023-12","2023-11-27"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics & Quant Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Super-resolution Imaging","Super-resolution Fluorescence Microscopy","Ampa Receptor"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en","eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2023 Rohit Vaidya"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/122134"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-12-01","The student, Rohit Vaidya, accepted the attached license on 2023-11-21 at 12:36.","The student, Rohit Vaidya, submitted this Dissertation for approval on 2023-11-21 at 12:47.","This Dissertation was approved for publication on 2023-11-27 at 16:18.","DSpace SAF Submission Ingestion Package generated from Vireo submission #19992 on 2024-03-01 at 13:30:54","Neurons in our brain communicate with each other through synapses. AMPARs are ion channels found in synapse and mediate the majority of the fast excitatory synaptic transmission. Their numbers, arrangement, and diffusion on the cell membrane of neurons act as important indicators of synaptic plasticity. Single molecule localization microscopy (SMLM) techniques have been used in the last 10 years to extensively study the nanoscale dynamics and organization of surface AMPARs (which are the functional form of AMPARs) and their clustering in synaptic nanodomains. The size of the fluorescent probe used to target AMPARs in the narrow (20-30 nm) synaptic cleft is crucial and using a large probe can affect the observations significantly. Moreover, most of these studies have been limited to dissociated neuronal cultures as opposed to brain tissue with intact circuitry, and as a function of aging and pathology. In the second chapter of this thesis, after introducing AMPARs and SMLM in the first chapter, the probe size dependence on observed AMPAR surface diffusion and synaptic distribution is carefully studied in dissociated hippocampal neurons using a host of quantum dots with varying sizes. The next part of this thesis (chapters 3-7) focuses on developing a strategy to study native, surface AMPARs in mouse brain slices with SMLM. The importance of studying AMPARs in brain slices and the biochemical and optical challenges involved are explained in Chapter 3. A recently developed small probe, CAM2, is used to specifically label native, surface AMPARs (specifically, GluA2-4 subunits) in live mouse brain slices. This approach avoids artifacts arising due to protein overexpression and modification. Chapter 4 focuses on tackling the optical challenges involved with imaging in thick brain slices. A custom-built imaging setup is developed in which light scattering caused by refractive index mismatch is reduced, HILO illumination is used for reducing out-of-focus fluorescence background and instrument-induced aberrations are corrected using a deformable mirror. The localization data is analyzed using a recently developed algorithm called INSPR that retrieves the in-situ PSF for better fitting. Chapter 5 explains all the methods and materials involved. With this combined labeling plus imaging strategy, localization precisions of <10 nm (lateral) and <30 nm (axial) could be achieved in 30 μm thick, fixed Thy1-YFP-H mouse brain slices that have a subset of excitatory pyramidal neurons filled with yellow fluorescent protein (YFP) for better delineation. The imaging results are shown in Chapter 6. With this resolution, synaptic AMPAR nanodomains ~70 nm in size could be visualized, which were previously only observed in dissociated neurons. In Chapter 7, AMPAR distribution is studied as a function of brain region. A large population of extra-synaptic AMPARs is observed in hippocampus compared to cortex where AMPARs are found mostly clustered in synapses, potentially indicating a larger synaptic plasticity exhibited by hippocampal neurons. AMPAR distribution is also studied in PS19 mice, an Alzheimer’s Disease mouse model exhibiting tauopathy, at 6 months of age. Synaptic plasticity is known to get hampered at this age in PS19 mice before the onset of actual neurodegeneration. Studying nanoscale organization of AMPARs as an indicator of synaptic strength in brain slices could thus help understand disease onset and identify potential drug targets."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Studying synaptic organization and dynamics of AMPA receptors in primary neuronal cultures and brain tissue using super-resolution fluorescence microscopy"]}]}],"canonical_facts":{"dc:contributor":["Selvin, Paul R","Chung, Hee Jung","Gruebele, Martin","Grosman, Claudio"],"dc:creator":["Vaidya, Rohit Mohan"],"dc:date":["2023-12","2023-11-27"],"dc:description":["Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-12-01","The student, Rohit Vaidya, accepted the attached license on 2023-11-21 at 12:36.","The student, Rohit Vaidya, submitted this Dissertation for approval on 2023-11-21 at 12:47.","This Dissertation was approved for publication on 2023-11-27 at 16:18.","DSpace SAF Submission Ingestion Package generated from Vireo submission #19992 on 2024-03-01 at 13:30:54","Neurons in our brain communicate with each other through synapses. AMPARs are ion channels found in synapse and mediate the majority of the fast excitatory synaptic transmission. Their numbers, arrangement, and diffusion on the cell membrane of neurons act as important indicators of synaptic plasticity. Single molecule localization microscopy (SMLM) techniques have been used in the last 10 years to extensively study the nanoscale dynamics and organization of surface AMPARs (which are the functional form of AMPARs) and their clustering in synaptic nanodomains. The size of the fluorescent probe used to target AMPARs in the narrow (20-30 nm) synaptic cleft is crucial and using a large probe can affect the observations significantly. Moreover, most of these studies have been limited to dissociated neuronal cultures as opposed to brain tissue with intact circuitry, and as a function of aging and pathology. In the second chapter of this thesis, after introducing AMPARs and SMLM in the first chapter, the probe size dependence on observed AMPAR surface diffusion and synaptic distribution is carefully studied in dissociated hippocampal neurons using a host of quantum dots with varying sizes. The next part of this thesis (chapters 3-7) focuses on developing a strategy to study native, surface AMPARs in mouse brain slices with SMLM. The importance of studying AMPARs in brain slices and the biochemical and optical challenges involved are explained in Chapter 3. A recently developed small probe, CAM2, is used to specifically label native, surface AMPARs (specifically, GluA2-4 subunits) in live mouse brain slices. This approach avoids artifacts arising due to protein overexpression and modification. Chapter 4 focuses on tackling the optical challenges involved with imaging in thick brain slices. A custom-built imaging setup is developed in which light scattering caused by refractive index mismatch is reduced, HILO illumination is used for reducing out-of-focus fluorescence background and instrument-induced aberrations are corrected using a deformable mirror. The localization data is analyzed using a recently developed algorithm called INSPR that retrieves the in-situ PSF for better fitting. Chapter 5 explains all the methods and materials involved. With this combined labeling plus imaging strategy, localization precisions of <10 nm (lateral) and <30 nm (axial) could be achieved in 30 μm thick, fixed Thy1-YFP-H mouse brain slices that have a subset of excitatory pyramidal neurons filled with yellow fluorescent protein (YFP) for better delineation. The imaging results are shown in Chapter 6. With this resolution, synaptic AMPAR nanodomains ~70 nm in size could be visualized, which were previously only observed in dissociated neurons. In Chapter 7, AMPAR distribution is studied as a function of brain region. A large population of extra-synaptic AMPARs is observed in hippocampus compared to cortex where AMPARs are found mostly clustered in synapses, potentially indicating a larger synaptic plasticity exhibited by hippocampal neurons. AMPAR distribution is also studied in PS19 mice, an Alzheimer’s Disease mouse model exhibiting tauopathy, at 6 months of age. Synaptic plasticity is known to get hampered at this age in PS19 mice before the onset of actual neurodegeneration. Studying nanoscale organization of AMPARs as an indicator of synaptic strength in brain slices could thus help understand disease onset and identify potential drug targets."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/122134"],"dc:language":["en","eng"],"dc:rights":["Copyright 2023 Rohit Vaidya"],"dc:subject":["Super-resolution Imaging","Super-resolution Fluorescence Microscopy","Ampa Receptor"],"dc:title":["Studying synaptic organization and dynamics of AMPA receptors in primary neuronal cultures and brain tissue using super-resolution fluorescence microscopy"],"dc:type":["text"],"thesis:degree_discipline":["Biophysics & Quant Biology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:00Z"}