Files in this item

FilesDescriptionFormat

application/pdf

application/pdfJIN-DISSERTATION-2019.pdf (3MB)Restricted to U of Illinois
(no description provided)PDF

Description

Title:Super-resolution imaging of glutamate receptors in cultured hippocampal neurons under basal and LTP conditions
Author(s):Jin, Chaoyi
Director of Research:Selvin, Paul R.
Doctoral Committee Chair(s):Selvin, Paul R.
Doctoral Committee Member(s):Grosman, Claudio; Chung, Hee Jung; Zhang, Kai
Department / Program:School of Molecular & Cell Bio
Discipline:Biophysics & Computnl Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):AMPAR
NMDAR
Tracking
Basal
LTP
Abstract:The locations and dynamics of glutamate synaptic receptors—the AMPA receptors (AMPARs) and NMDA receptors (NMDARs)—located on the postsynaptic membrane of nerve cells, are crucial for neuronal health, learning and memory formation. They can exist under basal condition where no synaptic strength change occurs, and chemically induced long-term potentiation (cLTP) condition, where synaptic strength is increased by activation of NMDARs from glycine that triggers changes in the number and structure of AMPARs. With three-dimensional super-resolution microscopy (7~16 nm resolution in x-y and 20~32 nm resolution in z), we study AMPARs and NMDARs under basal and cLTP conditions. Under basal condition, we compare two different size fluorophores attached to AMPAR or NMDAR: one is big quantum dots (bQDs), ~20 nm in diameter, which are widely used in the literature; the other is organic dyes, which together with its necessary binding protein, are ~ 4 nm in diameter. We find that organic dyes are superior to bQDs because organic dyes have better access to synapses, which itself is only 20-25 nm across, due to their smaller size. Our results with organics dyes (particularly, Atto647N) are shown to be different than the prevailing results based on bQDs. AMPARs are found to be mostly constrained in the synaptic region, which contradicts the ‘slots’ hypothesis using bQDs. NMDARs, however, are found in both synaptic and extrasynaptic regions. The ratio of synaptic/extrasynaptic population of the two NMDAR subunits (GluN2A and GluN2B) are found to be very different. This ratio is known to be significant in neuronal function/dysfunction. We also quantified this ratio during the maturation process and find redistribution of the synaptic/extrasynaptic NMDARs. Next, cLTP is induced. During this process, the activation of NMDARs is known to cause changes in number and structure of AMPAR on the postsynaptic membrane. In particular, the AMPAR subunits GluA1 and GluA2 are known to contribute to cLTP, but a quantitative understanding is missing in terms of the AMPAR insertion sites on the postsynaptic membrane; how AMPARs move after insertion; and if there are structural changes of AMPARs upon cLTP. We tracked the newly inserted AMPARs and find both synaptic and extrasynaptic insertion sites at 5 and 20 min after cLTP. The portion of synaptic AMPAR increases from 5 to 20 min (32% to 50%). Correspondingly, we also find an increase in synaptic AMPAR numbers. This is done by determining the number of AMPARs under basal condition and the number of the newly inserted ones at 5 and 20 min after cLTP. Taking GluA1-type AMPAR at 5 min after cLTP as an example, under basal conditions, there are 31.8±1.7 synaptic GluA1 on the membrane; when cLTP is induced, 8.1±0.23 internal AMPARs exocytose to the membrane: that is 22.1% ± 1.3% more synaptic GluA1 compared to basal level. Repeating the same method at 20 min after cLTP gives 28.9% ± 1.4% more synaptic GluA1 compared to basal level. This means there is a 7% (out of 22%) increase in synaptic GluA1 from 5 to 20 min. We determine that this occurs because of synaptic exocytosis, not the widely believed lateral diffusion. The conclusion for GluA2 is the same. Aside from the number change, we also observe the structure change of AMPAR during cLTP. We find a ~1.6x increase in AMPAR channel currents from 5 min to 20 min after cLTP. What structure change would cause this is unclear, but it is possible due to desensitization.
Issue Date:2019-07-01
Type:Text
URI:http://hdl.handle.net/2142/105771
Rights Information:Copyright 2019 Chaoyi Jin
Date Available in IDEALS:2019-11-26
Date Deposited:2019-08


This item appears in the following Collection(s)

Item Statistics