Graduation Date

Spring 5-6-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Anna Dunaevsky

Second Advisor

Hamid Band

Third Advisor

Kaustubh Datta

Abstract

Dendritic spines are the principal sites of excitatory synapses in the neurons of mammalian central nervous system. Spine are plastic, undergoing structural and functional changes under basal and experience dependent conditions. Spine properties are altered in a number of neurodevelopmental disorders including the Fragile X syndrome (FXS) which is the most common inherited form of intellectual disability. The structural reorganization of dendritic spines is thought to be associated with synaptic plasticity mechanisms that are deficient in FXS. A number of synaptic plasticity mechanisms involve modulation of synaptic strength via insertion or removal of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). However, the link between synaptic behavior and AMPAR dynamics has not been previously studied in vivo.

To investigate the role of AMPAR in spine dynamics in vivo we expressed AMPAR subunit GluA2 tagged to superecliptic phluorin (SEP), a pH sensitive GFP variant, in layer 2/3 neurons of the primary motor cortex (M1). Dendritic spines and sGluA2 were imaged in vivo using two-photon light microscopy over a period of ten days in both wild type and the fmr1 knock out (KO) mice, a mouse model of FXS. Repeated in vivo imaging revealed that in the fmr1 KO mouse dendritic spines were denser, smaller, contained less sGluA2 and had higher turnover rates compared to littermate controls (WT). Our data confirmed the relationship between synaptic strength and synaptic stability, with greater AMPAR containing spines being more stable in both WT and the KO mice. Additionally, we observed that AMPAR levels were dynamic in most stable spines, fluctuating over 10 days with larger proportion of spines showing multiple dynamic events of AMPAR in the KO. Directional changes in sGluA2 were also observed in subpopulation of spines, with new small spines gradually accumulating sGluA2. Moreover, sGluA2 levels dropped just prior to spine elimination with greater loss observed in the KO spines. To further investigate the role of AMPAR in experience dependent plasticity, we trained KO mice in a forelimb task and monitored behavioral learning and biochemically measured synaptic AMPAR levels. KO mice had mild motor deficits in a single forelimb reaching task compared to WT controls. Furthermore, after one day of motor skill training WT mice had a gradual increase in synaptic sGluA1 which was delayed in the KO. Thus we conclude that AMPAR levels within spines are continuously dynamic and are predictive of spine behavior. These dynamics are further modulated upon learning with impairments under basal and experience dependent conditions in the fmr1 KO mouse.

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