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Synaptic Vesicle Glycoprotein 2A (SV2A) As a Measure of Synaptic Plasticity in Animal Models of Depression

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Synaptic Vesicle Glycoprotein 2A (SV2A) As a Measure of Synaptic Plasticity in Animal Models of Depression Synaptic Vesicle Glycoprotein 2A (SV2A) as a Measure of Synaptic Plasticity in Animal Models of Depression Saba Ali & Mariam Labrouzi Masters in Biomedicine June 2019 Supervisors Professor, MD, DMSc, Jens D. Mikkelsen Professor, Ph.D., M.Sc. Cathy Mitchelmore Abstract Depression is associated with a loss of synapses, which is brought on by a downregulation in important synaptic proteins involved in maintenance of synapses. The presynaptic protein, Synaptic Vesicle Glycoprotein 2A (SV2A) is a proposed indirect measure of synaptic density and has been shown to be inversely correlated to the severity of depressive symptoms exhibited in humans. Quantification of synapses through various methods using radiotracers targeting synaptic proteins, such as SV2A, may be a non-invasive method for diagnosing, monitoring and an in depth understanding of depression mechanisms. The overall aims of this thesis were to validate techniques detecting SV2A in the rat brain determined by in vitro autoradiography using the radiolabeled ligand [3H]UCB-J and immunoblotting using specific antibodies for SV2A and synaptophysin. Further; in order to determine the distribution of SV2A across brain regions and in depression, various animal models were employed, namely: Flinders Sensitive Line, chronic mild stress and electroconvulsive stimulation. The examination of the regional SV2A distribution across various brain regions, revealed that differences in the amount of SV2A are observed between age groups; and further, that the SV2A expression is higher overall compared to that of synaptophysin. In the diurnal cycle experiments we found that the SV2A binding and the corresponding blood corticosterone levels were correlated in the corticosterone treated animals. In the electroconvulsive stimulation rat model a significant difference was observed between the ECS and control group in two brain regions. A decrease in SV2A binding was observed in the amygdala and ventral hippocampus in the ECS group. In the chronic mild stress rat model, the SV2A binding was significantly increased in both the prefrontal cortex and the orbitofrontal cortex; as a response to agomelatine treatment. In conclusion, conducting similar in vitro experiments as presented here and in vivo and clinical studies is necessary for the proper understanding of the involvement of SV2A in synaptic plasticity and depression. Page 2 of 67 Summary Depression associeres med synapsetab grundet en nedregulering af vigtige proteiner involveret i opretholdelsen af synapser. Et foreslået indirekte mål for synapsedensitet er det præsynaptiske protein SV2A. Proteinet har vist sig at være omvendt korreleret til sværhedsgraden af symptomer på depression i mennesker. Kvantificering af synapser gennem forskellige metoder der bruger radioligander målrettet mod synapseproteiner, såsom SV2A, er mulige non-invasive tilgange til diagnosticering og monitorering af depression samt til at udvide den molekylærbiologiske forståelse af lidelsen. De overordnede formål med dette speciale var at validere teknikker til detektion af SV2A i rottehjernen ved brug af in vitro autoradiografi med radioliganden [3H]UCB-J og immunoblotting med antistoffer, der specifikt targeterer vesikelproteinerne SV2A og synaptophysin. Derudover, at bestemme fordelingen af SV2A henover hjerneregioner og i forskellige dyremodeller af depression, nemlig: Flinders Sensitive Line, elektrokonvulsiv stimulation og kronisk mild stres. Undersøgelsen af den regionale SV2A-fordeling i forskellige hjerneregioner viste forskelle i proteinmængden imellem aldersgrupper. Dette forsøg viste ydermere, at SV2A-ekspressionen overordnet set er højere end ekspressionen af synaptophysin. Døgnrytmeeksperimentet med administration af corticosteron viste, at SV2A-bindingen og de korresponderende blod-corticosteronniveauer var korrelerede i dyr behandlet med corticosteron. I rotterne behandlet med elektrokonvulsiv stimulation blev en signifikant forskel observeret mellem ECS- og kontrolgrupperne, specifikt i to hjerneregioner. Et fald i SV2A bindingen blev set i amygdala og ventral hippocampus i ECS-gruppen. I kronisk mild stress rottemodellen var SV2A bindingen signifikant opreguleret i prefrontal og orbitofrontal cortex som respons til agomelatinbehandling. Lignende in vitro-eksperimenter, som præsenteret her, samt in vivo- og kliniske studier er nødvendige for den dybdegående forståelse af SV2A’s rolle i synaptisk plasticitet og depression. Page 3 of 67 Table of Contents Abstract ............................................................................................................................................................... 2 Summary.............................................................................................................................................................. 3 Table of Contents ................................................................................................................................................. 4 Abbreviations ....................................................................................................................................................... 7 1 INTRODUCTION ...................................................................................................................................................... 8 2 THEORETICAL BACKGROUND ...................................................................................................................................... 9 2.1 Major Depressive Disorder (MDD) ............................................................................................................................. 9 2.2 Synapse Physiology and Glutamatergic Neurotransmission.................................................................................... 12 2.3 Synaptic Plasticity ..................................................................................................................................................... 13 2.4 Synaptic Plasticity and Depression ........................................................................................................................... 14 2.5 Synaptic Vesicle Glycoprotein 2A (SV2A) ................................................................................................................. 15 2.6 Markers of Synaptic Density .................................................................................................................................... 19 2.7 Animal Models of Depression .................................................................................................................................. 20 2.7.1 Corticosterone ................................................................................................................................................... 21 2.7.2 Flinders Model................................................................................................................................................... 21 2.7.3 Electroconvulsive Stimulation (ECS).................................................................................................................. 22 2.7.4 Chronic Mild Stress (CMS) ................................................................................................................................. 23 2.8 Theory behind the methods ..................................................................................................................................... 24 2.8.1 Autoradiography ............................................................................................................................................... 24 2.8.2 Western Blot ..................................................................................................................................................... 25 3 STATE OF THE ART .................................................................................................................................................28 Main aims ....................................................................................................................................................................... 28 4 METHODS ...........................................................................................................................................................29 4.1 Animals and Brain Tissue for Autoradiography ....................................................................................................... 29 4.1.1 Tissue for Method Validation. ........................................................................................................................... 29 4.1.2 Tissue for Studying the Effect of Corticosterone on the Diurnal Cycle............................................................. 29 4.1.3 Flinders Resistant and Sensitive Line. ............................................................................................................... 30 4.1.4 Electroconvulsive Stimulation (ECS).................................................................................................................. 30 4.1.5 Chronic Mild Stress (CMS). ................................................................................................................................ 30 Page 4 of 67 4.2 In Vitro Autoradiography using [3H]UCB-J ............................................................................................................... 31 4.2.1 Data Analysis ....................................................................................................................................................
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