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|Title:||Fluorescent protein calcium sensor for monitoring synaptic transmission|
|Authors:||Okorocha, Albert Egwu|
|Presented at:||University of Leicester|
|Abstract:||Monitoring Ca²⁺ in intracellular compartments remains an uphill task. Fluorescent, organic calcium dyes and genetically encoded calcium indicators (GECIs) have been widely used to increase our understanding of neural activity. Notable among the GECIs is the GCaMP family. The first versions of this family showed potential but were relatively dim and had poor signal to noise ratios. To overcome these weaknesses, our laboratory developed three new ratiometric versions of the SyGCaMP2 family called SyGCaMP2-mCherry, SyGCaMP2-mCherry⁺ and GCaMP2-mCherry. The purpose of the work presented here was to establish whether these sensors were functional and to then use them to examine calcium signalling within the presynaptic terminals of cultured hippocampal and cerebral cortical neurones. The sensors were expressed in HEK 293T cells and their sensitivities to changes in intracellular free Ca²⁺concentration ascertained. The dissociation constants calculated for SyGCaMP2-mCherry⁺ and SyGCaMP2-mCherry were 140 ± 3.1 nM and 149 ± 3.3 nM respectively, but GCaMP2-mCherry was not accurately measured. Sensors were expressed in cortical and hippocampal neurones using lipofection based transfection methods and the expression patterns of each recorded. Both SyGCaMP2-mCherry and SyGCaMP2-mCherry⁺ were expressed in puncta that co-localised with presynaptic markers bassoon and VGlut1. Neurones were activated using field stimulation and the responses to different intensities and patterns of stimulation evaluated. Using the Thy1.2 promotor for neuronal expression, two transgenic mice (SyG 14 and SyG 37) were engineered in our lab that expressed SyGCaMP2-mCherry and responses to electrical stimulation were characterised in hippocampal brain slices. The roles of intracellular Ca²⁺stores in shaping the presynaptic Ca²⁺dynamics were examined and results demonstrated that inhibiting the sarcoendoplasmic reticulum calcium transport ATPase (SERCA pump) with Cyclopiazonic acid (CPA) and thapsigargin led to an enhancement in synaptic strength. In addition, activating ryanodine receptors (RYRs) with caffeine and low concentrations of ryanodine altered presynaptic Ca²⁺ dynamics.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Cell Physiology and Pharmacology|
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