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Ihring, Alexandra (2006): Characterization and Modification of Genetically Encoded Indicators to Monitor Neural Activity in Drosophila melanogaster. Dissertation, LMU München: Faculty of Biology
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Abstract

Genetically encoded fluorescent indicators of neural activity represent promising tools for systems neuroscience. In the first part of my thesis, a comparative in vivo analysis of ten different genetically encoded calcium indicators as well as the pH-sensitive SynaptopHluorin is presented. The calcium indicators are either based on a single chromophore (GCaMP variants, Camgaroo variants, Pericam variants) or on two chromophores (Yellow Cameleon variants, Troponeon variants). I expressed these indicators in the cytosol of presynaptic boutons of the Drosophila larval neuromuscular junction and analyzed their fluorescence changes upon stimulation. GCaMP 1.3, GCaMP 1.6, Yellow Cameleon 2.0, 2.3, and 3.3, Inverse-Pericam, the troponin C-based calcium sensor TNL 15 and SynaptopHluorin allowed reliable detection of presynaptic fluorescence changes at the level of individual boutons. However, the response characteristics of all of these indicators differed considerably from each other. TNL 15 exhibited the most stable and fastest rising signals at lower activity rates, whereas GCaMP 1.6 produced the fastest signals at high rates of nerve activity with largest fluorescence changes. GCaMP 1.6 and GCaMP 1.3 signals, however, were complicated by bleaching, as was the case for Inverse Pericam. The fluorescence signals of the double-chromophore indicators were in general smaller but more photostable and reproducible. Camgaroo-1 and Camgaroo-2 showed little or no response, and Flash Pericam did not result in any detectable fluorescence. GCaMP 1.3 and YC 3.3 revealed fairly linear fluorescence changes and a corresponding linear increase in the signal-to-noise ratio (SNR) over an expanded range of neural activity. As expected, the expression level of the indicator had an influence on the signal kinetics and the SNR, whereas the signal amplitude was independent. In the second part of my thesis work I fused several genetically encoded calcium indicators to different signal sequences. The targeting of the indicators to distinct parts of the cell such as the membrane, vesicles or ion channels allows detection of calcium ions before they disperse in the cytosol. Specific signals can be extracted more efficiently and in a more relevant physiological context. Tagging of YC 2.3, GCaMP 1.6 and TNL 15 to transmembrane domains or proteins involved in the synaptic vesicle cycle did not result in functional targeting. TN XL fused to the transmembrane domain mCD8 at the N-terminus and eight amino acids from a calcium channel subunit at the C-terminus resulted in membrane association at the NMJ. Fractional fluorescence changes up to 6.5 % were recorded upon stimulation. In cells of the fly visual system scattered fluorescent puncta were observed. This fusion protein has the potential for monitoring calcium dynamics in close proximity of ion influx. The presented data will be useful for in vivo experiments with respect to the selection of an appropriate indicator, as well as for the correct interpretation of optical signals.