Characterization and Modification of Genetically Encoded Indicators to Monitor Neural Activity in Drosophila melanogaster

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.