Genetic Tools for the Analysis of Neural Networks in Flies

Episode von Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 03/06 von Ludwig-Maximilians-Universität München · 11. Februar 2008

Motion vision is of fundamental importance for moving animals from arthropods to mammals. In this thesis I lay ground for the functional analysis of the neural circuit underlying visual motion detection in fruit flies by means of genetic tools. In Drosophila melanogaster transgenic tools allow for both experimental observation and manipulation of neural activity: genetically encoded calcium indicators (GECIs) can be used for the optophysiological characterization of neural activity and transgenes for the inhibition of neural activity can be used to determine these neurons' function. Combined, yet independent use of both tools is a powerful approach for the functional analysis of a neural network. However, GECI signals in vivo generally suffer from poor signal-to-noise ratios and GECI characteristics change dramatically and unpredictably when transfered from the cuvette into neurons of living animals, probably due to interactions with native cellular proteins.

Here, I quantified and compared the in vivo response properties of five new (Yellow Cameleon 3.60 & 2.60, D3cpV, TN-XL and TN-XXL) and two more established ratiometric GECIs (Yellow Cameleon 3.3, TN-L15). In addition, I included the single-chromophore probe GCaMP 1.6 in this comparison. The analysis was performed under 2-photon microscopy at presynaptic boutons of neuromuscular junctions in transgenic fly larvae. I quantified action potential induced changes of calcium concentrations by calibrating responses of a synthetic calcium indicator that was microinjected under 2-photon guidance. The observed cytosolic calcium concentration was 31 nM at rest and increased linearly

with stimulus frequency by 0.1 to 1.8 uM at sustained activity of 10 and 160 Hz, respectively.

This allowed for a quantitative comparison of the responses of GECIs in terms of their steady state response amplitudes, signal-to-noise ratio, response kinetics, calcium

affinities and hill coefficients in vivo. The results were then compared to in vitro properties of GECIs measured in cuvettes.

The data reveal that a new generation of GECIs retain improved signalling characteristics in vivo. Maximum

fluorescence changes were 2-3 fold increased in new compared to former ratiometric GECI variants. Small calcium changes in response to 10 Hz stimulation induced fluorescence responses with signal-to-noise ratio above 2 in Yellow Cameleon 2.60 & 3.60, D3cpv and TN-XXL. Kinetics were slowest in Yellow Cameleon 2.60 and fastest in TN-XL. The observed changes between in vitro and in vivo performance revealed systematic differences between GECIs of different types. GECIs in this study employ different calcium sensing molecules: calmodulin-M13 in Yellow Cameleons and GCaMP, a redesigned calmodulin-M13 in D3cpv, and troponin C in TN-indicators. Those indicators comprising calmodulin-M13 as calcium sensors displayed reduced maximum fluorescence changes and reduced hill coefficients in vivo, while troponin-based GECIs and D3cpv showed increased hill coefficients and increased maximum fluorescence changes in vivo. Calcium affinity of all GECIs was increased in vivo. The results demonstrate that there are now suitable

GECIs at hand for experimental questions at differing expected calcium regimes. However, in contrast to a synthetic calcium sensor, none of the tested GECIs reported calcium concentration changes related to single action potentials at presynaptic boutons of the neuromuscular junction.

In the visual system of Drosophila, optical recordings from motion sensitive neurons while selectively blocking certain classes of columnar neurons will allow for a network analysis of the motion detection circuit. The Gal4-UAS system can be used to express proteins that block neural activity. A similar two-part expression system, based on bacterial protein- DNA interaction (LexA and LexA-operator), can be used in parallel to drive the expression of GECIs. I generated flies expressing TN-XXL or Yellow Cameleon 3.60 under the control of the LexA-operator and demonstrated GECI expression in olfactory receptor neurons. In parallel, I cloned putative genomic enhancers that can be used to drive LexA expression in motion sensitive cells of the flies visual system.

Finally, adult fixed flies expressing TN-XXL in motion sensitive neurons were visually stimulated by large field moving gratings. Parallel fluorescence measurements from these neurons showed for the first time directional selective calcium responses in Drosophila. The next step will now be the combination of calcium imaging in these neurons and functional blocking of their presynaptic partners.

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