Implementation of an all-optical setup for insect brain optogenetic stimulation and two-photon functional imaging

Insect brain is a very complex but at the same time small, simplified and accessible model with respect to the mammalian one. In neuroscience a huge number of works adopt drosophila as animal model, given its easiness of maintenance and, overall, of genetical manipulation. With such a model one can investigate many behavioral tasks and at the same time have access to a whole brain in vivo, with improved specificity and cellular resolution capabilities. Still, a remarkable goal would be to gain a precise control over the neural network, in order to fully manipulate specific areas of the brain, acting directly on network nodes of interest. This is possible thanks to optogenetics, a technique that exploits photosensitive molecules to modulate molecular events in living cells and neurons. At the same time, it is possible to perform a neuronal readout with light, exploiting calcium-based reporters; in this way, neuronal response investigation can gain in temporal and spatial resolution. This is an all-optical approach that brings many advantages in the neural network study and an insight in the functional connectivity of the system under investigation. We present here a setup that combines a two-photon imaging microscope, capable of in vivo imaging with a sub-cellular resolution and an excellent penetration depth down to hundreds of microns, with a diode laser optogenetic stimulation. With such a setup we investigate the drosophila brain in vivo, stimulating single units of the primary olfactory system (the so-called glomeruli, about 20 µm of diameter). By our knowledge this is one of the first time a similar all-optical approach is used in such an animal model: we confirm, in this way, the possibility to perform these experiments in vivo, with all the advantages coming from the improved accessibility of our model. Moreover, we present the results using a sample co-expressing GCaMP6 and ChR2-XXL, optimal performing sensor and actuator, largely exploited in the field for their high efficiency: these were rarely used in combination, since their spectral overlap, nevertheless we are able to show the feasibility of this combined approach, enabling to take advantage from the use of both these performing molecules. Finally, we will show different approaches of data analysis to infer relevant information about correlation and time response of different areas of the brain, that can give us hints in favor of some functional connectivity between olfactory subunits.