Towards an all-optical optogenetic activation and readout system for insect brains

We present our first steps to build an all-optical setup to perform two-photon calcium imaging on optogenetically activated neurons: the goal is to add optogenetics to our experimental two-photon imaging platform and apply this technique first to the fruit fly Drosophila melanogaster and subsequently to the honeybee Apis mellifera at the level of the primary olfactory processing brain centers, the antennal lobes. Insect brains are an important model in neurobiology, thanks to their small sizes, tight structures, but rich performances. We use two-photon microscopy to image morphology and functions of the insect olfactory system in order to study various aspects of information coding. We extended functional response studies beyond mapping out average activity: the role of the temporal dimension in stimulus coding is investigated by analyzing also the dynamics of neuronal activity. Besides analyzing responses to natural odour stimuli, our objective is to develop an optogenetic toolbox to optically interfere with the honeybee brain with high temporal and spatial precision. Activating specific nodes of the first olfactory processing center, the antennal lobe, and imaging the propagation of activity in this brain region via two-photon microscopy, will allow to study connectivity in this neuronal network. The required hardware for optical activation was integrated in our two-photon microscope: a 473 nm laser for opsin activation, overlapped with the infrared imaging beam, making both sources scannable with the same galvo mirror system. The key point is fast switching between the two light sources, allowing a fast transition between optogenetic activation and calcium imaging.