Calcium imaging in the insect nervous system using an innovative dye loading technique

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Neuroscience aims at understanding the organisation and function of nervous systems focussing on vertebrate and invertebrate model animals. Recording the activity of neurons is classically achieved with extra- or intracellular electrodes which measure the electrical spike and synaptic activity with high temporal and amplitude resolution. Over the last decades optical recordings have been developed, based e.g. on calcium sensitive dyes, which are loaded into neurons or populations of neurons. Upon binding to Calcium ions these dyes change their fluorescence intensity or wavelength and generate optical signals that can be monitored with sensitive cooled CCD cameras or confocal microscopes. As calcium is an essential molecule for neuronal functioning and synaptic processing the optical signals report the excitatory activity of the labelled neurons - although not with the precision of electrophysiological measurements. Optical imaging however, allows to analyse the spatial dimensions of neuronal processing at the cellular and network level and has substantially contributed to our understanding of nervous systems. In some model systems, like the fruit fly Drosophila or the zebrafish, even genetically encoded calcium indicators are available, which can be expressed in specific cell lines of the nervous system. The corresponding molecular-genetic tools are not (yet) available in other organisms, which nonetheless are model systems to study a specific behaviour. One of these system are acoustically communicating insects like crickets or bush-crickets, which for decades have been in the focus of neuroscience research aiming to understand the neuronal mechanisms underlying hearing, auditory pattern recognition and sound pattern generation. Progress in analysing these systems was mainly based on electrophysiological and neuroanatomical techniques, as loading central or afferent neurons with calcium sensitive dyes, is difficult and required loading these neurons with intracellular dye injection via microelectrodes. Inspired by the iontophoretic application of drugs through the intact human skin, we recently developed a dye delivery method through the intact sheath of nerves or ganglia. Glass capillaries with 40-80 micro-meter tip diameter are filled with the calcium sensitive tracer and are attached to the neuronal sheath e.g. of an insect auditory nerve. Initially they are used as recording electrodes, and once a good signal has been established the circuit is switched into iontophoretic dye delivery mode. The calcium sensitive dye moves into the axons of the nerve and then travels in both directions from the injection site, labelling the peripheral sensory organ and the central afferent axonal arborisations in the nervous system. No other labelling method can achieve this. The method can also be used to specifically label auditory neuropil regions in the brain of crickets. Our preliminary imaging experiments prove the principle and demonstrate that sound evoked optical signals report the specific activity of the labelled auditory afferents and central neurons. We now aim to further take advantage of this technique to systematically study (1.) the spatial organisation of auditory processing in the cricket brain; (2) the activation of auditory afferents in the hearing organ and (3) the spatiotemporal activity patterns underlying the singing motor activity in male crickets. Besides using our sensitive CCD camera system, we will use more advanced confocal and two-photon imaging systems to enhance the resolution of the data. The chosen objectives represent central questions in insect neuroscience and should contribute to further our understanding of insect acoustic communication. Moreover the planned experiments will demonstrate the versatility of our method as a new tool in insect neuroscience and will be relevant to the wider community of researchers.