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

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.

We recently developed a versatile method for iontophoretic delivery of calcium sensitive dyes through the intact neural sheath which allows functional optical imaging of the labelled neurons. In brief, glass micropipettes with large tip openings are filled with a calcium sensitive dye and positioned on an intact nerve or ganglion. The micropipette is first used to record the activity at the attachment site. Once a good recording is established the circuitry is switched to iontophoresis with DC or current pulses of appropriate polarity, which deliver the dye through the sheath into the neurons. Importantly, the dye solution is prevented from leaking by increasing its viscosity and any gas generated during the iontophoresis is captured in a custom designed micropipette holder. The calcium sensitive dye spreads anterograde and retrograde and in case of auditory nerves labels the central axonal projections of the afferents as well as their cell bodies and dendrites in the hearing organ. Dye injection into a ganglion will specifically label the neurons at the injection site. With our sensitive CCD camera system we have already measured sound evoked calcium signals in thoracic auditory neuropils in locusts and in cricket brains. We now aim to demonstrate the general versatility of the labelling method by using it in crickets/bush-crickets to (1) image sound evoked activity related to temporal pattern recognition in the auditory neuropil of the brain; (2) by revealing the spatio-temporal activation pattern of auditory afferents in the crista acustica of the hearing organ and (3) by analysing the flow of neuronal activity in the abdominal ganglia while male crickets generate the singing motor pattern. Besides using our cooled CCD camera system, we will image the calcium signals with confocal microscopy and two photon microscopy to increase the spatial resolution. We are confident that our dye loading technique will expand the tool box for imaging in neuroscience research.

Science progresses by the development of new research techniques and methods. Our innovative method for iontophoretic loading of neurons with dyes for anatomical and functional imaging through surface electrodes will mainly be interesting and beneficial for other neuroscientist working in field. No other method allows to simultaneously label sensory or motor nerves in both directions, or to label local neuropils in brains and ganglia. Moreover the method can be used to deliver calcium sensitive dyes into the nervous system for optical imaging of neuronal activity in a variety of insects, in which genetically encoded calcium indicators are not yet available. It therefore boosts the importance of specimen, in which electrophysiological recordings can be achieved easily, but where advanced imaging techniques were difficult to apply.
A direct industrial or technical application of the method and the project results is not immediately evident, however, the method might find application in various neuroscience projects. We had several visitors from other labs who were interested to learn the technique hands on.
Our approach uses acoustically communicating insects as model systems to analyse fundamental principles of neural functions, sensory processing and/or motor control. As such our grant application covers a wide range of neuroscience topics. These will therefore be of particular interest to neuroscientists working on neural auditory processing, to scientists aiming to understand the function of miniature hearing organs and to those researchers who have an interest in the neuronal mechanisms underlying the generation animal behaviour. By analysing the functional properties of nervous systems, neuroscience contributes to a better and deeper understanding of animals in a similar way as genetics or molecular biology. These scientific endeavours reflect the inquisitive nature of humans and are embedded in the cultural and scientific framework of our society. We explore the unknown in order to obtain general and specific knowledge about organisms and matter, which will allow current and future generations to manage the resources and challenges of life on earth.
We see our contribution in securing social and economic return mainly in the field of higher education but also in public awareness for science. Our research takes place at the University of Cambridge, a top University for research and teaching within the UK. The Department of Zoology is a large, multi-disciplinary Department whose members conduct research and teaching in areas ranging from molecular biology to ecology. With the proposed project we will continue the track record of the Department and maintain the high quality in insect neurobiology. The project will contribute to training and education of promising students and young researchers in the field of neuroscience. The named RA will be trained in optical imaging techniques, and will be strongly involved the management of project objectives, time and resources, and in the publication process. The RA will present data at scientific conferences and contribute to any public science events. An essential part of the training will deal with job and grant applications to secure the next position for the RA. The PI will provide substantial support of these RA activities in form of discussions of research results, proof reading of manuscripts and applications, and with advice on career development. Cambridge University runs an excellent program career development and transferable skills. The RA will be strongly encouraged to attend such seminars.