Understanding neural integration of colour and motion cues to advance vision research and crop protection.

We know much about how light is transformed into electrical energy by the light sensitive photoreceptors located in the retina. Similarly, other neurons of the visual system have been studied in great detail and can be identified morphologically. However, our current understanding of how visual perception is achieved remains relatively poor. For example, we see colour and motion, but it is unknown how our brain translates this to seeing colourful moving objects with high resolution. The reason for this knowledge gap is that until recently we could not monitor many neurons simultaneously, with the detail necessary to understand how the information is transformed when it is transferred from one neuron to the next. Thus, the precise connectivity and computation that each neuron in the network performs remained to be discovered. Furthermore, monitoring neural responses has traditionally required the animal to be immobilized, but now we know that the response of the visual system is more robust when an animal is allowed to carry out the intended behaviour. Thus, it is exciting that new advances in microscopy and genetics now make it possible to simultaneously see the neural activity across hundreds of neurons (including tiny neurons previously inaccessible by classic electrophysiology) in awake behaving animals. These state-of-the-art tools allow us to detect the calcium increase that occurs within a neuron in response to the information received from other neurons. Such calcium change can be visualized because the cells are genetically programmed to make a calcium sensitive protein that increases in fluorescence (brightness) according to the calcium concentration. The newest version of such protein, which I co-developed and plan to use in these experiments, is named GCaMP6. The fluorescence changes of GCaMP6 are so bright, that we can identify calcium increases due to the arrival of single activation events called action potentials. In addition, for neurons that fall outside of our genetic tool kit, electrical stimulation can be used to coax the entry of fluorescent dyes into cells.

By using genetic manipulation and GCaMP6 neural activity imaging, I aim to solve how colour and motion signals are brought together in the Drosophila brain as part of visual perception. Unlike colour cameras, the colour receptors in our eyes are randomly distributed, meaning neural computations are required to assign each pixel of our visual perception the appropriate colour. New evidence suggests that this happens in the first few layers of information relay between neurons. I am excited to investigate such locations and report the neural computations that enable the combined perception of colour and motion. Thus, the aim of this proposal is to simultaneously deliver visual stimuli with a panoramic projection system, monitor activity via GCaMP6 imaging in the first few neural layers and record motor outputs in natural behaviour assays to determine neuron function.

Drosophila research remains important to society as it can provide discoveries for aiding crop protection and improving human health. For example, results from this proposal will help improve our understanding of crop pests and how colour object recognition is performed during insect flight. As a test case, I will investigate the neural basis of fruit detection by the crop pest Drosophila suzukii. The results will enhance trap design and natural predator selection for integrated pest management. Furthermore, Drosophila is now used widely for many pharmaceutical applications, and has led to the identification of potential reagents for Fragile X syndrome and Huntington's disease. Hence, the methods developed as part of this proposal should provide new behavioural assays for evaluating gene function.

This project addresses a fundamental question of visual neuroscience: how are components of visual stimuli translated into a functionally appropriate behaviour? Which neural circuits combine visual attributes such as colour and motion? Historically, colour and motion signals were considered to be processed separately. Yet, integration of visual attributes has now been shown to occur in the early stages of neural processing, both in vertebrates and in flies. To elucidate how different components of visual stimuli improve behavioural performance, I propose to trace the colour and motion signals, using advanced neural activity imaging, from the retina to the inner medulla of Drosophila while the animal performs flight behavioural testing with its head immobilized. Neural signals will be monitored using the latest genetically encoded calcium indicators, such as RCaMP and GCaMP6, and altered using targeted neural activators/effectors (e.g. Kir2.1, dTrpA1).

The project has the following aims; (1) Identify the function of specific medulla neurons in the visual processing pathway, (2) Use neuroimaging behavioural assays to assess the integration of visual features, (3) Develop a viral delivery system that expresses GCaMP in specific insect neurons and (4) Evaluate the visual cues that attract the crop pest, Drosophila suzukii. The research will be undertaken in the Physiology, Development and Neuroscience Department (University of Cambridge), and in collaboration with international researchers (Ian Meinertzhagen, Chi-Hon Lee, Loren Looger, Luke Lavis, Jerry Cross). The assays developed will be applied to determine how crop pests are able to find fruit or hosts. Drosophila suzukii will be used as a test case. The results will help us to understand how colour and motion cues are used behaviourally when insects locate food sources and provide quantitative colour preferences for pests to enhance trap design and crop protection.

Fostering global and UK economic competiveness:
I will develop a neuro-behavioural diagnosis system to rapidly evaluate a broad range of stimuli and quantify the relative preference of visual cues which attract or repel pests. Future versions which allow visual and olfactory testing of pest species will be invaluable. Especially for crop scientists (e.g. Monsanto) that engineer genetically modified food plants to reduce or prevent insect pests from damaging potential harvests. The methods developed in this proposal should also have direct medical applications for screening mutants or pharmacological preparations.

The cutting edge imaging and neural activity indicators used for this proposal will generate data to inspire next generation microscopes suitable for high speed 3D imaging of neural activity. This is because in vivo neuroimaging microscopes are designed based on academic and private research requirements. For example, Newport just released the InSight DeepSee laser to enable red indicators to be imaged using 2-photon systems.

The viral delivery methods that I will develop will be attractive to biotechnology companies, such as AB Vector, as they already produce custom viral products for molecular biology. Genetic alteration or protein over-expression in specific somatic insect cells is an expanding market because this is faster than producing traditional transgenic animals. Viral transduction is also used widely in agriculture as viral biopesticides for corn and orchard crop pests (e.g. Certis USA).

Increasing the effectiveness of public services and policy:
Replacement, Refinement and Reduction of animals in research (3Rs) is a critical goal of the UK Home Office and of concern to funding agencies and journal publishers. Established viral delivery methods will open the possibility to study more invertebrate species and enable the replacement of vertebrates in some cases. For example, for specific research questions that seek to understand brain function, invertebrate adaptations may be better suited than current vertebrate models. Viral delivery also removes the need to keep hundreds of transgenic strains, which are often highly inbred. Thus, the focus can be switched to improving the housing standards of "wild-type" invertebrate populations that need to be maintained.

Characterising the behavioural repertoire of invasive pest species, such as spotted wing Drosophila, provides valuable intelligence for national and international crop protection agencies (e.g. DEFRA, USDA). Not only can this information be used for modelling the potential spread of the pest, but also for integrated pest management reducing the need for insecticides and improving selection of natural predators and trap designs.

Enhancing quality of life, health and creative output:
A wealth of discoveries for improving human health have derived from Drosophila research. For example, the identification of Drosophila Hox genes has lead to a better understanding of human neural segmentation and development. The finding of a Drosophila transient receptor potential fly mutant opened a research direction into a whole family of human ion channels involved in pain, taste, stretch and vision, and related channelopathies. Drosophila is used widely for pharmaceutical applications, and has led to the finding of potential reagents for Fragile X syndrome and Huntington's disease. Furthermore, understanding how insects work, how brains function and predicting behaviour are all topics of general public interest. The proposed research will provide training and inspiration for new scientists, but will also will be delivered to the public during engaging demonstrations of how modern neuroscience and genetics is helping to improve the UK's economic competitiveness and quality of life.