Case Studies in Invertebrate Visual Processing: I. Spectral and Spatial Processing in the Early Visual System of Drosophila melanogaster II. Binocular Stereopsis in Sepia officinalis Rachael Claire Cashin Feord Pembroke College September 2019 This dissertation is submitted for the degree of Doctor of Philosophy Declaration This thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration with the exception of the work presented in Chapter 6, which is the product of a study conceived by Dr Paloma Gonzalez-Bellido and Dr Trevor Wardill, who carried out the initial experiments. My contributions to the work are nonetheless substantial and individual contributions to the project are detailed in the acknowledgment section of Chapter 6 (section 6.5) It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text It does not exceed the prescribed word limit for the relevant Degree Committee. 1 2 Abstract Case Studies in Invertebrate Visual Processing: I. Spectral and Spatial Processing in the Early Visual System of Drosophila melanogaster II. Binocular Stereopsis in Sepia officinalis Rachael Claire Cashin Feord This thesis addresses two aspects of visual processing in two different invertebrate organisms. The fruit fly, Drosophila melanogaster, has emerged as a key model for invertebrate vision research. Despite extensive characterisation of motion vision, very little is known about how flies process colour information, or how the spectral content of light affects other visual modalities. With the aim to accurately dissect the different components of the Drosophila visual system responsible for processing colour, I have developed a versatile visual stimulation setup to probe for the combinations of spatial, temporal and spectral visual response properties. Using flies that express neural activity indicators, I can track visual responses to a colour stimulus (i.e. narrow bands of light across the spectrum) via a two-photon imaging system. The visual stimulus is projected on a specialised screen material that scatters wavelengths of light across the spectrum equally at all locations of the screen, thus enabling presentation of spatially structured stimuli. Using this setup, I have characterised spectral responses, intensity-response relationships, and receptive fields of neurons in the early visual system of a variety of genetically modified strains of Drosophila. Specifically, I compared visual responses in the medulla of flies expressing either a subset or all photoreceptor opsins, with differing levels of screening pigment present in the eye. I found layer-specific shifts of spectral response properties correlating with projection regions of photoreceptor terminals. I also 3 found that a reduction in screening pigment shifts the general spectral response in the neuropil towards the longer wavelengths of light. I have also mapped receptive fields across the different layers of the medulla for the peak spectral response wavelength. My results suggest that receptive field dimensions match the expected size predicted by the conservation of a columnar organisation in the medulla, with little variation from layer to layer. In a subset of these cells, we see an elongated receptive field suggestive of static orientation selectivity with an apparent split in the preferred axis of orientation of these receptive fields, with a near-orthogonal angle between the summed vectors of the split populations. The camera type eyes of vertebrates and cephalopods exhibit remarkable convergence, but it is currently unknown if the mechanisms for visual information processing in these brains, which exhibit wildly disparate architecture, is also shared. I chose to investigate the visual processing mechanism known as stereopsis in the cuttlefish Sepia officinalis. Stereoscopic vision is used to assess depth information by comparing the disparity between left and right visual fields. This strategy is commonplace in vertebrates having evolved multiple times independently but has only been demonstrated in one invertebrate: the praying mantis. Cuttlefish require precise distance estimation during their predatory hunt when they extend two tentacles in a ballistic strike to catch their target. Using a 3D perception paradigm whereby the cuttlefish were fitted with anaglyph glasses, I show that these animals use stereopsis to resolve distance to their prey. Although this is not an exclusive depth perception mechanism for hunting, it does shorten the time and distance covered prior to striking at a target. Furthermore, stereopsis in cuttlefish works differently to vertebrates, as cuttlefish can extract stereopsis cues from anti-correlated stimuli. 4 Acknowledgments Firstly I would like to express my thanks to my supervisor, Dr Trevor Wardill, for the guidance, support and confidence he has shown me over the course of this project. I am grateful to the BBSRC Doctoral Training Partnership for funding me over the course of this four-year PhD programme. I would like to thank Dr Paloma Gonzalez-Bellido for being a mentor to me throughout my PhD, and for all of the invaluable advice she has provided. Thanks also go to Dr Kristian Franze for kindly stepping in as my Cambridge based supervisor once the lab had moved across the ocean. A huge thank you goes to my PhD siblings, Jack Supple, Sam Fabian and Sergio Rossoni, as well as Mary Sumner to whom I am very grateful for this shared journey. I am also very grateful to Dr Kathryn Feller and Dr Camilla Sharkey for their perpetual support, kindness, encouragement, wisdom and sense of fun throughout the project, and to Dr Jorge Blanco for his wizardry with genetics. I would like to thank the Department of Physiology, Development and Neuroscience for their constant support over the course of my PhD. Special thanks go to the Neural Oscillation Group: Prof Ole Paulsen, Dr Susanna Mierau, Dr Audrey Hay and Dr Ana González-Rueda for taking me under their wing. Thanks go to the University of Minnesota and to the Marine Biology Laboratory (Woods Hole, MA) for welcoming me into their respective institutions and giving me space and resources for my research. I would also like to say a special thank you to the community that is Pembroke College, as well as to all the music and choral communities of Cambridge for making my experience at this university much more special and valuable than I could have imagined. Very importantly, a huge thank you to my truly wonderful parents, Karan and Damian, without whom I would never have made it this far. This thesis is dedicated to them. And thank you to my siblings Helen, Elisabeth and Antony for always lending a sympathetic ear. And finally, I would like to extend my thanks to all of my friends for their moral and emotional support. 5 6 Table of contents Declaration ........................................................................................................... 1 Abstract ................................................................................................................ 3 Acknowledgments ............................................................................................... 5 Table of contents ................................................................................................. 7 List of figures ..................................................................................................... 13 List of tables ....................................................................................................... 15 Chapter 1 – Introduction ................................................................................... 17 1.1 STUDYING INVERTEBRATE VISION ................................................................ 17 1.1.1 Who wouldn’t want to study invertebrates? .................................................. 17 1.1.2 The valuable contributions of invertebrate research .................................... 17 1.1.3 The case for comparative neurobiology ........................................................ 19 1.1.4 Key contributions to visual research from invertebrates ............................... 20 1.1.5 Thesis outline ................................................................................................ 20 1.2 DROSOPHILA AS A MODEL FOR VISION RESEARCH .................................. 21 1.2.1 Drosophila holds many tools for studying neural circuitry ............................ 21 1.2.2 Functional imaging: visualising neural activity .............................................. 22 1.2.3 Drosophila has pioneered motion vision research ........................................ 24 1.2.4 Colour vision in Drosophila ........................................................................... 25 1.2.5 Drosophila white mutant ................................................................................ 25 1.3 COLOUR/SPECTRAL PROCESSING IN DROSOPHILA .................................. 26 1.3.1 Defining colour vision ...................................................................................