Neuroethology of the Zebrafish: Describing the Neural Circuits that Control Innate Behaviors A dissertation presented by Adam Raymond Kampff to The Department of Molecular and Cellular Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biology Harvard University Cambridge, Massachusetts October 2008 © 2008 – Adam Raymond Kampff All rights reserved. Florian Engert Adam Raymond Kampff Neuroethology of the Zebrafish: Describing the Neural Circuits that Control Innate Behaviors Abstract The complexity of the animal brain begs for simple tools that will assist scientists attempting to unravel the beautifully entangled mess. I begin by describing an experimental strategy for indentifying the components of neural circuits that control innate behaviors. My approach emphasizes the role of behavior analysis and the insights offered by neuroethology. While describing the neuroethological strategy, I will introduce the larval zebrafish and tools that I have developed to study the neural basis of innate visual behavior. The second chapter reports an implementation of the strategy that successfully revealed components of the circuit underlying the zebrafish optomotor reflex. We discovered distinct subsets of spinal projection neurons that were responsible for directing the swims and turns that constitute an important visual response to whole- field motion. The success of this study relied on the ability to record behavior-related neural activity in a restrained zebrafish, but this was found to be difficult for other visual behaviors. The third chapter addresses this problem by describing a novel technique to monitor neural activity in an unrestrained, behaving zebrafish. Employing the bioluminescent calcium reporter, Aequorin, we were able to detect the activity of Hypocretin neurons in freely swimming zebrafish. The success of this technique will allow an extensive survey of how identified populations of neurons collectively choreograph the complex behavior patterns of a developing vertebrate. iii Table of Contents Chapter 1: Introduction to the Neuroethology of Larval Zebrafish 1.0 - A neural description of behavior 1.1 - The neuroethological approach to identifying neural circuits 1.2 - The larval zebrafish as a model system for the neuroethological approach 1.3 - Implementing and augmenting the neuroethological strategy 1.4 - References Chapter 2: Neural Correlates of the Optomotor Response 2.0 - Introduction 2.1 - Behavioral responses to stabilized whole-field motion 2.2 - Motor patterns underlying the optomotor response 2.3 - Calcium imaging of responses in spinal-projection neurons 2.4 - Projection neuron responses to whole-field motion 2.5 - Laser ablation of turning neurons 2.6 - Discussion 2.7 - Methods 2.8 - References Chapter 3: Monitoring Neural Activity in Freely Behaving Zebrafish 3.0 - Introduction 3.1 - Potential strategies for neural recordings in unrestrained zebrafish 3.2 - Aequorin and its use as neural Ca2+-reporter 3.3 - Using GFP-Aequorin in freely-swimming larval zebrafish 3.4 - Neuroluminescent zebrafish 3.5 - Genetically-targeted recordings from hypocretin neurons during behavior 3.6 - Future Directions 3.7 - Methods 3.8 - References iv Acknowledgements I wish to thank Florian Engert; it is perhaps unfair to him and his amazing support to thank anyone else. So I won’t…at least not in the same paragraph. I wish to thank my collaborators in the Engert Lab, but in particular, Mike Orger and Eva Naumann. I wish to thank my advisory committee. I wish to thank my parents. Most of all, I wish to thank YOU, the reader, but you are very likely one of the people mentioned already. Oh, and the zebrafish, thanks for all the…fish. v Chapter 1 Introduction to the Neuroethology of Larval Zebrafish 1.0: A neural description of behavior The nervous system controls an organism’s behavior, and I want to understand how it does this. This is a daunting goal1 and rather than flounder in aimless disappointment, I have focused on answering a simplified question: what are the components of the neural circuit responsible for controlling an innate response to a sensory stimulus? In the following chapter, I describe an experimental strategy for addressing this question and a model preparation that is uniquely suited to this strategy. However, when combined with well designed experimental techniques, I believe this strategy can be used to identify many such circuits, underlying many different behaviors, at work in many different brains. I am not naïvely suggesting that we would ultimately achieve a satisfactory understanding of the brain, but I am proposing that we will have made a significant step towards this goal. Having described some of the brain’s functional circuits, we will be in a position to ask how they develop, how they change during learning, and how they fail when damaged. 1.1: The neuroethological approach to identifying neural circuits Attempts to describe the circuitry of the brain are complicated by the difficult task of identifying neurons that participate in a dedicated circuit. The experimental 1 neuroscientist must consider a tremendous range of sensory stimuli, any of which might evoke a response from the repertoire of an animal’s behavior. An uninformed search for related stimuli and responses would be futile. Fortunately, ethologists have taken a perspective informed by the natural ecology and evolution of animal behavior2. They have had remarkable success in identifying very specific stimuli that evoke unique behaviors3. Such behaviorally-relevant stimuli, sometimes termed ‘sign stimuli’ or ‘releasing mechanisms’3, provide an excellent tool for uncovering the components of a dedicated neural circuit. If a neuroscientist is able to present these relevant stimuli, or sufficiently accurate reproductions, to an experimental preparation from which neural activity can be recorded, then it is informative to simply record which neurons are activated (or inhibited). These specifically active neurons are possible participants in the circuit controlling the related behavior. Furthermore, subtle changes to the relevant stimulus often eliminate, or significantly alter, the behavioral response3. Comparing the neurons activated by the relevant stimulus and similar stimuli that have no effect, or different effects, on behavior, the neuroscientist can isolate subsets of neurons that might be specific to the behavior. Subsequent studies are needed to address the causal role of these isolated neurons in controlling the behavior; their necessity and sufficiency can be assayed with targeted ablation and stimulation experiments. This experimental strategy for circuit identification, which I will call the neuroethological approach because of its emphasis on detailed behavioral analysis, is outlined in Figure 1.1. 2 Figure 1.1 Figure 1.1- The Neuroethological Approach to Circuit Identification A successful implementation of the neuroethological strategy requires an appropriate experimental preparation. I will motivate my decision to work with larval zebrafish by comparing the preparation to an ideal neuroethological model system. 1.2: The larval zebrafish as a model system for the neuroethological approach The zebrafish (Danio rerio) is a freshwater teleost native to the rivers of India4. The larvae develop externally and are almost completely translucent, contributing to their popularity as a developmental model organism5. At only one day post fertilization (1dpf), zebrafish show behavioral responses to touch as well as spontaneous motor activity6. Visual responses appear by day three, almost immediately after the axons of ganglion cells leaving the eye reach their targets7, and remarkably, after just 5 days, they begin 3 visually hunting prey8. Such a rapid development has inspired many research groups to address the genetic basis of zebrafish behavior9, and the research community has produced a host of genetic tools useful in the investigation of the neural circuits controlling behavior10, 11. However, the criteria for a successful neuroethological system are often distinct from those of a general model system. In the following, I will discuss how the larval zebrafish, along with the tools that I have developed, provides an almost- ideal system for implementing the three stages of the neuroethological strategy: behavior analysis, neural recordings, and causality tests. Behavior Analysis (ideal): In a laboratory setting, assays for identifying behaviorally-relevant stimuli require precise control of the stimulus environment while simultaneously monitoring the animal’s behavior. Difficulties in controlling the sensory environment result primarily from the modality being controlled (e.g. it is currently much easier to produce a spatially patterned visual scene than a spatially patterned olfactory space), and in some cases, certain animal preparations will allow the use of more easily controlled stimuli12. However, it is often more challenging to accurately measure the behavior of an animal, and this will provide a more stringent criterion for evaluating potential neuroethological systems. For many neuroscience model systems: primates, rodents, birds and most insects, behavior consists of the intricate coordination of muscle contraction, directing the motion of multiple limbs, and resulting in complex movement through three-dimensional space. Reducing the description of behavior to a measurable variable usually produces a 4 grotesque simplification of the response, e.g. describing