An optimized optokinetic behavioral assay to assess central nervous system function in mice

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In humans, there are five general classes of eye movements: saccades, smooth pursuit, vergence, the vestibular-ocular reflex (VOR), and the optokinetic reflex (OKR). In vertebrates, the VOR and OKR are the most conserved eye movements and have been described in a number of mammals including mice, cats, rabbits, monkeys and humans. With the introduction of targeted and transgenic technology, there is a high premium on identifying phenotypes in targeted and transgenic mice. Here I describe a computer-assisted optokinetic behavioral assay to rapidly identify CNS defects in mice. Using this system, I quantified the basic stimulus-response properties in two different mouse strains - C57B1/6J and 129Sv/Ev. I showed that the mouse OKR is a conjugated response and it is direction selective - preferring stimuli moving in the temporal to nasal direction. With a series of targeted and transgenic mice, I determined the photoreceptors required for the OKR response. Furthermore, I identified the functional role two genes in vision - Brn3b and Frizzled-4. To address whether the OKR could be used to assess central nervous system deficits, I tested the effect of several CNS drugs on mouse eye movements. I observed that several drugs, including gabapentin and anandamide, disrupted OKR without disturbing general motor acitivity, as measured by the rotorod. Several other stereotyped eye movements were generated in drug-induced states - including changes in the slow component of the OKR (ketamine and baclofen) and spontaneous eye movements (cocaine and memantine). The final series of experiments utilized the OKR as a behavioral measure to assess restoration of vision. In these experiments, we tested mice with no functional light response before and after the delivery of a Channelrhodopsin-2 virus. We observed a very inefficient restoration of the direct light response (1/24) and no restoration of image forming responses with the OKR. In my thesis, I put forth a novel OKR technology to identify visual and central nervous system function in a variety of experimental paradigms including phenotyping visual defects in gene targeted and transgenic mice, identifying CNS deficits under drug-induced mental states, and quantifying the severity of CNS disease in mouse models.
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AN OPTIMIZED OPTOKINETIC BEHAVIORAL ASSAY TO ASSESS CENTRAL NERVOUS SYSTEM FUNCTION IN MICE By Hugh Cahill A dissertation submitted to The Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland September 2008 UMI Number: 3340040 Copyright 2009 by Cahill, Hugh All rights reserved. INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3340040 Copyright 2009 by ProQuest LLC. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 E. Eisenhower Parkway PO Box 1346 Ann Arbor, Ml 48106-1346 Abstract In humans, there are five general classes of eye movements: saccades, smooth pursuit, vergence, the vestibular-ocular reflex (VOR), and the optokinetic reflex (OKR). In vertebrates, the VOR and OKR are the most conserved eye movements and have been described in a number of mammals including mice, cats, rabbits, monkeys and humans. With the introduction of targeted and transgenic technology, there is a high premium on identifying phenotypes in targeted and transgenic mice. Here I describe a computer-assisted optokinetic behavioral assay to rapidly identify CNS defects in mice. Using this system, I quantified the basic stimulus-response properties in two different mouse strains - C57B1/6J and 129Sv/Ev. I showed that the mouse OKR is a conjugated response and it is direction selective - preferring stimuli moving in the temporal to nasal direction. With a series of targeted and transgenic mice, I determined the photoreceptors required for the OKR response. Furthermore, I identified the functional role two genes in vision - Brn3b and Frizzled-4. To address whether the OKR could be used to assess central nervous system deficits, I tested the effect of several CNS drugs on mouse eye movements. I observed that several drugs, including gabapentin and anandamide, disrupted OKR without disturbing general motor acitivity, as measured by the rotorod. Several other stereotyped eye movements were generated in drug-induced states - including changes in the slow component of the OKR (ketamine and baclofen) and spontaneous eye movements (cocaine and memantine). The final series of experiments utilized the OKR as a behavioral measure to assess restoration of vision. In these experiments, we tested mice with no functional light response before and after the delivery of a Channelrhodopsin-2 virus. We observed a very inefficient restoration of the direct light response (1/24) and no restoration of image forming responses with the OKR. In my thesis, I put forth a novel OKR technology to identify visual and central nervous system function in a variety of experimental paradigms including phenotyping visual defects in gene targeted and transgenic mice, identifying CNS deficits under drug- induced mental states, and quantifying the severity of CNS disease in mouse models. Thesis Advisor: Jeremy Nathans, M.D., Ph.D. Thesis Reader: Randall Reed, Ph.D, IV Acknowledgements. I would like to express my gratitude to my thesis advisor, Jeremy Nathans, for his support over the years in the lab. Jeremy has selflessly taught me how to design experiments and took the time to teach me even the simplest protocols. He is truly the definition of a great advisor. He played a very active role in my thesis project including designing experiments and analyzing data. He taught me to creatively and critically think about science. I feel incredibly fortunate to have been able to be a part of this wonderful and exciting lab. One important aspects of working in Jeremy's lab was the light- hearted jokes and fun atmosphere that he established in the lab. It was always an enjoyable pleasure to be in the lab. The larger Hopkins community donated biological reagents and helped with comments and discussions. These researchers include - David K. Ryugo, Paul Fuchs, King Wai Yau, Michael Lee, Donald Price, Alena Savonenko, Philip Wong, Tudor Badea, Matrin Biel, Janice Lem, His-Wen Liao, Yanshu Wang, Samer Hattar, V Huimin Yu, Phil Smallwood, Xin Ye, Chunqiao Liu, Nick Marsh-Armstrong, Hui Sun, Amir Rattner, Paul Worley, Christopher Ross, and Valina Dawson. Without their help and support none of these experiments would have been possible. I have also had the opportunity to collaborate with researcher outside the Hopkins community including - Dr. David Poulsen, Dr. Martina Biel and Drs. Janice Lem. These individuals provided reagents (Dr. Poulsen) and targeted mice (Dr. Biel and Dr. Lem) for this project. My thesis committee advisors guided the project with insightful comments that helped mold the final project. Dr. Timothy Moran, Dr. King-Wai Yau, and Dr. Randy Reed dedicated their time to patiently discuss all of the experiments and results in my project. These recommendations and discussions led to interesting follow up experiments that added great value to the project. I would like to thank past and present members of the Nathans' lab, including Huimin Yu, Xin Ye, Hui Sun, Jiachao Chen, Nini Guo, Tudor Badea, Amir Rattner, Yanshu Wang, Tom Rotolo, Leila Toulibui, John vi Williams and Phil Smallwood. They have been wonderful colleagues and close friends. Also, I would like to thank my family for support - including my parents, Marguaret and Daniel, and my brothers, Dan, Mike, and Chris. And finally, I would like to thank the Visual Neuroscience Training Grant for supporting these experiments. Vll Table of Contents Abstract ii Acknowledgements v Table of Contents viii General Introduction 1 Chapter One Summary 12 Introduction 14 Results 18 Discussion 35 Methods 37 References 45 Figures Legends 52 Figures 58 Chapter Two Summary 69 Introduction 71 Results 74 Discussion 94 Methods 99 References 101 Figure Legends 107 Table and Figures 110 viii Chapter Three Summary 123 Introduction 125 Materials and Methods 128 Results 131 Discussion 133 References 138 Figures Legends 141 Figures 143 Curriculum Vitae 145 ix General Introduction Quantifying behavior in the rapidly growing number of transgenic and target mouse models has created interest in developing fast and objective phenotyping assays. In general, mouse behavioral tests measure a motor output to sensory stimuli (i.e. auditory, olfactory, gustatory, vestibular, visual and/or somatosensory inputs) and in vision research, assays can roughly be divided into two broad categories — non-image and image forming behavioral tests. The first category tests the mouse's ability to capture light from the environment and produce a behavioral response that utilizes this information. The latter assay challenges the mouse visual system to organize this information into objects from edge, movement and contrast cues. Both of these types of visual behavioral tests have been successfully used to identify the role of genes in animal behavior [1, 2, 3, 4, 5] . The most common non-image forming behavioral tests are pupil constriction, light-dark avoidance and circardian rhythm measurements. Image forming behavioral assays include the classically conditioned forced choice tests, Morris water maze, visually induced head-tracking, and 1 optokinetic behavioral paradigms. These non-image and image forming tests have been optimized to evaluate an animal's ability at particular tasks (i.e. contrast discrimination or learning and memory). They require a large time investment and are not ideal for rapid measurements of CNS activity. For example, visual water tasks have the advantage that complex object based stimuli can be evaluated — including spatial acuity, contrast differences, luminance detection, and chromatic discrimination. But this test utilizes the animal's memory system in combination with the visual sensory system for successful completion of the behavioral task and requires many trials for statistically significant results [6]. Therefore, one disadvantage in developing a rapid CNS test of animals as illustrated by this example, is that these assays often involve multiple neural circuits and requiring training to arrive at a conclusive behavioral result. The second major disadvantage of several of the behavioral assays is that the motor response adapts over time to the stimulus. As a result, many trials with multiple mice are required to collect statistically significant data sets. This fact coupled with the requirement for human intervention to score multiple trials 2 limits the researcher's ability to evaluate experiments in a high-throughput manner. The optokinetic reflex (OKR) is an image forming behavioral response that is highly conserved in evolution and is not greatly influenced by learning and memory. During the assay a full-field image is moved around the animal and the experimenter records the eye position. The animal produces a stereotyped eye movement that follows the moving image — a movement to stabilize the visual field upon the retina. This behavioral response requires no training and therefore is ideal for quantitative analysis. The goal of my thesis project is to measure the OKR in a rapid behavioral assay that can be used to quantify central nervous system function in mice. To improve the current optokinetic assay, I used computer generated visual stimuli in combination with a rotating projector to streamline stimulus generation and presentation. I used this new optokinetic reflex apparatus to present a striped black and white visual stimulus to the mouse and optimized these parameters for bar thickness, image velocity and contrast. To assess the amount of variability in the mouse, I compared C57B1/6J and 129Sv/Ev 3 mice that are commonly used in the generation of transgenic and targeted lines. I observed very different, yet stereotyped, OKR in these strains as quantified by eye movement saccades per unit time. Through a breeding experiment, I found that this difference between strains is an inherited genetic trait with at least five genes involved. With the availability of several targeted and transgenic mice, I investigate the contribution of different retinal cell populations to the OKR, including rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs). I found that the OKR requires the image forming rods and cones and that the ipRGCs are not required for an OKR — consistent with the non-image forming role hypothesized for ipRGCs. Taking advantage of computer generated stimuli, I created a series of chromatic striped panels to evaluate color sensitivity in mice. For these experiments, I used mice with middle wavelength cone pigment (wildtype), mice possessing with an engineered human long-wavelength cone pigment or heterozygous female mice with a mosaic of these two types.. This last mouse takes advantage of the fact 4 that the locus for the middle and long wavelength pigment is located on the X-chromosome. As a result of X- inactivation, female mice with two X-chromosomes exclusively express only one of the two (long and middle wavelength) alleles. In these mice, patches of cones in the retina express the human long wavelength pigment while other patches express the mouse middle wavelength pigment [7]. I found that mice expressing the human long wavelength pigment make long wavelength detect long wavelength light (as determined by color matches) at higher levels of illumination than mice expressing the mouse middle wavelength pigment — consistent with the prediction that the former perceive long-wavelength light as brighter than do wildtype mice. The heterozygous Red-Green Opsin females appeared to average the responses from both types of cones for the OKR assay, resulting in an intermediate response. To assess whether OKR testing could identify visual defects in knockout mice, I screened nine knockout mouse lines — two lines from the family of Brn3 transcription factors and seven knockout lines from the family of frizzled receptors. I discovered that among the frizzled receptor knockout lines, only frizzled 4 knockouts had no 5 OKR responses. In screening the Brn3 transcription factor knockout mice, I discovered that Brn3b knockout mice had defects in horizontal OKR and pupil constriction. In both of these assay the behavioral response was reduced but still present. Furthermore, I documented that these mice completely lacked a vertical OKR. These behavioral measurements were consistent with anatomical observations that show that Brn3b null mice lack an accessory optic tract, a group of retinal ganglion cell (RGC) axons that do not follow the major projection to the thalamus. One of the nuclei that receives inputs from these accessory optic RGCs — the medial terminal nucleus (MTN) completely lacks input from the retina. In the rabbit, physiological experiments have implicated the MTN in responses to vertically moving stimulus [8], consistent with the observation that Brn3b null mice lack a vertical OKR. To determine whether the OKR can identify central nervous system deficits, beyond the retina, I tested mice under two additional experimental paradigms. First we tested mice with known genetic mutations that effect the central nervous system — such as alpha-synuclein transgenic mice, mGluRl knockout mice, and Huntington's disease mice. We also investigated the ability of the OKR assay to identify 6 drug-induced CNS dysfunction. I observed that several putative neurological disease mice and approximately one dozen drugs (including cocaine, gabapentin, memantine, baclofen) disrupted eye movements. These results suggest that the OKR can be used to identify changes in central nervous system function and perhaps, most interestingly, identify disease progression. Several methods have been proposed for restoring visual responses in blind mouse models. I tested whether the OKR can be used to evaluate therapeutic restoration of vision in the context of one of these methods: infecting retinal ganglion cells with a virus encoding Channelrhodopsin-2 (ChR2) to confer light sensitivity [9]. To accomplish this, I engineered an adeno-associated virus vector to express Channelrhodopsin-2, a light gated ion channel. After infecting retinal ganglion cells with the engineered virus, I performed two assays of visually dependent behaviors — a pupil constriction assay and the OKR. One out of twenty four mice had a modest pupil constriction after infection with the virus, and none of the infected mice show an OKR. These results demonstrated that the optokinetic reflex could be used to assess the 7 restoration (or lack thereof) of visual function in experimental therapeutic treatments. In conclusion, I believe that the OKR is a formidable addition to the collection of behavioral assays used to phenotype mice. This assay can be performed rapidly with a diverse range of different visual stimuli and it evaluates central nervous system function under a range of experimental contexts — including, but not limited to, visual system dysfunction. In summary, I present a novel computer assisted assay and quantification of the OKR as a fast and reliable behavioral test that is worthy of inclusion in the standard repetitoire of central nervous system diagnostic procedures. 8 References: 1. Crawley JN (1999) Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Research 835:18-26. 2. Prusky GT, Alam NM, Beekman S, Douglas RM (2004) Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system. Invest Ophthalmol Vis Sci 45:4611-6. 3. Redlin U, Hattar S, Mrosovsky N (2005) The circardian Clock mutant mouse: impaired masking response to light. Journal of Comparative Physiology 181:51-59. 4. Hattar S, Lucas RJ, Mrosovsky N, Thompson S, et al. (2003) Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424:76-81. 5. Jacobs GH, Williams GA, Cahill H, Nathans J (2007) Emergence of Novel Color Vision in Mice Engineered to Express a Human Cone Photopigment Science 315:1723-1725. 6. Prusky GT, Douglas RM (2004) Characterization of mouse cortical spatial vision. Vision Res 44:3411-8. 7. Smallwood PM, Olveczky BP, Williams GL, Jacobs GH, et al. (2004) Genetically engineered mice with an additional class of cone photoreceptors: implications for the 9 evolution of color vision. Proc Natl Acad Sci USA 100:11706-11. 8. Simpson JI (1984) The accessory optic system. Annu Rev Neurosci. 7:13-41. 9. Bi A, Cui J, Ma YP, Olshevskaya E, et al. (2006) Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron 50:23-33. 10 CHAPTER ONE The optokinetic reflex as a tool for quantitative analyses of nervous system function in mice: application to genetic and drug-induced variation PLoS One, 2008 11 Summary The optokinetic reflex (OKR)f which serves to stabilize a moving image on the retina, is a behavioral response that has many favorable attributes as a test of CNS function. The OKR requires no training, assesses the function of diverse CNS circuits, can be induced repeatedly with minimal fatigue or adaptation, and produces an electronic record that is readily and objectively quantifiable. We describe a new type of OKR test apparatus in which computer-controlled visual stimuli and streamlined data analysis facilitate a relatively high throughput behavioral assay. We used this apparatus, in conjunction with infrared imaging, to quantify basic OKR stimulus-response characteristics for C57BL/6J and 129/SvEv mouse strains and for genetically engineered lines lacking one or more photoreceptor systems or with an alteration in cone spectral sensitivity. A second generation (F2) cross shows that the characteristic difference in OKR frequency between C57BL/6J and 129/SvEv is inherited as a polygenic trait. Finally, we demonstrate the sensitivity and high temporal resolution of the OKR for quantitative analysis of CNS drug 12 action. These experiments show that the mouse OKR is well suited for neurologic testing in the context of drug discovery and large-scale phenotyping programs. 13 Introduction The rapid growth in the number and variety of behavioral studies of mice — in the contexts of forward genetic screens, targeted mutagenesis, or preclinical drug testing - has put a premium on developing methods for quantifying nervous system function in this species [1-4]. In humans, the classic neurologic examination relies on eliciting specific motor responses to assess not only the motor system itself but also sensory and cognitive processes upstream of the motor system [5]. In mice, simple motor tasks such as grip strength and facility on a rotorod are routinely used to monitor basic neuromuscular function, and in the latter case, also cerebellar and vestibular functions [6]. However, many behaviors, such as the amount and pattern of movement within a cage, show significant variability on repeated trials and/or between genetically identical mice and can only be reliably quantified by averaging over a large number of observations [7]. Other behaviors, such as those involved in learning and memory, can only be reliably assessed after a period of training. 14 In mice, several visually-evoked physiologic and behavioral responses have been used to assess motor function, cognition, and memory, as well as visual system function itself. In anesthetized mice, the light response of the outer retina, includi
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