Optomotor Response Reduced by Procaine Injection in The
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OPTOMOTOR RESPONSE REDUCED BY PROCAINE INJECTION IN THE CENTRAL COMPLEX OF THE COCKROACH, BLABERUS DISCOIDALIS BY: MALAVIKA KESAVAN Submitted in partial fulfillment of the requirements For the degree of Master of Science Dissertation Adviser: Dr. Roy Ritzmann Department of Biology CASE WESTERN RESERVE UNIVERSITY January, 2014 1 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Malavika Kesavan candidate for the Master of Science degree *. (Committee Chair) Dr. Jean Burns Dr. Roy Ritzmann Dr. Mark Willis Dr. Barbara Freeman (date) 08/28/2013 2 TABLE OF CONTENTS Abstract ……………………………………………………………………………….. 8 INTRODUCTION ...………………………………………………………………….. 9 Basic anatomy of the B. discoidalis …………………………………………………. 11 External Anatomy …………………………………………………………….. 11 Internal Anatomy ……………………………………………………………... 11 The Head Ganglia in Insects ………………………………………………….. 12 The Central Complex: Anatomy ……………………………………………… 14 Focus of project ……………………………………………………………………… 16 Basic Visual Information Flow in Insects ………………………………………….. 16 Anatomy ……………………………………………………………………… 17 Basic Visual Movement Detection ………………………………………….... 18 The Central Complex ……………………………………………………………….. 19 Comparative Approach lends Insight into function of Central Complex .......... 19 Overview of Central Complex Development ………………………………… 19 The Central Complex's influence on Spatial Orientation …………………….. 21 The Central Complex Role in Changing Motor Behavior……………………… 22 The Central Complex and Memory …………………………………………... 25 Motor Behavior ……………………………………………………………………… 26 Locomotion …………………………………………………………………… 26 Escape Behavior ……………………………………………………………………... 28 Procaine ……………………………………………………………………………… 30 Significance of Project ………………………………………………………………. 31 3 METHODS …………………………………………………………………………... 31 Housing ……………………………………………………………………….. 31 Phase 1: Pre-trial …………………………………………………………….. 32 Phase 2: Injections ……………………………………………………………. 32 Phase 3: Trial ………………………………………………………….……… 33 Phase 4: Histology .…………………………………………………………... 34 Electrophysiology …………………………………………………………….. 36 Escape Behavior ……………………………………………………………… 37 Data Processing ………………………………………………………………. 37 Escape Response Analysis ………………………………………….. ………. 40 Post Injection Behavior ………………………………………………………. 41 Errors in Data Analysis ……………………………………………………….. 41 RESULTS …………………………………………………………………………… 43 Controls ……………………………………………………………………….. 43 The Procaine Effect on Optomotor Response ………………………………... 45 Activity Levels ................................................................................................... 50 Escape Response ……………………………………………………………… 51 Extracellular Brain Recording ……………………………………………….. 53 DISCUSSION ……………………………………………………………………….. 54 Central Complex Influence on Optomotor Response ………………………... 55 Effect of Procaine on Central Complex ………………………………. 55 Effect of Procaine on Behavior ………………………………………. 56 Activity Levels ………………………………………………………………... 57 4 Escape Response ……………………………………………………………… 58 CONCLUSION ……………………………………………………………………... 60 Future Work for Next Student ……………………………………………………... 61 Acknowledgments …………………………………………………………………… 62 References ……………………………………………………………………………. 63 5 LIST OF FIGURES AND TABLES Figure 1: Cockroach Central Nervous System ………..……………………………….. 12 Figure 2: Representation of the central complex images in 3D ………..….………….. 14 Table 1: Summary Table of central complex lesions ………………………………….. 25 Figure 3: Schematic of experimental set up (side-view) . ..…………………………… 31 Table 2: Histological Process …………………………………………………………. 35 Figure 4: Confocal Images of B. discoidalis brain with dye ………………………….. 36 Figure 5: Example of raw output ……………………………………………………… 38 Figure 6: Data processing method ……………………………………………………... 38 Figure 7: Example Angular Outputs ………………………………………………….. 40 Table 3: Summary of Experimental Groups ………………………………………….. 40 Figure 8: Examples of False Negatives ………………………………………………. 42 Figure 9: Examples of False Positives ………………………………………………... 43 Figure 10: Optomotor Responses for untreated cockroaches …………………..…… 44 Figure 11: Optomotor Responses for cockroaches with sham surgery ……….…….... 45 Table 4: Turn Response comparison between saline and 20% Procaine ……………... 46 Figure 12: Optomotor Responses for cockroaches injected with saline …………….... 47 Figure 13: Optomotor Responses for cockroaches injected with procaine …………… 48 Figure 14: Optomotor for cockroaches injected with 10% procaine …………………. 49 Table 5: Turn response comparison between saline and 10% procaine trials ………... 49 Figure 15: Comparison of optomotor response in all experimental groups …………... 50 Table 6: Average time spent Active during trial ……………………………………… 51 Figure 16: Average speed of escape for cockroaches under the influence of 20%....….. 52 6 Figure 17: 20% Procaine influence on central complex spiking activity …………….. 54 Figure 18: 10% Procaine influence on central complex spiking activity …………….. 54 7 Optomotor Response Reduced by Procaine Injection in the Central Complex of the Cockroach, Blaberus Discoidalis By: MALAVIKA KESAVAN Abstract The central complex is a group of midline neuropils found in all arthropods. In insects, circuits within the central complex play a role in processing visual and tactile information. Activity recorded in the central complex is correlated with and often precedes changes in step frequency and turning movement. Electrolytic lesions in the central complex adversely affect various locomotory behaviors. However, questions exist about collateral damage to areas outside of the central complex as the lesion probe was inserted. To address this issue, we developed a reversible chemical block of neural activity in the central complex using the anesthetic procaine. Black and white moving stripes induced an optomotor turning response in the cockroach, Blaberus discoidalis. After documenting the response in a normal individual, we injected a solution of 10% or 20% procaine into the central complex. Subjects with anesthetized central complex had a significant decrease in optomotor response up to 30 minutes after injections. Controls injected with saline showed no deficit. As the effects of the anesthetics diminished, the cockroach regained the ability to turn. While the optomotor response processing happens mostly within the cockroach brain, most escape circuitry is located in the thoracic ganglia. Nevertheless, the brain has been shown to influence escape response. We, therefore, extended our analysis to examine the effects of the procaine injection into the central complex on escape. Subjects with reduced output from the central complex 8 showed little deficit in escape speed. Electrophysiological recording showed that procaine reduced firing activity in the central complex. Introduction Cockroaches such as Blaberus discoidalis, a member of the arthropod phylum, are notoriously good at moving quickly in unpredictable and novel terrain. Despite their reputation as “simple” creatures most robotic work has been unable to span behavioral adaptability of these animals. How is the cockroach able to incorporate the vast complexity of its environment into its choices? The central complex has emerged as a leading contender as a higher order motor planning area in insects (Strauss & Heisenberg, 1993). The central complex receives multimodal sensory information ultimately outputting to the lateral accessory lobes where it can affect the neurons descending to the thoracic ganglia (Martin et al., 1999). The aim of my thesis project was to adapt a reversible block for examining the role of the central complex in controlling specific aspects of locomotor behavior. This technique was used to complement work done with electrolytic lesion in the central complex by a previous student in the laboratory. Electrolytic lesions in the central complex adversely affect various locomotory behaviors including turning; however, questions existed about collateral damage to areas outside of the central complex when the lesion probe was inserted(Harley & Ritzmann, 2010;Ritzmann et al., 2012). Though several controls were done to ensure that the surgery was not the cause of the behavior deficit, having a reversible neural effect would confirm these results. To address this issue, a reversible block of neural activity in the central complex was developed for this preparation using the local anesthetic, procaine. We believe refined versions of this 9 technique can be used to subtly manipulate central complex circuits allowing us to map information flow and ultimately further the understanding of the function of the central complex. In my experiments, an optomotor turning response was induced by displaying vertical black and white stripes that were moving horizontally in the visual field of B. discoidalis cockroaches while they were tethered on an air suspended ball. After documenting this response in normal individuals, a solution of 20% procaine was injected into the central complex. Subjects treated with procaine retained the ability to walk, though they rarely responded to moving stripes stimulus. On average, cockroaches with the anesthetized central complex showed a significant decrease in response to the visual stimuli up to 30 minutes after the injection. Cockroaches injected with saline showed no such deficit. Furthermore, cockroaches with no surgery performed on them acted in a similar manner to those injected with saline. As the effects of the anesthetic in the central complex diminished the animal regained the ability to respond to the visual stimuli