ELUCIDATING THE NEURAL CIRCUIT RESPONSIBLE FOR CHEMOTAXIS IN TRITONIA EXSULANS BERGH, 1894 _________________ A University Thesis Presented to the Faculty of California State University, East Bay _________________ In Partial Fulfillment of the Requirements for the Degree Master of Science in Biological Science _________________ By Samantha Zacarias May 2021 Copyright © 2021 by Samantha Zacarias ii Abstract The sea slug, T. exsulans (synonymous with Tritonia diomedea in the literature) is an ideal model organism for understanding neural pathways through which olfactory sensory information is transduced into a motor response. The goal of this research was to describe the chemosensory neural pathway, beginning with the olfactory nerves that relay information about odorant contact with sensory receptors on the rhinophores and ending with a motor response in the form of a change in direction of the slug’s movement. It was initially hypothesized that sensory fibers in Lateral Cerebral Nerve 1 (LCN1) that receive sensory cues from the rhinophores come into direct contact with the neurites of the potentially turn-inducing Pedal 3 Motor Neuron (Pd3). Microscopy results show that in all successful preparations, the fluorescent dyes introduced into LCN1 and Pd3 do not colocalize when imaged under confocal microscopy. A single preparation showed a <0.5µm distance between the two fluorophores while the remainder showed an average distance of 257µm ±193. It can be stated that there may be a monosynaptic connection between Pd3 and LCN1, despite the absence of extensive colocalization. Backfills done on LCN1 show cell body clusters in the pleural (Pl) and cerebral (Ce) ganglia with a single cluster of 1-3 cell bodies and neurites in the anterior medial edge of the pedal (Pd) ganglion in the neuropil near the commissure between the pedal and pleural ganglia Fluorophore injections of Pd3 have shown the axon exiting the Pd ganglion via Pedal Nerve 3 (PdN3) and sometimes show local neurites extending ventrally into the neuropil. iii Our experiments show that the sensory circuit for chemotaxis may be a monosynaptic sensory transmission pathway. iv ELUCIDATING THE NEURAL CIRCUIT RESPONSIBLE FOR CHEMOTAXIS IN TRITONIA EXSULANS BERGH, 1894 By Samantha Zacarias Approved: Date: Electronic Signatures Available May 14, 2021 ______________________________ ________________________ Dr. James A. Murray ______________________________ ________________________ Dr. Maria Gallegos ______________________________ ________________________ Dr. Brian Perry v Acknowledgments Thanks to my advisor Dr. Murray and my committee members Dr. Gallegos and Dr. Perry for your academic support. Thanks to my significant other, DJ Schuessler Jr. and our dog Merlin for keeping me sane and always being there for me. This project was directly funded by the CSUEB Center for Student Research and the confocal microscope used for this research was funded by the Keck Foundation. vi Table of Contents Abstract iii Acknowledgments vi List of Figures viii List of Tables ix Introduction 1 Materials and Methods 5 Overview of Procedure 5 Animals and Their Care 10 Tritonia Cerebral Nerve Backfill Protocol 12 Tissue Preparation for Slide Mounting Protocol 15 Imaging and Analysis 17 Results 20 Pedal 3 Verification 20 Dye Injection and Backfill Imaging Results 24 Discussion 40 References 43 vii List of Figures Figure 1 Semi-intact whole animal preparation of T. exsulans. 9 Figure 2 Two T. exsulans housed at the CSU East Bay campus. 11 Figure 3 T. exsulans brain after undergoing backfill procedure. 14 Figure 4 Electrophysiological verification of Pd3. 22 Figure 5 Experiment 20151210 movement characterization of Pd3. 23 Figure 6 Maximum intensity z-projection of a typical successful right and 27 left Pd3 dye injection. Figure 7 Maximum intensity z-projection of a typical successful right and 29 left LCN1 backfill. Figure 8A Maximum intensity z-projection for experiment 20140723. 31 Figure 8B Maximum intensity z-projection inset of region highlighted with 33 arrow in figure 8A from experiment 20140723. Figure 8C Magnified maximum intensity z-projection inset of touching 34 neurites from experiment 20140723. Figure 8D Maximum intensity z-projection for experiment 20151208. 35 Figure 8E Maximum intensity z-projection for experiment 20151210. 36 Figure 8F Maximum intensity z-projection for experiment 20160209. 37 viii List of Tables Table 1 Leica SP8 confocal microscope scan setting details listed by 19 experiment ID. Table 2 Measurements of distance between neurites of LCN1 and Pd3. 38 Table 3 Pearson’s correlation coefficients (r) for all experiments. 39 ix 1 Introduction All brains are composed of the same fundamental cellular building blocks— neurons and glia—that use the same signaling mechanisms—neurotransmitters and action potentials—whether they be vertebrate or invertebrate brains. This makes it possible for research to be conducted on simpler invertebrate brains with fewer cells and connections, and for the conclusions derived from that research to be usefully applied to more complex vertebrate systems. The sea slug, T. exsulans (synonymous with Tritonia diomedea in the literature; Korshunova & Martynov, 2020) has been used as a neuroethological model organism for the past 50 years due to a simpler nervous system that lends itself to research with large, colorful, identifiable cell bodies that are consistent in location across individuals (Willows et al., 1973). The Tritonia brain contains relatively few cells (~7,000 cells) which are very large (up to 800 µm), re-identifiable, and have distinct coloration that allows for single cell discernibility, making Tritonia’s nervous system a prime candidate for electrophysiological study. T. exsulans has historically been used to gain insight into the neuroscientific fundamentals of locomotion, feeding, escape response, and sensory systems (Dorsett et al., 1973; Field & Macmillan, 1973; McCullagh et al., 2014; Murray et al., 2006; Murray et al., 1992, 2011; Redondo & Murray, 2005; Willows, 1978; Wyeth & Willows, 2006a, 2006b). Prior research on locomotion, sensory systems, and behavioral field observation has shown that Tritonia uses a combination of odor and 2 water flow as navigational guidance cues in the first confirmed instance of an odor-gated rheotactic navigational strategy in gastropods (McCullagh et al., 2014; Wyeth & Willows, 2006a, 2006b). This unique navigational method and the convenient suitability of the model organism highlight the need to unravel the underlying neural circuitry involved. Understanding the neural circuit uniting olfaction and directional changes in locomotion of Tritonia exsulans is a first step in the direction of understanding one of the fundamental aspects of Tritonia’s unique and complex navigational strategy. Tritonia use antennae-like structures called rhinophores to sense odors in seawater and have been shown to respond to the odors of prey, predators, and conspecifics with an appropriate change in direction of locomotion (Field & Macmillan, 1973; Willows, 1978; Wyeth et al., 2006; Wyeth & Willows, 2006a). Medial Cerebral Nerve 1 carries motor neurons to the rhinophores while sensory information is sent from the rhinophores to the brain via Lateral Cerebral Nerve 1 (LCN1) (Willows et al., 1973). This input from LCN1 produces output from the brain in the form of a change in direction of motion which is thought to be mediated by a pair of neurons known as Pedal Motor Neuron 3 (Pd3) (Redondo & Murray, 2005). The Pedal 3 motor neuron has been found to be active during turning in response to water flow and may also be necessary to elicit turning (Murray et al., 2006; Redondo & Murray, 2005). Due to its essential role in turning, it is suspected that Pd3 plays a role in the neural circuit responsible for chemotactic navigation. However, Pd3 has not yet been directly associated with turning in response to odorants in the chemotactic pathway. 3 It is currently not known how odor directionality is encoded at the neural level and how motor neurons respond to that encoded direction. Thus, this thesis project generally aims to clarify the neural circuity involved in the processing of sensory information to reach adaptive goals. Tritonia exsulans orients to tidal flow direction, the geomagnetic field, and to odor sources, so we expect this work to contribute to the development of T. exsulans into a model system for marine navigation (Lohmann & Willows, 1987; J.A. Murray & Willows, 1996; Willows, 1978; Wyeth & Willows, 2006b). In addition, since behavior drives ecology, understanding how this slug’s behavior is created at the level of neurons builds a foundation for further discovery of the ecological role that this sea slug and others like it occupy. Understanding the ecology of all marine organisms is essential towards achieving the preservation of a marine ecosystem that is undergoing rapid changes in temperature, pH, nutrients, and dissolved gasses. The specific focus of this thesis project was to understand the neural mechanism by which olfactory input affects locomotor output in the opisthobranch slug, Tritonia exsulans. We hypothesized that the sensory afferents with axons in LCN1 and Pd3 together form a monosynaptic junction in the sensory transmission pathway of the nudibranch’s chemotactic neural circuit. We utilized confocal microscopy along with two different fluorophores to image the neurites of LCN1 and Pd3 and predicted that at low resolution, LCN1 and Pd3 should show extensive co-localization of the two