Elucidating the Neural Circuit Responsible For

ELUCIDATING THE NEURAL CIRCUIT RESPONSIBLE FOR

CHEMOTAXIS IN TRITONIA EXSULANS BERGH, 1894

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A University Thesis Presented to the Faculty

California State University, East Bay

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In Partial Fulfillment

of the Requirements for the Degree

Master of Science in Biological Science

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Samantha Zacarias

May 2021

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.

ELUCIDATING THE NEURAL CIRCUIT RESPONSIBLE FOR

CHEMOTAXIS IN TRITONIA EXSULANS BERGH, 1894

Samantha Zacarias

Approved: Date:

Electronic Signatures Available May 14, 2021 ______Dr. James A. Murray

______Dr. Maria Gallegos

______Dr. Brian Perry

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.

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

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

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.

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 different fluorophores introduced into LCN1 and Pd3. If the neurites were found to be close, but sufficiently resolved to not colocalize, it would still be possible for synapses to exist. In

this case, our ability to depict the synapses would be limited by our visualization techniques.

It is important to note that LCN1 not only contains sensory afferents, but also motor neuron axons that cause rhinophore withdrawal (Willows et al., 1973). Despite this, any potential synapses with Pd3 would more likely be from LCN1’s sensory afferents. Since it is not possible to confidently identify dendrites and axon collaterals specifically from just morphology in Tritonia, we did not aim to identify the types of neurites, so any neurite overlap would be deemed a potential synapse. If colocalization occurred as predicted, it could indicate that the two fluorophores could not be identified as occupying distinctly separate locations within the xyz resolution limits of the microscope, or it could result from overlap in two fluorophores that are adequately resolved. The smallest resolution implemented in this research was 0.260µm x 0.260 µm x 1.38 µm. This is much larger than the likely size of a Tritonia synapse (20nm in

Aplysia), therefore, detecting both fluorophores in the same location would have implied the possibility of a synaptic junction between LCN1 and Pd3 neurites, but would not be definitive evidence of synaptic contact (Lloyd & Church, 1994). Synapses would then have had to be confirmed through electrophysiological methods, or morphologically with a super resolution or TEM follow up imaging study.

Materials and Methods

Overview of Procedure

Semi-intact, whole-animal preparations as described in Willows et al. (1973a) were used for this thesis project. For this type of live animal preparation, a small posterior to anterior incision was made just above the brain with sharp dissection scissors, taking care to only penetrate through the body wall, avoiding damage to the layers of connective tissue and muscle that encase and adhere to the brain. Hooks anchored to the inner plexiglass tank walls of the double-walled tank were then used to spread the incision and suspend the nudibranch in Instant Ocean (Spectrum Brands; Virginia, USA) artificial sea water (28-30 ppt). Artificial sea water was cooled to 4-10°C with icy tap water flowing between the double walls of the tank. Degree of cooling varied depending on how long the ice water pump system had been running. Allowing the sea water temperature to drop to 5°C and lower helped to cool the nudibranchs’ tissue enough to make them slightly less active and less prone to attempt escape swims which would dislodge an intracellularly embedded glass pipette, destroy the cell body of interest, and even completely impale the brain on the glass electrode. This degree of cooling did not

damage the tissue of the slug or compromise identification of cells via motor response to positive current (Katz et al., 2004).

Next, another small incision was made of the connective tissue and muscle, dorsal and posterior to the brain. A small, oval, wax-covered, non-magnetic, stainless steel platform was then slipped ventral to the brain and dorsal to the esophagus. The platform was wide enough to support the ~6 mm brain and was attached to a micromanipulator outside of the tank by a long, stainless steel arm. Once in position, the platform was raised slightly to pull the brain taught before pinning it to the wax covered platform with stainless steel insect pins. Special care was taken to not stick pins through nerves or portions of the ganglia, but instead through the connective tissue that envelopes the nerves and brain. Using this method allowed the brain to be sufficiently stable to permit intracellular recording and fluorophore injection for periods of many hours, despite the frequent escape attempts made by the animal. See Figure 1 for clarifying images of the semi-intact preparation set-up.

With the slug’s brain immobilized, Pd3 was then identified by location and morphology as previously established in the literature (Murray et al., 1992). The suspected Pd3 cell body was then delicately pierced with a glass microelectrode loaded with AlexaFluor hydrazide (Invitrogen Corporation; California, USA) diluted in 200 mM

KCl. Glass microelectrodes were made with a Sutter P-87 puller (Sutter Instrument;

California, USA) and thin-walled borosilicate glass capillaries. Filled electrode resistance ranged from 1-25 MΩ (million Ohms). Higher resistance corresponded to narrower, sharper glass tips. If tips were made too narrow, they could break or produce

reduced flow of the fluorophore into the cell. 2.5 nA of negative current was immediately administered to the cell to stop injury spiking and to allow for the phospholipid bilayer to seal around the tip of the electrode. If injury spiking was successfully controlled, intracellular recordings of the transmembrane electrical potential of motor neuron Pd3 were done using a PowerLab 26T and LabChart v.7 software

(ADInstruments; Colorado, USA), paired with a Dagan IX2-700 Dual Intracellular

Preamplifier (Dagan Corporation; Minnesota, USA). The goal was to confirm that the putative Pd3’s intracellular activity matched the established Pd3 activity as is described in the literature (Murray et al., 1992; Redondo & Murray, 2005). Positive current was administered with the Dagan IX2-700 through the glass electrode to induce action potentials in the hopes of capturing the characteristic motor effects of Pd3 on a Canon

VIXIA HV30 HD Camcorder mounted on a small flexible tripod just outside of the sea water tank. The purpose of capturing the cell’s motor effects was to again verify the identity of the putative Pd3 cell body.

After Pd3’s identity was established, it was then iontophoretically injected with

AlexaFluor hydrazide diluted in 200 mM KCl. AlexaFluor hydrazide consists of a negatively charged molecule conjugated to a fluorophore, in our case with peak excitation at either 488 nm or 555 nm. The purpose of the intracellular fluorophore injection was to highlight all of Pd3’s neurites under confocal microscopy.

Once the motor output portion of the neural circuit was prepared as described above, the entire brain, consisting of the pleural, cerebral, and pedal ganglia was dissected out of the slug for the preparation of the sensory input portion of the neural

circuit. During dissection, the cerebral nerves were cut as distally as possible to ensure sufficient length for Lateral Cerebral Nerve 1 (LCN1) to be backfilled. When possible, both left and right LCN1 of the same brain were backfilled with 10% neurobiotin.

Preliminary experiments used 2% neurobiotin, only to find that concentration was not sufficient to get penetration into the nerve and neurites were not well defined when imaged. Neurobiotin was then labeled for imaging with Streptavidin conjugated to

AlexaFluor 633 (Invitrogen Corporation; California, USA), an excitation wavelength distinct from that of the fluorophore injected into Pd3. The purpose of the backfill was to illuminate all of LCN1’s neurites once imaged under confocal microscopy (see backfill protocol below). After backfilling was completed, T. exsulans brains were processed according to the protocol below for preparation of tissue for slide mounting.

The injected fluorophore and the fluorescent streptavidin were finally imaged with a Leica SP8 confocal microscope (see Imaging and Analysis section for details).

Images were analyzed for colocalization with ImageJ. The entire procedure was replicated over 50 times with varying degrees of success. Portions of the procedure were practiced countless times on many individuals of various sea slug species, including

Armina californica, Tochuina gigantea, and Dendronotus sp. All portions of the procedure were eventually successfully completed on a single brain only a few times due to the extreme difficulty of the various techniques required. The purpose of this was to determine if interaction between Pd3 and LCN1 neurites occurred as was hypothesized.

A C

Figure 1. Semi-intact whole animal preparation of T. exsulans. A. Overview of lab set up for semi-intact animal preparation. B. Detail of hook set up on double walled plexiglass tank. C. Detail of pinned T. exsulans brain on wax covered platform. All images taken by S. Zacarias.

Animals and Their Care

T. exsulans were collected via SCUBA by J.A. Murray from the waters of Ilahee and Langley, Washington, or from Tofino, British Columbia by Living Elements, LTD

(British Columbia, Canada). The animals were housed on the CSU East Bay campus in an aquarium outfitted with a UV sterilizer and carbon and biological filters.

Temperatures were maintained around 9℃-10℃. Artificial sea water was prepared with

Instant Ocean and salinity was maintained at 28±2‰. Animals were kept for up to 3 months on a diet consisting mostly of frozen sea pen slices (Ptilosarcus gurneyi) (live sea pens were occasionally delivered with the sea slugs) and were only sacrificed once they were deteriorating and near the end of their life (Figure 2).

Figure 2. Two T. exsulans housed at the CSU East Bay campus. A portion of a live sea pen can be seen in the background. Image taken by S. Zacarias.

Tritonia Cerebral Nerve Backfill Protocol

The dissected brain was first pinned out by the connective tissue in a Sylgard

(Dow Chemical Company; Michigan, USA) elastomer-lined petri dish with insect pins while bathed in cold artificial seawater (ASW) from the semi-intact preparation tank.

The brain was pinned such that the nerves and ganglia would lay flat and stretched out as they would inside of the sea slug. The outer connective tissue encasing the brain and the nerves was then very carefully removed with fine forceps and Vannas scissors. Fresh, cold ASW was added to the dish as needed to keep the brain alive. All nerves except for the right and left LCN1s were trimmed short while the nerves of interest remained long.

The medial branch of Cerebral Nerve 1 was also trimmed short.

An unbroken line of a Vaseline (Unilever, England) and mineral oil mixture was dispensed via a blunt syringe needle along the bottom of a 30x10mm petri dish bottom

(or top) such that the dish was divided into two parts by the hydrophobic barrier. A droplet of ASW was placed on each side of the barrier, with the drop being placed close enough to almost touch the hydrophobic barrier.

The freshly cleaned Tritonia brain was then added to the droplet on one side, making sure to break the surface tension of the ASW droplet to fully submerge the brain and nerves. The distal end of each LCN1 was firmly gripped with a coarse pair of forceps and gently lifted over and across the hydrophobic barrier to the ASW droplet on

the other side. Most of the ASW was then wicked away with a Kimwipe (Kimberly-

Clark Corporation; Texas, USA) on both sides.

Another layer of the Vaseline and mineral oil mixture was then applied in an unbroken line on top of the first layer, making sure to sandwich the nerve that was pulled across. This was done to ensure a watertight seal around the nerve to prevent the neurobiotin from leaking onto any other parts of the brain. A couple of drops of ASW were quickly added again to both sides of the barrier.

The distal end of the LCN1 that was damaged from being grasped by the forceps was then trimmed in one fluid cut with Vannas scissors. The ASW was gently wicked away from the nerve side and a drop of DI water was added. The nerve soaked in DI water for 30-60 seconds to fray open the freshly cut nerve ending for proper absorption of the neurobiotin. Once the nerve ending was frayed, the DI water was wicked away and a couple of drops of ASW were added to re-stabilize the nerve.

The corner of a Kimwipe was cut off and dipped in 10% neurobiotin diluted in

100mM KCl with 2% fast green. Most of the ASW was again wicked away from the nerve, ensuring that enough was left behind to keep the nerve moist without diluting the neurobiotin. The neurobiotin soaked Kimwipe was then finally placed on top of the nerve ending. If completed correctly, this point in the backfill procedure looked like the image in Figure 3.

The entire petri dish half was placed in a sealed, lidded plastic container with a moist paper towel lining the bottom to maintain moisture of the tissue. The container was

then refrigerated overnight, being careful to not subject it or the refrigerator to any jarring motions that could cause spillage of neurobiotin over the Vaseline barrier.

On the next day, the tissue was removed from the refrigerator and left in the plastic container at room temperature for 1-4 hours. The backfill was then ended by cutting the LCN1 off from the rest of the brain on the side of the barrier without neurobiotin.

Figure 3. T. exsulans brain after undergoing backfill procedure. The Vaseline and mineral oil barrier clearly separate the ends of right and left LCN1 (bathing in neurobiotin) from the rest of the brain. Great care was taken to shield the dish from jarring motions that would cause the neurobiotin to spill over the barrier. Image taken by

S. Zacarias.

Tissue Preparation for Slide Mounting Protocol

Once the backfill was completed, the brain was pinned out in a Sylgard bottom petri dish and rinsed 3 times with artificial sea water (ASW), for 30 minutes each while on an orbital shaker. Any remaining connective tissue was removed from the surface of the brain and nerves. ASW was then replaced with 4% paraformaldehyde in Tris- buffered ASW to fix the brain. The petri dish with the tissue was then placed back into the sealed, lidded plastic container and returned to the refrigerator to soak overnight for a period of no more than 24hrs. If time allowed, the tissue was left to soak in the container at room temperature for 4-6hrs.

Once sufficient time had passed, the tissue was removed from the paraformaldehyde solution and rinsed by placing on an orbital shaker in a polypropylene microcentrifuge tube with 0.1M phosphate buffered saline (PBS) at a pH of 7.2–7.4. This was done a total of three times for 30 minutes each.

Once the brain was thoroughly rinsed, the PBS solution in the microcentrifuge tube was replaced with 4% Triton X-100 (Roche; Indiana, USA) in PBS (PBTX). The tissue was then refrigerated overnight and allowed to soak to remove the hydrophobic, orange pigmentation with the detergent solution. On the next day, the 4% PBTX was replaced with a 0.3% PBTX rinse solution. The brain was rinsed with 0.3% PBTX a total of three times for 30 minutes on an orbital shaker.

Once the detergent rinses were completed, the brain was removed from the microcentrifuge tube containing the 0.3% PBTX. The brain was then transferred to a microcentrifuge tube containing either 1:100 or 1:200 Streptavidin conjugated with

AlexaFluor 633, diluted in 0.3% PBTX for three days. For experiment 20140723, an additional fourth day was done of soaking in Streptavidin solution.

After 3 days, the tissue was removed from the Streptavidin solution and rinsed on an orbital shaker in a new microcentrifuge tube with 0.3% PBTX a total of six times for

30 minutes. Care was taken to cover the tube containing the tissue with foil while on the shaker to minimize fluorophore photo-bleaching. Once the rinses were completed, the tissue was soaked in a fresh rinse of 0.3% PBTX while refrigerated overnight. If the next set of rinses could not be completed on the next day, the tissue could stay refrigerated in

0.3% PBTX for several days.

Next, the brain was again rinsed on an orbital shaker with 0.3% PBTX a total of six times for 30 minutes. After this second day of rinses, the brain was again soaked in a fresh rinse of 0.3% PBTX overnight while refrigerated. The tissue was again permitted to remain in this state for several days if necessary.

To prepare the brain for dehydration, it was next rinsed by replacing the PBTX in the microcentrifuge tube with DI water twice for 15 minutes each on the orbital shaker.

The dehydration of the tissue with Ethanol (EtOH) then began with 9 consecutive, 10- minute rinses in each of the following EtOH dilutions: 30%, 50%, 50%, 70%, 90%, 95%,

95%, 100%, and 100%.

After dehydrating, the brain was cleared with one 10-minute soak in a 1:1 solution of xylene and 100% EtOH, then soaked for another 10 minutes in xylene only. Finally, the tissue was mounted in DPX (Sigma-Aldrich; Missouri, USA; refractive index of 1.52) between two #1.5 thickness glass coverslips for imaging under a Leica confocal microscope. Coverslips were used on both sides since using a slide on one side would prevent imaging of the tissue from that side due to the thickness of the glass.

Imaging and Analysis

All imaging was completed with a Leica SP8 confocal laser scanning microscope

(Leica Microsystems, Germany). Due to the double fluorescent labeling and large size of the tissue of interest, images were created through sequential tile scans with lower magnification objective. Scan settings varied from one preparation to the next (refer to

Table 1 for imaging details that varied.), but all scans used an HC PL APO CS2 20x objective with oil immersion (refractive index of 1.518) and a numerical aperture of 0.75.

A pinhole aperture of 1AU for the smallest wavelength was used on all scans, along with laser intensity and photomultiplier tube (PMT) gain settings such that the full dynamic range of the detector was used with as low a laser intensity as possible. Image tiles were set to automatically merge in Leica Application Suite X (LAS X) software after scans

were completed. Images were processed in LAS X software to crop unnecessary layers of the Z stack and to remove any remaining cross-talk before colocalization analysis was completed in FIJI, running ImageJ2 (Rueden et al., 2017; Schindelin et al., 2012).

In FIJI, images for experiments 20151208, 20151210, and 20160209 were converted to maximum intensity z-projections for measurement of the 2-dimensional xy distance between fluorophores and the addition of 100 µm scale bars. Maximum intensity z-projections were used for ease of analysis when there was no overlap of fluorophores. Images then had a manual threshold applied to them to eliminate voxels with background and autofluorescence intensities. Images were then analyzed for colocalization with FIJI’s Coloc 2 plug-in. Coloc 2 determined Pearson’s correlation coefficients which were deemed sufficient to show lack of colocalization.

The image for experiment 20140723 was analyzed for measurement of fluorophore distance in LAS X instead of FIJI due to how close Pd3 and LCN1 neurites were. LAS X was used to determine a 3-dimensional distance between the two fluorophores and to create 3D renderings. Coloc 2 in FIJI was then used to evaluate a

Costes p-value in addition to a Pearson’s r value.

Table 1. Leica SP8 confocal microscope scan setting details listed by experiment ID.

Experiment Zoom Number Laser PMT Voxel Size Calculated XY

ID of Tiles Line Range XYZ (µm) and Axial

(nm) (nm) Resolution Limit

(µm)

20140723 1.25x 8 552, 562-580; 0.455 x 0.296x0.296x1.57;

638 700-775 0.455 x 0.338x0.338x1.80

1.041

20151208 1x 6 488; 498- 0.132 x 0.260x0.260x1.38;

638 541; 0.132 x 0.338x0.338x1.80

648-680 1.041

20151210 1x 3 488; 498- 0.173 x 0.260x0.260x1.38;

638 551; 0.173 x 0.338x0.338x1.80

658- 701 1.041

20160209 1x 4 488; 498- 0.284 x 0.260x0.260x1.38;

638 603; 0.284 x 0.338x0.338x1.80

648-748 1.041

Results

Pedal 3 Verification

It was rare to get both the movement associated with stimulation of Pedal 3 and the characteristic intracellular electrophysiological activity associated with Pd3 (most notably the square like post synaptic potentials) recorded on a preparation that also had the intracellular dye injection and backfill succeed. These results are limited to the experiments that had both successful dye injections and backfills.

Experiment 20140723 had a video file that was corrupted, so images of the movement characterization were lost. Despite the inability to depict the movement characterization, successful electrophysiological verification of the cell’s identity was completed as seen in Figure 4, in addition to visual verification by location, color, and size. It was noted that the noise levels in the electrophysiological recording for this experiment were just under 5mV versus the 1mV noise levels seen in the other experiments.

Experiment 20151208 suffered from severing of the pedal nerves when the initial incision to access the brain was made. Pedal Nerve 3 which contains Pd3’s motor axon, was amongst those damaged, so movement characterization could not be completed.

Despite the inability to characterize the movement, electrophysiological verification was successful as seen in Figure 4.

Experiment 20151210 was the only of the 4 successful preparations that had successful capture of the movement associated with stimulation of Pd3 (Figure 5) in addition to successful electrophysiological verification (Figure 4). The mid-posterior foot margin, as described in the literature, can be seen lifting approximately 2 seconds after stimulation began in a delayed response to approximately 10 seconds of stimulation of Pd3 with 5nA of positive DC (Zazay et al., 2012). Spike rate reached a maximum of

9.4 Hz and dropped over the stimulation period to 4.4Hz, displaying the spike frequency accommodation as described by Zazay et al., 2012. Peak foot margin lift was maintained for approximately 2.7 seconds. Total distance that the foot margin moved was estimated from the video taken to be approximately 19mm.

Experiment 20160209 was unique in that it was not possible to characterize Pd3’s movement since it was an isolated brain preparation. At times, slugs died overnight, and the brain was completely dissected out in the hopes that some electrical activity remained to make good use of the tissue. In this instance, the brain was so far gone that even though Pd3 remained alive, it was not in the best condition and piercing of the cell resulted in injury spikes that could not be controlled, so electrophysiological confirmation of Pd3’s identity was also not possible. We chose to include this experiment despite failing all but visual identification due to the rarity of having both the dye injection and backfill procedures succeed, which they did in this case despite the deteriorated state of the tissue.

Intracellular Intracellular (mV)

-40 Time (s) Pedal 3 3 Pedal Tt_20140723.adicht

07/23/2014 5:07:02.380 1 PM 2 3 4 5 6 7 5050 40 B

L intraL (mV) (mV) 0

20140723 20140723 0 -10

Pedal 3 Intracellular Intracellular 3 Pedal -20-20 Time (s) 43190 4320 1 4321 2 4322 3Tt_20151208.adicht 4323 4 4324 5 4325 6 4326 7 4327 12/8/2015 2:24:32.975 PM

-160-160 C

-180

My RIGHT (mV) -200

(mV) 20151208 20151208 -220-220

Time (s) Pedal 3 Intracellular Intracellular 3 Pedal 518730 51874 1 51875 2 51876 3 Tt_20151210.adicht 51877 4 51878 5 518796 518807 51881 12/10/2015 3:18:12.008 PM -60-60 D

-80

-100

MyRIGHT (mV)

151210 151210 (mV)

20 -120-120

Time (s) Pedal 3 Intracellular Intracellular 3 Pedal 0 55093 1 550942 55095 3 55096 4 55097 5 55098 6 55099 7 55100

Figure 4. Electrophysiological verification of Pd3. Segments of comparable duration were acquired from each experiment, rendering similar x axes. Y axes were highly variable from one experiment to the next due to variation in the health of the cell post

piercing and injection of negative current to help stabilize the cell. A. This panel shows the characteristic spontaneous activity associated with Pedal Neuron 3 and was adapted from Redondo and Murray, 2005. B, C, and D. Intracellular spontaneous activity recorded during experiments 20140723, 20151208, and 20151210. Traces show the

‘square-like’ PSPs that are typical of Pd3.

Stimulus Tt_20151210.adicht Stimulus 12/10/2015 3:25:35.000 PM 350350 Start End 300 250

200 150

100 (mV)

My RIGHT (mV) 50 0 0 -50

-100-100 Pedal 3 Intracellular Intracellular 3 Pedal

10 10

4 Channel 3 (Hz) 2

0 Time (s)

Spike Rate (Hz) SpikeRate 0

55535 55536 55537 55538 55539 55540 55541 55542 55543 55544 55545 55546 A 0 1 2 3 4 5 6 7 8 9 10 11

B C

Figure 5. Experiment 20151210 movement characterization of Pd3. A. Approximately

10 seconds of 5nA DC stimulation was administered to Pd3. Top panel shows intracellular spike activity under stimulation. Bottom panel shows concurrent spike rate

calculation in Hz. B. Foot margin highlighted with green brackets before lift occurs.

Left is anterior and right is posterior. Black rod is motion detector and is ~2mm wide. C.

Foot margin highlighted with green brackets at peak lift. Peak lift maintained for ~2.7 seconds. Total distance traveled by the foot margin was ~19mm.

Dye Injection and Backfill Imaging Results

Dye injection and backfill procedures were eventually successfully completed on the same brain only 4 times due to the difficulty of the various techniques required.

Figure 6 shows a full brain scan of a typical independently successful Pd3 dye injection and figure 7 shows a full brain scan of a typical independently successful LCN1 backfill.

These images are shown here to clarify Tritonia brain anatomy and for greater perspective of the pedal ganglion scans done of the four experiments highlighted in this thesis.

In 3 of 4 of the tissue preparations, fluorophores delineating the neurites of LCN1 and Pd3 were not sufficiently close to each other for 3-dimensional colocalization to even be a possibility since the fluorophores did not even overlap in the 2-dimensional xy plane when maximum intensity z-projections were made (Figure 8D-F).

In experiment 20140723, measurements made with LAS X showed no measurable distance between the two fluorophores but did not show fluorophore overlap in 3 dimensional renderings. Due to a voxel measuring 0.455 µm x 0.455 µm x 1.041 µm, we can say that the distance between Pd3 and LCN1 fluorophores was <0.5 µm. Figures 8A-

C show how very close neurites were in a maximum intensity z-projection.

Colocalization analyses with Coloc 2 in FIJI completed on the maximum intensity z-projections yielded Pearson’s r values (no threshold, since the images had manual thresholds set) ranging from 0.00 to -0.01, supporting the lack of visual fluorophore overlap in all four preparations (Table 3). The 3-dimensional z-stacks of experiment

20140723 were further analyzed with Coloc 2 in FIJI to yield a Costes p-value of 0.69, indicating a lack of significant colocalization despite there being no discernible distance between Pd3 and LCN1 fluorophores.

Distance between neurites of LCN1 and Pd3 were measured with FIJI and LAS X

(Table 2). Measurements show a large amount of variation in the xy distance between the respective neurites with an average of 257 µm with a standard deviation of 193 µm, and a range of 0-449 µm. Backfills of LCN1 show large clusters of associated cell bodies

(since LCN1 is a nerve) in the cerebral and pleural ganglia of the brain along with a smaller cluster of up to 3 tightly grouped cell bodies in the pedal ganglion. Some of these are likely sensory receptor cell bodies while others may be motor neuron cell bodies as described in the literature (Audesirk & Audesirk, 1980a, 1980b; Willows et al., 1973).

The backfills of LCN1 showed most fibers in the pedal ganglion extending ventrally

(away from the more dorsally located Pd3 neurites) into the neuropil in those locations.

Notably in experiment 20140723, a single backfilled neurite from an LCN1 associated cell extended dorsally into Pd3’s neurite field. Three punctata associated with the LCN1 neurite were within 500 nm of Pd3’s neurites. Dye injections of Pd3 consistently resulted in a single primary neurite, presumably tapering into an axon, extending out of the pedal ganglion via Pedal Nerve 3 and only local dendritic extensions if at all present. This is as expected since Pd3 is a single neuron, and the majority of Tritonia’s CNS neurons are unipolar (Willows et al., 1973).

Ce Pl

Figure 6. Maximum intensity z-projection of a typical successful right and left Pd3 dye injection. Anterior is towards the left edge. Scale bar in lower left corner is 300 µm wide. Dye injection of right and left Pd3 shown in bright magenta. Brightness and contrast have been adjusted to show detail. Straight lines are scan and mosaic artifacts.

The cerebral ganglia are labeled Ce, the pleural ganglia are labeled Pl, and the pedal ganglia are labeled Pd.

Figure 7. Maximum intensity z-projection of a typical successful right and left LCN1 backfill. Anterior is towards the left edge. Scale bar in lower left corner is 300 µm wide.

Backfill of right and left LCN1 shown in bright green. Brightness and contrast have been adjusted to show detail. Straight lines are scan and mosaic artifacts. White arrows point to right and left LCN1.

Figure 8A. Maximum intensity z-projection for experiment 20140723. Right pedal ganglion shown. Anterior is towards the top right corner. Scale bar in lower left corner is 100 µm wide. Dye injection of Pd3 shown in magenta. Backfill of LCN1 shown in green. Brightness and contrast have been adjusted to emphasize lack of colocalization.

Straight lines are scan and mosaic artifacts. The stub of LCN1 can be seen at the top of the image along with a cluster of associated cell bodies in the cerebral ganglion. Neurites from LCN1 and Pd3 are at their closest here, at <0.5 µm apart (white arrow). The axon of Pd3 fails to extend into Pedal Nerve 3; this is likely an artifact of fluorophore penetration failure.

Figure 8B. Maximum intensity z-projection inset of region highlighted with arrow in figure 8A from experiment 20140723. Anterior is towards the top right corner. Scale bar in lower left corner is 100 µm wide. Dye injection of Pd3 shown in magenta. Backfill of

LCN1 shown in green. Brightness and contrast have been adjusted to make detail more discernible. White arrow in this image draws attention to the location where the fluorophores from Pd3 and LCN1 make contact.

Figure 8C. Magnified maximum intensity z-projection inset of touching neurites from experiment 20140723. Anterior is towards the top right corner. Scale bar in lower left corner is 20 µm wide. Dye injection of Pd3 shown in magenta. Backfill of LCN1 shown in green. Brightness and contrast have been adjusted to make detail more discernible.

White arrow in this image again draws attention to the location where the fluorophores from Pd3 and LCN1 make contact.

Figure 8D. Maximum intensity z-projection for experiment 20151208. Right pedal ganglion shown. Anterior is towards the top of the image. Scale bar in lower left corner is 100 µm wide. Dye injection of Pd3 shown in magenta. Backfill of LCN1 shown in green. Brightness and contrast have been adjusted to emphasize lack of colocalization.

Straight lines are scan and mosaic artifacts. Autofluorescence makes visualization of the unlabeled tissue possible, clearly showing the axon from LCN1 crossing into the pedal ganglion through the commissure and the axon from Pd3 exiting the ganglion through

Pedal Nerve 3. A secondary cell body from the LCN1 backfill can also be seen.

Figure 8E. Maximum intensity z-projection for experiment 20151210. Left pedal ganglion shown. Anterior is towards the top of the image. Scale bar in lower left corner is 100 µm wide. Dye injection of Pd3 shown in magenta. Backfill of LCN1 shown in green. Brightness and contrast have been adjusted to emphasize lack of colocalization.

Pedal Nerve 3. A single cell body in the anterior pleural ganglion can also be seen.

Figure 8F. Maximum intensity z-projection for experiment 20160209. Left pedal ganglion shown. Anterior is towards the top of the image. Scale bar in lower left corner is 100 µm wide. Dye injection of Pd3 shown in magenta. Backfill of LCN1 shown in green. Brightness and contrast have been adjusted to emphasize lack of colocalization.

Straight lines are scan and mosaic artifacts. Autofluorescence makes visualization of the

medial boundary of the pedal ganglion along with visualization of the lateral boundary of the pleural ganglion possible.

Table 2. Measurements of distance between neurites of LCN1 and Pd3. Measurements were made in FIJI with a straight-line tool. Distance for 20140723 was confirmed in 3D using LAS X. Values were rounded to the nearest micron to account for human error in freehand measurement.

Smallest XY distance between Experiment ID LCN1 and Pd3 neurites (µm)

20140723 <0.5

20151208 449

20151210 347

20160209 232

Average 257

Standard Deviation 193

Table 3. Pearson’s correlation coefficients (r) for all experiments. Coloc 2 plug-in in

FIJI was used to determine no threshold values after a manual threshold had been applied to eliminate background from analysis. All values indicate a lack of colocalization in all experiments.

Pearson’s correlation Experiment ID coefficient (r)

20140723 0.00

20151208 -0.00

20151210 0.00

20160209 -0.01

Discussion

The lack of distance between the two fluorophores used in experiment 20140723 implies that there may be a monosynaptic chemotaxis circuit within the brain of Tritonia exsulans. This potential sensory transmission circuit supplements the recent findings that

Tritonia simultaneously uses a combination of odor and water flow sensory input 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). Tritonia must integrate sensory information to appropriately enact modulation of motor neuron output. Since our potential chemotactic circuit only takes up a small part of

Pd3’s neurite field, we believe that there are likely rheotactic sensory neurons synapsing on the remainder of Pd3’s neuritic field. This grouping of neurons likely facilitates integration of the chemotactic information from LCN1 along with rheotactic information from other sources by synapsing onto Pd3 to then modulate its motor output to effect turning.

The absence of a monosynaptic link between LCN1 and Pd3 in three out of the four successful preparations may be an artifact of the backfill procedure not having sufficient penetration into the tissue to highlight all the neurites that may be there. The large variation in measured distance between LCN1 and Pd3 associated neurites along with the variation in the number of cell bodies in the pedal ganglion from LCN1 may be evidence for insufficient fluorophore penetration. We can confidently state that inputs to

the pedal ganglion from LCN1 just do not make it past the medial, anterior portion of the ganglion as seen in the results section. The LCN1 associated cell bodies show neurites that extend ventrally into the neuropil mostly away from where Pd3’s neurites can be found axially. Since Pd3’s neurites do extend in one preparation (20140723) toward

LCN1’s cell bodies in the axial direction, and it is in the neuropil that synaptic connections between cells are typically made, then it is possible that our findings in three out of four preparations could also be an artifact of the dye injections not having sufficient penetration to highlight neurites that may bridge the axial and xy gap between

LCN1 and Pd3. Some preparations completely lacked or had diminished detail of Pd3’s neuritic extensions from the primary neurite and those can be definitively written off as being artifactual results.

The electrophysiological and neuroimaging techniques required for the completion of this thesis were extremely difficult to complete successfully. We highly recommend that the results of this thesis should be replicated by a different approach. It would be best to begin by taking an electrophysiological approach to determine if there might be direct interaction between the soma of the pedal ganglion that are associated with LCN1 and Pd3. We recommend simultaneous intracellular recordings be done on

Pd3 and on any of the soma in the pedal ganglion that our backfills indicate are associated with LCN1.

There is also another possible source of experimental error that would account for absent and truncated dendritic extensions from the primary neurite. Tritonia were only sacrificed for this experiment once their health deteriorated at the end of their normal

captive life span. Backfills and dye injections rely on intracellular mechanisms to be dispersed within a neuron. Once neural tissue begins to degrade, there is a decreased likelihood of these techniques succeeding to the highest extent. Perhaps if healthier tissue is used for a follow up study, greater detail of dendritic extensions will be seen.

Overall, the work completed for this thesis supports our hypothesis and suggests that a monosynaptic circuit within the brain indeed governs chemotaxis via turning effected by Pd3. This puts us one step closer towards mapping the circuit for a complex navigation behavior in the unassuming gastropod, Tritonia exsulans.

References

Audesirk, G., & Audesirk, T. (1980a). Complex mechanoreceptors in Tritonia diomedea

I. Responses to mechanical and chemical stimuli. Journal of Comparative

Physiology, 141(1), 101–109. https://doi.org/10.1007/BF00611883

Audesirk, G., & Audesirk, T. (1980b). Complex mechanoreceptors in Tritonia diomedea

II. Neuronal correlates of a change in behavioral responsiveness. Journal of

Comparative Physiology, 141(1), 111–122. https://doi.org/10.1007/BF00611884

Dorsett, D. A., Willows, A. O. D., & Hoyle, G. (1973). The neuronal basis of behavior in

Tritonia. IV. The central origin of a fixed action pattern demonstrated in the

isolated brain. Journal of Neurobiology, 4(3), 287–300.

https://doi.org/10.1002/neu.480040309

Field, L. H., & Macmillan, D. L. (1973). An electrophysiological and behavioural study

of sensory responses in Tritonia (Gastropoda, Nudibranchia). Marine Behaviour

and Physiology, 2, 171–185.

Katz, P. S., Sakurai, A., Clemens, S., & Davis, D. (2004). Cycle period of a network

oscillator is independent of membrane potential and spiking activity in individual

central pattern generator neurons. Journal of Neurophysiology, 92(3), 1904–1917.

https://doi.org/10.1152/jn.00864.2003

Korshunova, T., & Martynov, A. (2020). Consolidated data on the phylogeny and

evolution of the family Tritoniidae (Gastropoda: Nudibranchia) contribute to

genera reassessment and clarify the taxonomic status of the neuroscience models

Tritonia and Tochuina. PLOS ONE, 15(11), e0242103.

https://doi.org/10.1371/journal.pone.0242103

Lloyd, P. E., & Church, P. J. (1994). Cholinergic neuromuscular synapses in Aplysia

have low endogenous acetylcholinesterase activity and a high-affinity uptake

system for acetylcholine. Journal of Neuroscience, 14(11), 6722–6733.

https://doi.org/10.1523/JNEUROSCI.14-11-06722.1994

Lohmann, K. J., & Willows, A. O. (1987). Lunar-modulated geomagnetic orientation by

a marine mollusk. Science, 235(4786), 331–334.

https://doi.org/10.1126/science.3798115

McCullagh, G. B., Bishop, C. D., & Wyeth, R. C. (2014). One rhinophore probably

provides sufficient sensory input for odour-based navigation by the nudibranch

mollusc Tritonia diomedea. Journal of Experimental Biology, 217(23), 4149–

4158. https://doi.org/10.1242/jeb.111153

Murray, J. A., Estepp, J., & Cain, S. D. (2006). Advances in the neural bases of

orientation and navigation. Integrative and Comparative Biology, 46(6), 871–879.

https://doi.org/10.1093/icb/icl037

Murray, J. A., & Willows, A. O. D. (1996). Function of identified nerves in orientation to

water flow in Tritonia diomedea. Journal of Comparative Physiology A, 178(2).

https://doi.org/10.1007/BF00188162

Murray, J. A., Hewes, R. S., & Willows, A. O. D. (1992). Water-flow sensitive pedal

neurons in Tritonia: role in rheotaxis. Journal of Comparative Physiology A,

171(3), 373–385. https://doi.org/10.1007/BF00223967

Murray, J. A., Jones, A. P., Links, A. C., & Willows, A. O. D. (2011). Daily tracking of

the locomotion of the nudibranch Tritonia tetraquetra (Pallas 1788 = Tritonia

diomedea Bergh 1894) in nature and the influence of water flow on orientation,

crawling, and drag. Marine and Freshwater Behaviour and Physiology, 44(5),

265–288. https://doi.org/10.1080/10236244.2011.629463

Redondo, R. L., & Murray, J. A. (2005). Pedal neuron 3 serves a significant role in

effecting turning during crawling by the marine slug Tritonia diomedea (Bergh).

Journal of Comparative Physiology A, 191(5), 435–444.

https://doi.org/10.1007/s00359-005-0604-1

Rueden, C. T., Schindelin, J., Hiner, M. C., DeZonia, B. E., Walter, A. E., Arena, E. T.,

& Eliceiri, K. W. (2017). ImageJ2: ImageJ for the next generation of scientific

image data. BMC Bioinformatics, 18(1), 529. https://doi.org/10.1186/s12859-017-

1934-z

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T.,

Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J.,

Hartenstein, V., Eliceiri, K., Tomancak, P., & Cardona, A. (2012). Fiji: An open-

source platform for biological-image analysis. Nature Methods, 9(7), 676–682.

https://doi.org/10.1038/nmeth.2019

Willows, A. O. D. (1978). Physiology of feeding in Tritonia I. Behavior and mechanics.

Marine Behaviour and Physiology, 5(2), 115–135.

https://doi.org/10.1080/10236247809378528

Willows, A. O. D., Dorsett, D. A., & Hoyle, G. (1973). The neuronal basis of behavior in

Tritonia. I. Functional organization of the central nervous system. Journal of

Neurobiology, 4(3), 207–237. https://doi.org/10.1002/neu.480040306

Wyeth, R. C., & Willows, A. O. D. (2006a). Field behavior of the nudibranch mollusc

Tritonia diomedea. The Biological Bulletin, 210(2), 81–96.

https://doi.org/10.2307/4134598

Wyeth, R. C., & Willows, A. O. D. (2006b). Odours detected by rhinophores mediate

orientation to flow in the nudibranch mollusc, Tritonia diomedea. The Journal of

Experimental Biology, 209(8), 1441–1453. https://doi.org/10.1242/jeb.02164

Wyeth, R. C., Woodward, O. M., & Willows, A. O. D. (2006). Orientation and

navigation relative to water flow, prey, conspecifics, and predators by the

nudibranch mollusc Tritonia diomedea. The Biological Bulletin, 210(2), 97–108.

https://doi.org/10.2307/4134599

Zazay, R., Morrison, J. B., Redondo, R., & Murray, J. A. (2012). Structure and function

of pedal neurons controlling muscle contractions in Tritonia diomedea. Impulse:

The Premier Journal for Undergraduate Publications in the Neurosciences, 1–16.