OPTIMIZATION OF A MUSHROOM BODY ABLATION TECHNIQUE IN MARGINEMACULATUS

Brittany Cordova

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

December 2019

Committee:

Daniel Wiegmann, Advisor

Verner Bingman

Paul Moore © 2019

Brittany Cordova

All Rights Reserved iii ABSTRACT

Daniel Wiegmann, Advisor

The primary purpose of this study was to optimize a standard microsurgical

technique to effectively ablate the mushroom bodies of . Lesions

were conducted on twenty-nine Phrynus marginemaculatus at varying electrode insertion points,

angles of insertion, and electrode depth into the protocerebrum. Morphometrics were determined

for twelve of these subjects, five of which had lesions that successfully impacted the mushroom

body. It appears that the ideal lesion target location can be approximated based on the data

acquired from successful surgeries. The results of this study showed that the mushroom body

can be successfully targeted by insertion of the electrode at a distance D = -0.8651 + 0.3586L

anterior of the central apodeme on the midline of the , where L is the carapace length (D

and L measurements in millimeters), with an insertion angle of 70° and maximum depth of 1.0-

1.3 mm. The long-term goal of this project was to develop a procedure that could be used to ascertain whether homing and navigation are affected following ablation of the mushroom bodies of P. marginemaculatus. The lesion process needs to be further refined, however, because, while the mushroom bodies were successfully targeted, no successfully lesioned survived longer than a day following the surgical procedure. iv

Dedicated to my family. v ACKNOWLEDGMENTS

I’d like to thank my committee Daniel Wiegmann, Verner Bingman, and Paul Moore for their guidance and knowledge. I’d also like to thank my labmates, in particular Patrick Casto for always being willing to answer any questions and providing assistance as needed and Meghan

Moore for her help in setting up the lesion paradigm. This work could not have been made possible without the help of Skye Long and Beth Jakob who helped design the lesion protocol and taught me how to histologically analyze samples. In addition, I would like to acknowledge my good friend Carol Heckman who provided assistance and the equipment used for confocal microscopy. Finally, I’d like to thank my friends and family who have supported me and motivate me to accomplish my academic and personal goals.

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TABLE OF CONTENTS

Page

INTRODUCTION……………………………………………………………………….... . 1

Arthropod Navigation ...... …………… 1

STUDY ORGANISM: AMBLYPYGIDS .………………………………………………… 3

Habitats………………………...... 3

Sensory Physiology ...………………………………………………………………. 3

Amblypygid External Brain Morphology………………………………………… .. 4

Amblypygid Internal Brain Morphology…………………………………………… 5

The Arcuate Body ...... …………………………………………………. 6

The Mushroom Bodies……………………………...... 7

MUSHROOM BODY INHIBITION ....……………………………………………………. 9

Visual Learning……………………………………………………………………. . 9

Olfactory Learning ...... ……………………………………………………. 9

Tactile Learning ...... …………………………………………………. 10

Learning Involving Combinations of Sensory Stimuli …………………………… . 10

PURPOSE ...... ……………………………………………………………………………… 11

METHODS…………………………………………………………… ...... 12

Subjects……………………………………………………… ...... 12

Lesion Procedure ...... ……………………………………………………. 12

Histology……………………………………………………………………...... 14

Brain Dissection ...... ……………………………………………………. 14

Embedding ...... …………………………………………………. 15 vii

Sectioning …………………………… ...... 15

Brain Section Mounting …………………………… ...... 16

Imaging Lesion Damage …………………………… ...... 17

RESULTS AND DISCUSSION……………………………...... 18

Electrode Accuracy ...... ……………………………………………………. 18

Depth of Electrode Insertion………………………………………………………. . 20

Post-Surgical Mortality ...... ……………………………………………………. 21

Hemolymph Loss ...... …………………………………………………. 22

CONCLUSIONS……………………………...... 24

REFERENCES…………………………… ...... 26

APPENDIX A. TABLES……………………………………………………...... 31

APPENDIX B. FIGURES…………………………… ...... 33

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Running head: MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

INTRODUCTION

Arthropod Navigation

Arthropods, like other animals, are motivated to navigate through their habitats in search of mates, shelter, and resources such as food and water. They are equipped with specialized sensory mechanisms that allow them to employ specific strategies to navigate the terrain. Some , including scorpions (Gaffin, 2012), bees (Dyer, 1991), fiddler (Layne, 2003), and desert ants (Muller, 2010) rely on a navigational strategy known as path integration to forage for resources before returning to a central nesting site. The efficacy of this strategy is contingent upon evaluating idiothetic cues derived from egocentric space, including proprioception as well as optic flow or the pattern of perceived motion between an animal and their environment, to generate a sense of speed, angles, and trajectory. Assimilation of both the forward moving speed and the angular turning rate generate an internal system of egocentric Cartesian coordinates that allow the animal to orient itself to return directly home (Merkle, 2006).

Other arthropods rely on allothetic cues, which are external cues provided from the environment, such as olfactory (Jacobs, 2012), visual (Marshall, 2011), tactile (Seidl, 2006), auditory (Bernardet, 2008), celestial (Warrant, 2010), and magnetic fields (Arendse, 1978) that provide directional information that guides animals to a goal. Allothetic homing is dependent upon the spatial orientation of sensory landmarks regardless of an animal’s position in space.

Goal oriented navigation can be based on route following, a landmark based navigational strategy that develops a familiar path based on a set of landmarks along a given homing path

(Cheng, 2006), image matching (Collett, 1996), or can utilize compass cues such as light polarization, the sun, celestial cues, and magnetic fields. 2

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

Environments with more cluttered terrain may impede perception of some sensory stimuli.

Cues such as compass cues, olfactory, and auditory cues may become patchy in time and space when there are more obstacles such as trees, rocks, and hills present. Complex terrain imposes more selective pressures that require modified navigational strategies that may require a reliance on multimodal sensory input to facilitate successful navigation (Hebets, 2014). Specialized sensory input must be accompanied by corresponding brain regions to process the stimuli. For example, the optic nerves provide input to the optic lobe, and likewise, the antennal nerves project onto the antennal lobe. In some arthropods, input from both of these discrete sensory modalities have been observed to converge upon the mushroom bodies, which are well conserved across taxa (Nilsson, 1997). These mushroom bodies are predicted to be a locus for processing and integration of multimodal sensory input. One such organism that is predicted to rely on multimodal sensory input for navigation and possesses a highly developed mushroom body is the amblypygid.

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STUDY ORGANISM: AMBLYPYGIDS

Habitats

Amblypygids, colloquially referred to as whip , inhabit densely cluttered tropical and subtropical rainforest environments (Weygoldt, 2000). They exhibit preference for particular microhabitats where they take refuge in crevices of rocks (Chapin, 2015), buttresses of trees

(Hebets, 2002), and among leaf litter and debris (Fowler-Finn, 2006). Amblypygids express site fidelity; they will leave a refuge to hunt for prey, and though they may inhabit several refuges over the course of several days, they regularly return to their home shelter (Hebets, 2002).

Displacement studies conducted in a tropical rainforest understory demonstrated that amblypygids were able to successfully return to a home site, which nullifies pathfinding as the primary navigational strategy employed by amblypygids (Beck, 1974). Radio telemetry experiments have confirmed that amblypygids do not return via a direct path as would be expected when pathfinding, but rather wander indirectly, often visiting other refuges on the way to their home shelter (Hebets, 2014). It has been hypothesized that amblypygids utilize a more integrative approach, relying on multiple sensory modalities (primarily olfaction) to create a cognitive map when homing (Wiegmann, 2016).

Sensory Physiology

Despite the name, whip spiders do not have four sets of walking legs like true spiders, but have evolutionarily modified their first pair of ambulatory appendages to be utilized for sensory input. These antenniform legs contain segmented annuli that extend from the anterior portion of their prosoma. Tarsal annuli are densely packed with specialized club, porous, and trichobothria sensilla to process olfactory, chemosensory, and chemotactile information, but more sensilla are sporadically distributed along the length of the tibia (Weygoldt, 2000). As their name suggests, 4

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS whip spiders will “whip” these antenniform legs in the air to maximize exposure to odorants in the environment (Santer, 2011). Amblypygids have eight simple eyes arranged into a pair of anterior medial eyes and two groups of three posterior lateral eyes. Previous displacement experiments conducted in the field have demonstrated that these appendages, but not their eyes, are necessary for successful navigation and homing in a complex two-dimensional terrain

(Bingman, 2017). Since amblypygids are nocturnal organisms with relatively primitive eyes, they do not rely solely on vision for nighttime navigation. It has been postulated that they utilize a more integrative approach that incorporates information from their modified sensory appendages, perhaps with visual inputs, and that they are equipped with an intricate brain structure that would allow them to successfully navigate and return to a home site after each night (Wiegmann, 2016).

Amblypygid External Brain Morphology

Similar to other , the central nervous system of amblypygids resides within the prosoma and is composed of two major components, the supraoesophageal ganglion and the suboesophageal ganglionic mass (Babu, 1984). The supraoesophageal ganglion, which is considered the brain proper, contains both the protocerebrum and the deutocerebrum

(collectively known as the syncerebrum). The protocerebrum constitutes the first neuromere of the brain. It receives input from the four pairs of optic nerves that correspond to the anterior median, posterior, median, posterior lateral, and anterior lateral eyes (Babu, 1984). The second neuromere, the deutocerebrum, is fused directly to the protocerebrum, but does not receive any innervation. The cheliceral nerves are located inferior to the optic nerves, just lateral to the midline attached to the tritocerebrum on either side of the esophagus. The suboesophageal 5

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS ganglionic mass on the other hand, contains the pedipalpal, antenniform, ambulatory, and fused opisthosomal ganglia (Babu, 1984).

The entirety of the central nervous system, including both the suboesophageal and supraoesophageal ganglion, is contained within a neurilemma. This neurilemma is composed of approximately six to ten layers of connective tissue which tends to be thicker on the ventral side.

The central nervous system contained within this neural lamella is then surrounded by a layer of nephrocytes and endocrine cells known as the anterior organ (Streble, 1966, Legendre, 1959).

Measurements taken from the wandering Cupiennius salei estimated that the ventral neurilemma is nine to eighteen micrometers, and the surrounding tissue layer is roughly 500-600 micrometers around the pedipalpal and opisthosomal ganglia (Babu, 1984). Digestive diverticula line the external portion of the nephrocyte/endocrine cell layer, separating it from the carapace.

Amblypygid Internal Brain Morphology

Arachnid neuroanatomy has been well documented in such as wandering spider

Cupiennius salei, and parallels in general brain structure and morphology are assumed to be true for the brains of amblypygids (Babu, 1984). Individual ganglia of arachnids can be identified based on the arrangement of cellular groups and fiber tract organization, which is confined within the sheath of the neurilemma (Barth, 2013). This sheath creates a cellular rind surrounding the interior fibrous core. Cell bodies are not found around the root of the peripheral nerve, the dorsal and dorsolateral areas, or the mid-central core of the ganglia, but are localized within the frontal, lateral, and dorsal regions (Babu, 1984). Groups of these cells give rise to fiber bundles that can be easily traced in the suboesophageal ganglia to the motor somata and interneurons. However, fiber tracts localized within the supraoesophageal mass are much more abstruse, and little is known about these pathways in spiders. Association centers used for 6

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS integration of sensory information in amblypygids, much like those seen in other wandering spiders, are compartmentalized within the protocerebrum; optic centers, the mushroom bodies, and glomeruli are located in the anterior portion, with a fiber tract that runs to the posterior protocerebrum, linking them to the arcuate body. It is possible that the mushroom bodies receive olfactory input through this fiber tract from the olfactory glomeruli in the ventral ganglia

(Wiegmann, 2016). Integration of visual, tactical, and olfactory sensory information from external stimuli requires a higher order processing system and two loci, the arcuate body and the mushroom bodies, have both been considered potential sites for sensory integration.

The Arcuate Body. The central complex, an area of sensory integration in insects, is made up of four components: the protocerebral bridge, the fan shaped body, the ellipsoid body, and the noduli. A homologous structure has been identified in arachnids and is known as the arcuate body (Napiórkowska, 2018). In insects, direct visual input produces predictable motor output

(B.Turner-Evans, 2016). Fruit flies were used in a Buridan paradigm experiment in which the flies were allowed to walk back and forth between two vertical landmarks. The landmark would disappear and reappear adjacent to the original target location, causing the fruit flies to reorient towards the new stimuli. The novel adjacent landmark would then disappear and the flies had to redirect themselves to the original target location. Fruit flies with ellipsoid body defects failed the spatial orientation short-term memory task as they were unable to redirect back to the original target (Neuser, 2008). Another experiment conducted on fruit flies was modeled after the Morris water maze. It consisted of an arena with visual cues along the wall that acted as a landmark for flies to identify a cool spot to seek solace on an otherwise hot plate. After training, the cool spot was removed and the flies were allowed to roam freely. Control flies showed a robust preference for the learned location of the cool spot, but were unable to successfully navigate upon silencing 7

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS of ellipsoid body neurons (Ofstad, 2011). Additionally, single neuron recordings in neurons acquired from the protocerebral bridge of locusts demonstrated that neurons of this region preferentially respond to specific light polarizations (Heinze, 2009). These data suggest that the central complex relies heavily on visual input for memory formation, but seeing as amblypygid navigation is likely guided by olfactory cues, the arcuate body is not likely imperative for homing.

The Mushroom Bodies. Extensive studies have been conducted on the involvement of the mushroom body in sensory integration and learning in arthropods such as honey bees (Scheiner,

2001), cockroaches (Mizunami, 1998), and Drosophila (Heisenberg, 1985). In these animals, the mushroom bodies are present as paired lobed peduncles and calyces that are made up of thousands of densely packed Kenyon cells (Heisenberg, 2003). Olfactory input is transmitted from the inner antennocerebral tract onto the protocerebrum and calyces of the mushroom body

(Wong, 2002). Furthermore, studies conducted on honey bees have found that calyces of the mushroom body not only receive projection fibers from the antennal lobes, but in addition synapse with input fibers from the optic neuropils (Mobbs, 1982). The anatomical convergence of two distinct sensory pathways is indicative of a potential locus for sensory integration. In addition, the highly elaborate network of neuropil arborizations create feedback pathways, suggesting that the mushroom body is involved in higher order processing of this sensory information. Mushroom body output neurons likely facilitate motor responses, but the precise mechanisms and pathways have yet to be elucidated.

The mushroom bodies of amblypygids are significantly larger that the mushroom bodies of other arthropods, occupying the majority of the brain proper (Hanström, 1928). The highly convoluted lobes and overall structural complexity of the mushroom bodies could hint at 8

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS evolution of a higher order processing system that would be sufficient to accommodate sensory integration and memory formation hypothesized to be necessary to navigate in complex environments. Interestingly, not all arachnid brains are equipped with mushroom bodies.

Sedentary spiders that rely on web-building to trap their prey such as , Drassidae, and

Dysderidae completely lack any type of mushroom body structure. Mushroom bodies of wandering spiders (such as wolf spiders, the wandering desert spiders, and amblypygids), however, are significantly more prominent, suggesting they may play a role in navigation (Barth,

2013). Notably, the highly sophisticated and convoluted mushroom bodies of amblypygids occupy nearly fifty percent of the brain mass (Hanström, 1928). The mushroom body as well as antennal lobe size, in theory, should be larger in arthropods where odorants are the primary guiding factor in navigation (Jacobs, 2012). This, and their robust navigation abilities, suggest that amblypygids are a favorable candidate for a lesion study to determine the consequences of mushroom body impairment on navigation behavior.

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MUSHROOM BODY INHIBITION

Visual Learning

Despite having such a prominent mushroom body, amblypygids have been vastly underrated as a model organism for studies of sensory control and its brain organization. A learning paradigm modified from rat hippocampal studies was used to study place memory in cockroaches (Mizunami, 1998). This approach conducted lesions by placing a sliver of foil into the protocerebrum of the subject to inhibit synaptic transmission. The subjects were placed into an arena with an assortment of visual cues designed to promote navigation to a cool target area on an otherwise hot plate. Subjects were unable to find their target zone after bilateral ablation of the beta lobe-pedunculus junction when the target was hidden beneath the arena.

Interestingly, the subjects were able to navigate to a target if the target was visible. These data suggest that the medial lobes and pedunculi are essential for integrating visual sensory information to reach an invisible target, but they are not required for learning an association between two separate sensory stimuli (in this case, visual and temperature stimuli).

Olfactory Learning

Genetically eradicated mushroom bodies extinguished the ability to associate an odor with an electric shock in Drosophila as well as significantly hindered learning an association of a particular odor with a sucrose reward (Heisenberg, 1985). Likewise, a similar study conducted chemical ablations to remove embryonic neuroblasts that would later develop into the mushroom bodies of Drosophila. Here, de Belle further demonstrated that these subjects were not able to be conditioned using olfactory cues (J. De Belle, 1994). Apis mellifura has provided further evidence to support the mushroom body’s involvement in olfactory learning. Unilateral ablations were inflicted upon the middle calyces of the mushroom bodies using hydroxyurea. 10

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

This treatment eliminated discriminatory ability when a conditioned odorant was presented to either antenna independently and significantly hindered learning when odorants were delivered simultaneously to both antennae (Komischke, 2005). These findings collectively suggest that the mushroom bodies play an integral role in olfactory based learning.

Tactile Learning

The mushroom body’s role in tactile learning has been studied in Apis mellifera.

Scheiner (2001) demonstrated that unilateral hydroxyurea induced mushroom body lesions, but not bilateral lesions, completely disrupted the associative learning process that trained subjects to expect a reward when their antennae came in contact with a metal plate. However, both unilateral and bilateral lesions completely abolished reversal learning (Scheiner, 2001).

Antennal tactile thermoreception in Drosopila, however, remained unaltered after total early developmental hydroxyurea induced mushroom body ablation (Wolf, 1998). The discrepancy in these findings suggest, perhaps, that the mushroom bodies are selectively involved in tactile learning, but are not involved in thermoreception.

Learning Involving Combinations of Sensory Stimuli

Previous studies have observed the consequences of mushroom body lesions on visual, olfactory, and tactile learning independently. It is hypothesized that the mushroom bodies act as a site for convergence of multimodal sensory input in amblypygids, but no current studies have observed how a lesioned mushroom body affects learning and memory when multiple sensory stimuli are present (Wiegmann, 2016).

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MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

PURPOSE

The primary purpose of this study was to optimize a standard microsurgical technique to effectively ablate the mushroom bodies of Phrynus marginemaculatus. Ultimately, this technique could then be utilized to determine the behavioral consequences of disrupting synaptic transmission within the mushroom body. Specifically, the goal was to establish a procedure under which the mushroom bodies can be reliably lesioned and later be tested for deficits in their navigation behavior.

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METHODS

Subjects

The lesions were conducted on twenty-nine Phrynus marginemaculatus collected from the National Key Deer Refuge (USFWS Permit Number FFO4RFKD-2015-06) as well as subjects ordered from tarantulaspiders.com that were collected from Northwest Florida in Citrus

County. During training and testing, each subject was isolated in a plastic deli cup (diameter 17.1 cm, height 10.8 cm) containing a coconut fiber substrate and a piece of bark for shelter. They were fed crickets once a week and misted with water periodically for hydration. A 12:12 hour light: dark cycle (19:00-07:00 dark phase) was maintained by overhead broad-spectrum fluorescent lights (400–750 nm). The room humidity was maintained at a range between 60-70% and the temperature between 21-26 ºC.

Lesion Procedure

This study was designed to develop a lesion procedure intended to inflict damage upon the mushroom bodies in order to ultimately investigate the involvement of the mushroom bodies for exploration and navigation by comparing the behavior of subjects whose mushroom bodies have been targeted for lesion to a sham lesion group. Lesions were made using a Grass D.C. constant current lesion maker. A 0.005 mm diameter epoxy coated tungsten electrode with an impedance of 5 milliohm and an eight-degree tip was used to deliver current to the subject, and a silver wire was used as a ground. The coating of the distal 1 - 2 mm portion of the electrode was removed, and the 34-gauge silver ground wire will be sharpened with a razorblade. The electrode was sterilized before each surgery and coated with immunofluorescent dye to act as a marker for identifying the lesioned area. To label the lesion site, 5 µl of DiI (1, 1’-diotadecyl-3,

3, 3’,3’-tetramethylindocarocyanine perchlorate) was pipetted onto the electrode tip and allowed 13

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS to dry. Once the DiI had dried, one more drop was added to ensure the electrode has been adequately coated. Prior to a lesion surgery, subjects were pinned to a Styrofoam base to ensure stability during the procedure (see Figure 1A). Insect pins will be cut and bent to form five

“staples” that were used to restrain the subjects; one to confine each pedipalp, one for each paraxial set of legs, and one that will lie transverse across the . The subject was further restricted by wrapping parafilm across the limbs to limit movement. A sharpened #1 insect pin was used to perforate the central apodeme located on the ventromedial portion of the prosomal exoskeleton. A second hole was punctured in the prosoma just above the trochanter of the most posterior leg, and the silver ground wire was inserted and secured in place.

Initially, a David Kopf Instruments electrode manipulator was used to insert an epoxyinsulated tungsten microelectrode (A-M Systems Catalog #577200; diameter 0.005 inches,

12 MΩ resistance, 12 ° tapered tip) at a 45° angle 1-2 mm into the hole created in the central apodeme for eight subjects, and one subject was impacted at an angle of 40° (Figure 1B and C).

A current of one milliamp was directly administered for five consecutive seconds. Preliminary dissections indicated the electrode had passed anterior to the brain and completely missed the intended target, often solely impacting the esophagus. The angle of electrode insertion was then increased to 70 °, and the electrode insertion point was moved anterior to the central apodeme.

The current was then adjusted to either 1, 0.5, 0.1, or 0.0 to determine if a lower current would increase the chance of survival.

Morphometric measurements were only obtained from twelve of the twenty-nine animals, and of these, measurements were taken from eight extracted brains due to dissection complications (Table 1). Lesion locations were translated into a coordinate system with the origin centered on the central apodeme. Negative values on the x-axis represent a lesion to the 14

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS left of a subjects midline and positive x-values indicate the lesion was oriented toward the right of the midline. All y-coordinates were either zero (at the apodeme) or positive (anterior to the apodeme) because no lesions were conducted posterior to the central apodeme.

Histology

Brain Dissection. A live subject was placed in the 2° refrigerator for approximately thirty seconds to be mildly anesthetized. It was important not to cool the subject too long as tissue would become too fragile to utilize. After cooling, the subject was still be able to move but was significantly less responsive when touched. After cooling, iris scissors were used to quickly remove the opisthosoma, thus severing the heart from the prosoma. The walking and antenniform legs were removed last.

The brain-containing prosoma was then transferred into a petri dish filled with 1x PBS solution for dissection. Dissection was performed with the ventral portion of the prosoma facing upwards. One pair of #5 forceps was used to grasp appendages while he iris scissors were used to sever the walking legs, the antenniform legs, the pedipalps, and the chelicerae as close to the sternum as possible. The subject’s carapace was trimmed under the chelicerae, and tissuetrimming will continue around the surrounding prosoma.

Using both pairs of forceps, pieces of the carapace were carefully broken away. One set of forceps was used as an anchor to stabilize the sample, and the other set was used to pull away extraneous carapace pieces. Fatty tissue was pulled against the forceps to avoid putting any unnecessary strain on the brain and ultimately damaging the sample. Once enough tissue had been removed using the iris scissors, the carapace was gently pulled away intact, thus exposing the diverticula. Then the diverticula were removed along with any surrounding tissue or fat to expose the brain. 15

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

The sclerotized, flanged esophagus transects the brain, and cannot be directly removed without inflicting damage to the surrounding brain. Therefore, to remove the esophagus, iris scissors were used to snip the pink/peach fan-shaped region near the mouth parts, thus removing the flanged portion of the esophagus. Then forceps were used to pull the remaining esophagus from the posterior portion of the brain.

The now isolated brain sample was placed in a microcentrifuge tube filled with a 3% paraformaldahyde/3% glutaraldehyde mixture (Figure 2). Samples were fixed by refrigeration overnight.

Embedding. To facilitate sectioning of the brain to determine lesion damage, a 20% gelatin-5% agar solution made in PBS was be heated in a 60-65oC water bath until the mixture has homogenized and was free of bubbles. A small, flat silicone embedding mold was filled with gelatin, and using forceps, the brain was be suspended in the middle of the gelatin with the protocerebrum facing up. The ventral ganglia was on the bottom, parallel to the work surface.

The mold was chilled in a refrigerator for thirty minutes to an hour. If the brain-containing mold was to be placed in the refrigerator for more than an hour, the silicone mold was wrapped with a moist paper towel. The gelatin had solidified when the brain-containing mold was removed from the refrigerator. The solid gelatin block was then transferred into a 3%glutaraldehyde-3% paraformaldehyde containing microtube and refrigerated overnight.

Sectioning. The paraformaldehyde/glutaraldehyde mixture was completely pipetted from the conical tube containing the embedded brain sample and replaced with PBS. The conical tube with brain was be placed in the refrigerator to rinse for fifteen minutes. This process was be repeated three times. Then, the gelatin block containing the brain was be removed from the PBS and oriented so that the protocerebrum faces upward. The corners of the block were cut at an 16

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS angle using a sharp blade with a notch on one side to mark the orientation of the brain. The block containing the brain formed a trapezoidal prism, with the base to be attached to the vibratome chuck (see below).

The base of the trapezoidal prism shaped gelatin block was mounted onto the vibratome chuck using superglue. The chuck was mounted on a Lancer Series 1000 vibratome sectioning system and drops of PBS were continuously added liberally to the top of the sample to maintain temperature and moisture conditions. The chuck was oriented so it was positioned just below the blade with the speed and amplitude both set to 2-3 on the vibratome’s gauge. The brains were cut at 100-micron slices. A fine tipped paintbrush was used to remove each section, which was then placed in a well of a tissue culture plate filled with PBS kept on ice. This process was repeated until sectioning had been completed.

Forty, sixty, eighty, and one hundred percent glycerol solutions were prepared in PBS and used to process the brain sections before mounting. The glycerol permeated the gelatin agar mold to make each section transparent, which allowed clearer imaging. First, the PBS was removed from each well by pipetting, being careful not to scratch the surface of the gelatin. The wells were then re-filled by pipetting forty percent glycerol solution. The wells were covered with a low evaporation lid and wrapped in tin foil and were placed on an orbital shaker for 15 minutes. Subsequently, the forty percent glycerol solution was pipetted out, and the process was repeated with the sixty, eighty, and one hundred percent glycerol preparations. The gelatin progressively became more transparent and became clear once the process has been completed.

A drop of EMS Glycerol Mounting Medium was added to each well containing a section and

100% glycerol solution. Samples were returned to the refrigerator until mounting.

Brain Section Mounting. A clear page reinforcement protector was placed on top of a 17

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

22x50 mm rectangle cover slip to act as a spacer that contained the sample and glycerol.

Forceps were used to position a brain section on the cover slip, removing any bubbles in order to maximize clarity when imaging. The 22x22 cover slip was laid carefully over the top of each page reinforcer containing the brain sample. Glycerol solution was added as needed to keep the sample moist. The brain section was mounted on the cover slip and placed over a microscope slide. The cover slip edges were sealed with clear nail polish.

Imaging Lesion Damage. Slides were imaged with confocal microscopy using a Leica

DM13000B inverted microscope equipped with a Lumencore Spectra X LED light engine, an X-

Light spinning-disk confocal unit, and a Rolera Thunder cooled-CCD camera with a

Photometrics backthinned, back-illuminated, electron-multiplying sensor (Photometrics). Z stackes were acquired using Metamorph software using the 10X objective lenses and standard

DAPI, rhodamine and Cy5 filters (Figure 5). Brain images were loaded into ImageJ to quantify the extent of lesion damage. The location and an estimate of the percentage of the mushroom body affected was to be documented. However, due to the high magnification settings of the available microscopes, it was impossible to measure the actual extent of the lesion damage inflicted.

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RESULTS AND DISCUSSION

A total of twenty-nine lesions were conducted on large, fully grown subjects as well as one immature subject. Large subjects were used almost exclusively in order to standardize a procedure to effectively impact the mushroom bodies. Morphometric analysis was conducted in

ImageJ on twelve subjects, five of which had successful mushroom body lesions. Large animals were defined as having an average prosomal length (measured anterior to posterior through the central apodeme) of 6.00 mm and an average prosomal width (measured laterally through the central apodeme) of 8.73 mm (see Table 1).

Electrode Accuracy

Lesions conducted at a 70° angle were observed to determine if a clustering pattern appeared for a successful lesion site. A successful lesion was defined as one in which the electrode tip impacted the mushroom body; this could be visualized by the presence of DiI surrounding the electrode impact wound (Figure 5C). Lesions conducted directly at the central apodeme reliably failed to puncture the protocerebrum, but rather impacted the fat that lies directly posterior to the brain. A ratio was created by taking y-coordinate values for each lesion was divided by the distance between the central apodeme and the anterior of the carapace in order to standardize location regardless of subject size. The distances were ordered and a runs test was conducted to test for randomness of successful lesion groupings. Successful lesions were found to be clustered where the ratio lies somewhere between 0.1 and 0.15.

The prosoma was translated into a two-dimensional rendering of the dorsal portion of the subject upon which the electrode insertion points could be graphically translated into distances of lesion points from the apodeme (Figure 3). Experimental dissection determined that the protocerebral target could be imagined as an oval located somewhere just anterior to the central 19

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS apodeme. From the dorsal perspective, the average prosomal area of a large subject was found to be 51.52 mm2 and the average protocerebral area was 1.81 mm2. Previous research has documented that the mushroom bodies occupy almost 50% of the protocerebral brain mass and dissections have demonstrated that this also holds true for the two dimensional projection

(Hanström, 1928). If the prosoma was lesioned at a random location, represented by the variable

Z, then the resulting probability that the mushroom bodies would be impacted could be represented as the following equation

P(Z M) = M/(M P) where variable M indicates the area of the∈ mushroom bodies∪ and P represents the area of the prosoma. In this case, the probability that an electrode randomly inserted on the prosoma would affect the mushroom bodies would be approximately 1.7%. However, as aforementioned, data reliably show that the protocerebrum resides just anterior to the central apodeme, and thus provides some insight on where to pierce the carapace. Further experiments demonstrated that the mushroom body was impacted when the electrode was inserted just anterior to the central apodeme of large subjects. Additionally, the mushroom body was affected when the electrode insertion point was 0.22 mm anterior to the apodeme on the immature subject (carapace length

4.46 mm and width 6.33 mm). The protocerebrum is not anchored into a reliable position within the carapace; it can move anteriorly or posteriorly based on when the subject last ate, as the stomach rests just behind the brain proper. This information, in conjunction with the known average width of the protocerebrum (1.77 mm), can create a much smaller target area on the carapace. The goal of this study was to optimize a standard lesion location that increased chances of affecting the mushroom body. 20

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

The distance from the electrode point to the central apodeme and the distance between the apodeme and the anterior of the carapace were plotted on a scatterplot. A linear regression was fit solely to the successful lesions to determine whether there was a significant correlation between the electrode insertion point and subject size. The resulting p-value for the regression with the data set of five successful lesions was 0.001. The sample size was small, but this suggests that the linear model could be a valid indicator to successfully target the mushroom bodies. Based on these data, it is possible that the size of a subject can be used to approximate a target area for electrode insertion. The equation D = - 0.8651+0.3586L can be used to determine a location for electrode insertion, where D represents the distance anterior to the central apodeme and L denotes the length of the carapace (both measurements taken in millimeters).

The lateral position of the electrode insertion could be modified as needed. As stated earlier, the average protocerebral width was 1.77 mm, and the protocerebrum was oriented centrally at the midline. The lesion location could be altered laterally to the left or right as needed in order to attempt to increase subject survival. If the lesion was conducted directly upon the midline, there is a chance that the electrical current may cause inadvertent damage to the esophagus that transects the brain, which would affect the subject’s ability to eat and potentially lead to a premature death.

Depth of Electrode Insertion

The electrode was inserted at various depths between 0.8-2.0 mm for all lesions, both successful and non-successful. Of these variations, it appeared that any depth greater than 1.5 mm would directly impale the ventral surface of the brain. Based on observation and measurements obtained from the eight successful brain dissections, I hypothesize that the ideal insertion depth lies somewhere between 1.0 and 1.3 mm into the prosoma as any lesion inflicted 21

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS at 1.5 mm or greater had significantly passed through the target location and frequently perforated the ventral portion of the brain. The five successful lesions were conducted at electrode depths of 1.0, 1.0, 1.3, 1.5, and 1.5 mm respectively. Of these, only the first three did not impact the ventral portion of the brain. The exact desired depth is difficult to ascertain due to varying amounts of fatty tissue surrounding the brain between subjects.

Post-Surgical Mortality

Subject mortality following the lesion procedure was the most significant impediment to overcome, with eight out of twenty-one subjects dying within the first two day recovery period.

Of these, there were three lesions that successfully impacted the mushroom body, but none of the subjects survived long enough to partake in behavioral testing.

The initial nine lesions were conducted at either 40° or 45° angles with the electrode insertion targeting the central apodeme. Upon dissection, it appeared that these lesions were just directly anterior to the protocerebrum, sometimes impacting the flanged esophagus that transects the amblypygid brain. The DiI is lipophilic and dispersed across the fatty tissue surrounding the brain, but the protocerebrum remained unaffected. These subjects survived the initial surgery and remained alive for at least ten days post-surgery.

Based on this information, the angle of electrode insertion was increased to 70° and the hole in the carapace was shifted anterior to the central apodeme in order to more directly impact the protocerebrum. The aim of this alteration in procedure was to mitigate the inadvertent damage to surrounding structures. The central apodeme is a convergence site for several pedal muscles, such as the tergocoxal muscles which attach to the carapace at the valleys and indentations that extend radially across the dorsal surface as well as the pharyngeal muscles.

Notably, the musculature appears to be less dense just anterior to this depression, making it a 22

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS more appealing target so as not to have unintentional behavioral consequences resulting from muscle disfiguration (Shultz, 1999). In addition, electrical stimulation at the central apodeme resulted in pedipalpal spasms and paralysis that lasted for roughly five minutes following the lesion procedure. The first dorsal endosternal suspensor muscle attaches to the carapace at the central apodeme, and the palpal anterolateral endosternocoxal muscle projects anteriorly from the surface of this tendinous process to the pedipalps (Shultz, 1999). Consequently, electrical stimulation at this locus is a likely explanation for the post-surgery tetanic contractions.

Hemolymph Loss. Additionally, hemolymph loss proved to be a significant deficit following the lesioning procedure. Regulation of the hemolymph vascular system relies upon the heart, which is positioned dorsally in the opisthosoma. The anterior aorta projects anteriorly into the prosoma and lies just beneath the carapace at the central apodeme where it extends further to vascularize the protocerebral artery (Klußmann‐Fricke, 2016). Damage to this thick anterior aorta would result in the loss of a significant amount of hemolymph due to the high fluid flow and vessel diameter. Targeting the lateral and dorsal supraesophageal perineural arterial network, which consist of significantly less dense vasculature in comparison to the rest of the protocerebrum as well as thinner vessel diameter than the anterior aorta, may be beneficial in regard to hemolymph conservation.

The hemolymph vascular system is essential for standard life processes including oxygen, fluid (Truchot, 1992), protein, and hormone transport (Terwilliger, 1999), as well as regulation of coagulation factors for clotting wounds (Theopolda, 2002). However, it also serves an additional role to facilitate movement. The ambulatory legs of amblypygids contain several flexors, but only a few joints contain extensors. They rely primarily on hydraulic pressure regulation to mediate leg extension and elevation (Parry, 1959). Excessive post-operational 23

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS hemolymph loss could consequently have behavioral implications, limiting the animal’s ability to perform basic motor functions. The two day post-surgery recovery period allows for stabilization of internal fluids as well as rehabilitation from the shock of invasive surgery, but there is a high chance of mortality within this time period if too much hemolymph has been lost.

A couple of techniques significantly lessened the loss of hemolymph. Firstly, moving the electrode insertion point more anterior along the dorsal carapace avoided impacting the anterior aorta, and thus prevented heavy fluid flow leaving the body. Secondly, a significant decrease of subjects’ body temperature reduced hemolymph movement throughout the body. Subjects were placed in the freezer for a few seconds to anesthetize and were kept on ice throughout the duration of the lesioning process. These subjects retained a significant amount of fluid in comparison to subjects that received the same lesion protocol at room temperature. Third, the wound inflicted by the initial insertion of the insect pin to expose the underlying tissue may take several minutes to clot, depending on the location and depth of the damage. Topical administration of super glue directly to the afflicted area sealed the wound within seconds and prevented excess fluid loss with the intent to extend subjects’ post-lesion life span.

24

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

CONCLUSIONS

It appears that the ideal lesion target location can in fact be approximated based on the data acquired from successful surgeries. The hypothesized insertion point can be estimated by measuring the length of the prosoma L (mm) to determine the appropriate distance D (mm) anterior to the central apodeme using the equation D = - 0.8651+0.3586L. The electrode should only be inserted at a 70° angle 1.0-1.3 mm maximum into the carapace to avoid puncturing the entirety of the protocerebrum. The insertion point should be skewed slightly from the midline in order to avoid damaging the esophagus. Several precautions can be taken to decrease subject post-surgery mortality, but they do not guarantee the long-term survival of the subject. Firstly, subjects chilled and kept on ice during surgery had significantly more hemolymph retention than subjects that were lesioned at room temperature. Secondly, application of a topical sealant such as super glue will further reduce the loss of additional fluid. Subjects were sacrificed the day after surgery to determine whether they could survive the initial lesion, but it is unknown how long they would survive following the procedure. Further experiments would need to be conducted in order to determine if their post-surgical lifespan would be sufficient to undergo behavioral testing.

The long-term goal of this project was to develop a procedure to ascertain whether homing and navigation are affected following ablation of the mushroom bodies of P. marginemaculatus. Ideally, subjects would survive long enough post-surgery to undergo classical conditioning trials to determine whether they could learn to associate an odor to a home shelter, and then consequently be able to navigate home in an arena trial following displacement.

To reiterate, the mushroom bodies are the hypothesized site of sensory information involved in 25

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS navigation and, hence, lesioned subjects should not be able to navigate as effectively as sham control subjects.

26

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

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MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

APPENDIX A. TABLES

Table 1

Lesion Parameters

Mean Standard Deviation N

Prosoma

Length (mm) 6.00 0.50 12

Width (mm) 8.73 0.25 12

Protocerebrum

Length (mm) 1.33 0.04 8

Width (mm) 1.77 0.04 8

Height (mm) 1.35 0.17 8

Morphometric analysis was derived from the prosoma pre-surgery as well as the protocerebrum following dissection. The length was measured across the dorsal portion of the carapace from the anterior to the posterior region along the vertical line created by the central apodeme. The width was measured dorsally from left to right along the horizontal bar of the central apodeme.

32

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Table 2

Summary of all Lesions Conducted

A summary of all lesions conducted. Sham lesioned subjects were denoted as “S” followed by a number; these subjects had their carapace pierced, but there was no electrical current administered. Electrically lesioned subjects were denoted by “L” followed by a number.

Lesioned subjects L10, L14, L16, L28, L29, and L30 successfully targeted the mushroom bodies.

The parameters for these lesions were indicated in the column headers. Date: date surgical procedure was performed; Subject: denotes whether the subject was a lesion or sham and the identifying number of the subject; Current: the electrical current administered through the electrode (mA); Duration: the duration of current administered (seconds); Angle: the angle the electrode was inserted into the carapace; Depth: the distance the electrode was inserted into the carapace Procedure Notes: general notes about the procedure. 33

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

APPENDIX B. FIGURES

Figure 1. Diagrams are shown to demonstrate general anatomy referenced by the surgical procedure. A. Schematic illustrating how a subject was mounted onto a Styrofoam block with staples made from bent insect pins. Staples were placed over each pedipalp, one over each 34

MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS paraxial set of legs, and one was placed across the opisthosoma. The electrode is denoted in the figure by the red cone placed near the central apodeme, and the silver ground wire was inserted just superior to the trochanter just below the carapace. B. A midsagittal view depicts the initial electrode angle of insertion into the amblypygid brain and mushroom bodies target. The electrode was inserted at a 70° angle into the superior ridge of the central apodeme to, ideally, selectively damage to the mushroom body lobes. MB, mushroom body; 1 antenniform leg nerve; roots of the 2-4 ambulatory nerves; OPIN, root of the opisthosomal nerves. C. A dorsal view of

P. marginemaculatus denoting the location of the central apodeme which acted as a reference point for electrode insertion. The silver ground wire was placed just above the trochanter under the carapace of the last ambulatory leg on the left side.

35

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Figure 2. The DiI was visible to the naked eye upon dissection as a vibrant pink dot on the surface of the brain if the mushroom bodies are accurately targeted. The lesion target region is within the protocerebrum. The dorsal (left) and lateral (right) photo-images depict major superficial landmarks of the brain. 1, antenniform leg nerves; 2-4 ambulatory nerves; ON, optic nerves; SOG, suboesophageal ganglia; OPIN, opisthosomal nerves.

36

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Figure 3. A schematic of the prosoma drawn to proportion based on the average length and width dimensions of subjects. Points were plotted with the origin centered around the apodeme. Due to size variability among animals, the x coordinates were divided by the distance of half the width and the y coordinates were divided by the distance from the apodeme to the anterior portion of the carapace. Green dots (14, 16, 28, 29, and 30) indicate lesions which successfully impacted the mushroom body. All other lesions indicated in red (15, 20, 24, 25, 26, and 27) did not hit the mushroom body target. All lesions pictured above were conducted with an electrode angle of 70. Successful lesions were inflicted where the ratio was approximately 0.1-0.15 directly anterior to the central apodeme.

38

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Figure 4. A plot relating the distance from the center of the apodeme to the anterior most point of the prosoma and the y-coordinates of the electrode insertion of lesioned subjects (insertion angle of 70°). The linear regression shown was calculated based only on successful lesions where the electrode impacted the mushroom body (D = - 0.8651+0.3586L ; P = 0.001, R2 =

0.98).

39 MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS

A B

C

Figure 5. Transverse sections were sliced every 100 microns and were imaged using confocal microscopy in order to verify that the lesion impacted the mushroom body. A. The apical surface of the dorsal portion of the mushroom bodies can be seen here. Sections were taken along the transverse plane. This particular image was taken from the second section of subject

22. The convoluted lobes are very distinct and can be seen in the white highlighted region, but the mushroom bodies were not affected by surgical manipulation in this case. The white arrow denotes the anterior portion of the animal. B. The highlighted region once again indicates the

40 MUSHROOM BODY ABLATION OF P. MARGINEMACULATUS mushroom body lobes. The glomeruli of the calyces can be seen surrounding the convoluted lobes. C. The lesion procedure for subject 14 was successful. The dark grey point circled in white indicates the point of the electrode. The damage induced by the current can be visually determined by the dispersal of the fluorescent DiI across the damaged brain tissue.