Optomotor Response Reduced by Procaine Injection in The

OPTOMOTOR RESPONSE REDUCED BY PROCAINE INJECTION IN THE

CENTRAL COMPLEX OF THE COCKROACH, BLABERUS DISCOIDALIS

BY:

MALAVIKA KESAVAN

Submitted in partial fulfillment of the requirements

For the degree of Master of Science

Dissertation Adviser: Dr. Roy Ritzmann

Department of Biology

CASE WESTERN RESERVE UNIVERSITY

January, 2014

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of Malavika Kesavan candidate

for the Master of Science degree *.

(Committee Chair) Dr. Jean Burns

Dr. Roy Ritzmann

Dr. Mark Willis

Dr. Barbara Freeman

(date) 08/28/2013

TABLE OF CONTENTS

Abstract ……………………………………………………………………………….. 8

INTRODUCTION ...………………………………………………………………….. 9

Basic anatomy of the B. discoidalis …………………………………………………. 11

External Anatomy …………………………………………………………….. 11

Internal Anatomy ……………………………………………………………... 11

The Head Ganglia in Insects ………………………………………………….. 12

The Central Complex: Anatomy ……………………………………………… 14

Focus of project ……………………………………………………………………… 16

Basic Visual Information Flow in Insects ………………………………………….. 16

Anatomy ……………………………………………………………………… 17

Basic Visual Movement Detection ………………………………………….... 18

The Central Complex ……………………………………………………………….. 19

Comparative Approach lends Insight into function of Central Complex ...... 19

Overview of Central Complex Development ………………………………… 19

The Central Complex's influence on Spatial Orientation …………………….. 21

The Central Complex Role in Changing Motor Behavior……………………… 22

The Central Complex and Memory …………………………………………... 25

Motor Behavior ……………………………………………………………………… 26

Locomotion …………………………………………………………………… 26

Escape Behavior ……………………………………………………………………... 28

Procaine ……………………………………………………………………………… 30

Significance of Project ………………………………………………………………. 31

METHODS …………………………………………………………………………... 31

Housing ……………………………………………………………………….. 31

Phase 1: Pre-trial …………………………………………………………….. 32

Phase 2: Injections ……………………………………………………………. 32

Phase 3: Trial ………………………………………………………….……… 33

Phase 4: Histology .…………………………………………………………... 34

Electrophysiology …………………………………………………………….. 36

Escape Behavior ……………………………………………………………… 37

Data Processing ………………………………………………………………. 37

Escape Response Analysis ………………………………………….. ………. 40

Post Injection Behavior ………………………………………………………. 41

Errors in Data Analysis ……………………………………………………….. 41

RESULTS …………………………………………………………………………… 43

Controls ……………………………………………………………………….. 43

The Procaine Effect on Optomotor Response ………………………………... 45

Activity Levels ...... 50

Escape Response ……………………………………………………………… 51

Extracellular Brain Recording ……………………………………………….. 53

DISCUSSION ……………………………………………………………………….. 54

Central Complex Influence on Optomotor Response ………………………... 55

Effect of Procaine on Central Complex ………………………………. 55

Effect of Procaine on Behavior ………………………………………. 56

Activity Levels ………………………………………………………………... 57

Escape Response ……………………………………………………………… 58

CONCLUSION ……………………………………………………………………... 60

Future Work for Next Student ……………………………………………………... 61

Acknowledgments …………………………………………………………………… 62

References ……………………………………………………………………………. 63

LIST OF FIGURES AND TABLES

Figure 1: Cockroach Central Nervous System ………..……………………………….. 12

Figure 2: Representation of the central complex images in 3D ………..….………….. 14

Table 1: Summary Table of central complex lesions ………………………………….. 25

Figure 3: Schematic of experimental set up (side-view) . ..…………………………… 31

Table 2: Histological Process …………………………………………………………. 35

Figure 4: Confocal Images of B. discoidalis brain with dye ………………………….. 36

Figure 5: Example of raw output ……………………………………………………… 38

Figure 6: Data processing method ……………………………………………………... 38

Figure 7: Example Angular Outputs ………………………………………………….. 40

Table 3: Summary of Experimental Groups ………………………………………….. 40

Figure 8: Examples of False Negatives ………………………………………………. 42

Figure 9: Examples of False Positives ………………………………………………... 43

Figure 10: Optomotor Responses for untreated cockroaches …………………..…… 44

Figure 11: Optomotor Responses for cockroaches with sham surgery ……….…….... 45

Table 4: Turn Response comparison between saline and 20% Procaine ……………... 46

Figure 12: Optomotor Responses for cockroaches injected with saline …………….... 47

Figure 13: Optomotor Responses for cockroaches injected with procaine …………… 48

Figure 14: Optomotor for cockroaches injected with 10% procaine …………………. 49

Table 5: Turn response comparison between saline and 10% procaine trials ………... 49

Figure 15: Comparison of optomotor response in all experimental groups …………... 50

Table 6: Average time spent Active during trial ……………………………………… 51

Figure 16: Average speed of escape for cockroaches under the influence of 20%....….. 52

Figure 17: 20% Procaine influence on central complex spiking activity …………….. 54

Figure 18: 10% Procaine influence on central complex spiking activity …………….. 54

Optomotor Response Reduced by Procaine Injection in the Central Complex of the

Cockroach, Blaberus Discoidalis

By:

MALAVIKA KESAVAN

Abstract

The central complex is a group of midline neuropils found in all arthropods. In

insects, circuits within the central complex play a role in processing visual and tactile

information. Activity recorded in the central complex is correlated with and often

precedes changes in step frequency and turning movement. Electrolytic lesions in the

central complex adversely affect various locomotory behaviors. However, questions exist

about collateral damage to areas outside of the central complex as the lesion probe was

inserted. To address this issue, we developed a reversible chemical block of neural activity in the central complex using the anesthetic procaine. Black and white moving

stripes induced an optomotor turning response in the cockroach, Blaberus discoidalis.

After documenting the response in a normal individual, we injected a solution of 10% or

20% procaine into the central complex. Subjects with anesthetized central complex had a

significant decrease in optomotor response up to 30 minutes after injections. Controls

injected with saline showed no deficit. As the effects of the anesthetics diminished, the

cockroach regained the ability to turn. While the optomotor response processing happens

mostly within the cockroach brain, most escape circuitry is located in the thoracic

ganglia. Nevertheless, the brain has been shown to influence escape response. We,

therefore, extended our analysis to examine the effects of the procaine injection into the

central complex on escape. Subjects with reduced output from the central complex

showed little deficit in escape speed. Electrophysiological recording showed that

procaine reduced firing activity in the central complex.

Introduction

Cockroaches such as Blaberus discoidalis, a member of the arthropod phylum, are

notoriously good at moving quickly in unpredictable and novel terrain. Despite their

reputation as “simple” creatures most robotic work has been unable to span behavioral

adaptability of these animals. How is the cockroach able to incorporate the vast

complexity of its environment into its choices? The central complex has emerged as a

leading contender as a higher order motor planning area in insects (Strauss & Heisenberg,

1993). The central complex receives multimodal sensory information ultimately outputting to the lateral accessory lobes where it can affect the neurons descending to the thoracic ganglia (Martin et al., 1999).

The aim of my thesis project was to adapt a reversible block for examining the role of the central complex in controlling specific aspects of locomotor behavior. This technique was used to complement work done with electrolytic lesion in the central

complex by a previous student in the laboratory. Electrolytic lesions in the central

complex adversely affect various locomotory behaviors including turning; however,

questions existed about collateral damage to areas outside of the central complex when

the lesion probe was inserted(Harley & Ritzmann, 2010;Ritzmann et al., 2012). Though

several controls were done to ensure that the surgery was not the cause of the behavior

deficit, having a reversible neural effect would confirm these results. To address this

issue, a reversible block of neural activity in the central complex was developed for this

preparation using the local anesthetic, procaine. We believe refined versions of this

technique can be used to subtly manipulate central complex circuits allowing us to map

information flow and ultimately further the understanding of the function of the central complex.

In my experiments, an optomotor turning response was induced by displaying vertical black and white stripes that were moving horizontally in the visual field of B. discoidalis cockroaches while they were tethered on an air suspended ball. After documenting this response in normal individuals, a solution of 20% procaine was injected into the central complex. Subjects treated with procaine retained the ability to walk, though they rarely responded to moving stripes stimulus. On average, cockroaches with the anesthetized central complex showed a significant decrease in response to the visual stimuli up to 30 minutes after the injection. Cockroaches injected with saline showed no such deficit. Furthermore, cockroaches with no surgery performed on them acted in a similar manner to those injected with saline. As the effects of the anesthetic in the central complex diminished the animal regained the ability to respond to the visual stimuli in both behavioral and electrophysiological assays. 10% procaine injections also showed a decrease in activity in the central complex; however the behavior was variable in the first

30 minutes of injection especially in comparison to the 20% procaine. Escape response under the influence of the 20% procaine was also examined and it was determined that the central complex does not significantly influence the speed of tactile induced escape responses.

This thesis will start with an Introductory section that provides a background on the central complex and the optomotor response in order to allow for detailed

understanding presented in the Results section. The Discussion will present our thoughts

on the ramifications of my data on the more general field.

I. Basic anatomy of B. discoidalis

External Anatomy

There are three basic anatomically sections to an insect: the head, the thorax and

the abdomen. The head of the B. discoidalis contains the brain and other sensory

structures such as the eyes and antennae. The thorax, or the middle part, holds the wings

and legs. Like all insects, B. discoidalis have three pairs of legs: the prothoracic (the front

legs), the mesothoracic (the middle legs) and the metathoracic (back legs) which in

conjunction can be used to climb, tunnel, and turn on nearly any surface including against

gravity (Wessnitzer & Webb, 2006). The cerci are located on the posterior of the insect

and look like two short antennas. Cerci have directionally-sensitive hairs on the ventral

side that can detect displacement of air that is generated when a predator is attacking.

Cerci are critical to eliciting the escape response in cockroaches (Nicklaus et al., 1967).

However, escape can also be induced via touch detected by tactile receptors on the

abdomen and thorax (Pollack et al. 1995) or antenna(Ye et al., 2003).

Internal Anatomy

The central nervous system spans the head, thorax, and abdomen. The

supraesophageal ganglion which is a concentration of neurons and their synapses making

up local networks, and the subesophageal ganglion are connected to the three thoracic

ganglion in the thorax (Wessnitzer & Webb, 2006)(Figure 1). Each abdominal segment

contains a ganglion that is connected together by axons grouped into the connectives.

Together with the ganglia make up the ventral nerve cord.

The Head Ganglia in Insects

Figure 1: Cockroach nervous system. There are two main ganglia in the head: the brain is located at the top of the diagram, the subesophageal ganglia is represented by the black box. There are three ganglia in the thorax and 7 sets of ganglia in the abdomen (not shown).

There are two ganglia in the head of the insect: the supraesophageal and subesophageal ganglia(Wessnitzer & Webb, 2006). The insect brain is the supraesophageal ganglion which is made up of three parts: the protocerebrum, the deutocerebrum, and the tritocerebrum(Aubele & Klemm, 1977; Hömberg et al., 1989;

Strausfeld, 2012; Wessnitzer & Webb, 2006). The protocerebrum contains the mushroom bodies, the central complex and the optic lobes (Wessnitzer & Webb, 2006). The mushroom bodies have been strongly associated with memory processing and sensory integration(Devaud, et al., 2007; Mizunami et al., 1998). The central complex is made up of several connected midline neuropils and has been implicated as an area responsible for motor planning and sensory integration( Ritzmann et al., 2012;Strausfeld, 1999;

Strausfeld & Hirth, 2013). The optic lobes receive direct input from both the compound

eyes and the ocelli(Heinrich et al., 2001; Wessnitzer & Webb, 2006). The deutocerebrum

contains the antennal lobes and the areas which process mechanosensory input from the

antennae of the animal as well initiatory signals for antenna movement(Homberg et al.

1989). The tritocerebrum and subesophageal ganglion receive sensory information and

produce motor response for the mouth parts of the insect(Aubele & Klemm, 1977).

The subesophageal ganglion is located between the supraesphogael ganglion and

the thoracic ganglia creating a two way highway for sensory information such as

proprioceptive information from the joints and sensory information from the hair cells to

send feedback to the brain as well as modulatory commands from the brain to influence

the motor centers of the thoracic ganglia. There is evidence that the subesophageal

ganglion contains neurons that modulate the thoracic circuits (Schaefer & Ritzmann,

2001; Delcomyn, 1999). Bilateral lesions to the circumesophageal connectives resulted in

signals still being sent from the subesophageal ganglion, but not the brain. This caused

the animal to walk for extended periods in the stereotyped tripod gaits (“walking

forever”)(Ridgel & Ritzmann ,2005). However, animals with bilateral lesions in the neck

connectives which stops descending inputs from the brain and the subesophageal

ganglion showed a hyperextended posture and produced insects that did not walk without

external injections of octopmine or pilocarpine which are compounds that are naturally

present in the insect nervous system or tactile stimulus (Ridgel & Ritzmann, 2005). From

these experiments it is clear that the subesophageal ganglion is also important in the regulation of the locomotor circuits. In addition, the subesophageal ganglion can

influence escape locomotory behavior (Schaefer & Ritzmann, 2001) (see Escape

Behavior section for more details).

The Central Complex: Anatomy

Figure 2: Representation of the locust central complex images in 3D. (a) Dorsal view of a 3D model of the central complex. MB: mushroom bodies, PI: pars intercerbralis, PB: protocerebral bridge, GB: Central body (fan shaped body), EB: ellipsoid body, LAL: lateral accessory lobe (b) Axonal connections. Note the axonal crossings that allow for interhemisphere communication. All columnar elements are connected(Boyan & Reichert, 2011).

The central complex is a region of the insect brain that has been highly conserved

among species. The central complex is located between the peduncles of the mushroom

bodies and is laterally surrounded by the antenna-glomerulus tract(Hanesch et al. 2002).

The shape and size of the different subregions of the central complex varies from species

to species. Much of the anatomical work cited in this document has been done in

Drosophila but most of the structures are grossly similar in most insects. The central

complex consists of four connected structures: ellipsoid body, fan-shaped body, paired

noduli and the protocerebral bridge. Some laboratories, particularly who work with

locusts, refer to the fan–shaped body and the ellipsoid body as the central-body upper and

central-body lower neuropils respectively. In Drosophila, the ellipsoid body resembles a

sphere with a hole in the center (ellipsoid body canal) and is shaped like a torus (though

from some angles it appears like an ellipse) (Hanesch et al., 2002). In other species such

as cockroaches, there is no ellipsoid body canal and the ellipsoid body is similar to a

saucer shape. The fan-shaped body is the largest of the central complex regions. It is

shaped like a saucer with the concave side pointing ventrally (toward the back of the

animal) and has a crowning neuropil known as the superior arch. The protocerebral

bridge can be visualized as a “rod curved like the handlebars of a bike"(Hanesch et al.,

2002) with a dip in the middle and the lateral sections pointing ventrally as well. The

paired noduli lie below the ellipsoid body and are essentially stacks of two to four

neuropils that form a near spherical shape(Hanesch et al., 2002). All these structures except the noduli are unpaired, but mirrored across the midline. The lateral accessory lobes are bilaterally symmetrical structures that are the main output region of the central complex (Hanesch et al., 2002).

There are sixteen columnar elements in the protocerebral bridge projecting to the fan-shaped and ellipsoid bodies. These columns intersect the horizontal cell layers of the central complex substructures (Heinze & Hömberg, 2008). The number of horizontal layers varies between species and subregions. The columns in the protocerebral bridge

connect to sixteen sub-columnar elements (two sub-columns make eight main columns)

in the fan-shaped body via histologically visible fibers. These columns continue down into the ellipsoid body(Hanesch et al., 2002).

There are many types of neurons in the central complex. The horizontally oriented layers are composed of tangential large-neuron cells while the vertical columns are composed of small-columnar elements. Anatomical studies suggest that the tangential neurons are the input neurons of the central complex and the small-field neurons project to the output regions of the insect brain from the central complex (Hanesch et al., 2002).

Tangential cells are generally located in the protocerebral bridge, the ellipsoid body, or the fan-shaped body(Hömberg, 1991; Strausfeld, 1976). The central complex outputs to the lateral accessory lobe (Wessnitzer & Webb 2006; Strausfeld et al., 2002).

FOCUS OF PROJECT

The optomotor response, as the word implies, is a motor response to a visual stimulus. Stereotyped behavioral pattern of movement in the direction of the visual stimulus motion has been documented since the 1960s (Strausfeld, 2012). This reflex process will be examined in this project. In this background, we will follow the proposed flow of information through the cockroach nervous system. The next section of the background will provide an overview of the information flow of this signal from the eyes to central complex. Then, the information from the central complex is processed and descends to the lower thoracic circuits to influence the leg movements. The last section will be an overview of the locomotive turning response.

II. Basic Visual Information Flow in Insects

Insect visual systems have been shown to be exquisitely sensitive to specific stimuli (Srinivasan et al., 1999). Visually mediated motion has been well studied in many insect systems. Moving black and white stripes can elicit curved walking (a “turn”)

(Burrows & Rowell, 1973;Reichardt, 1969)(for overview of optomotor response with

regards to moving stimuli see: Srinivasan et al., 1999). But how does the animal detect

this stimulus?

Anatomy

There are two types of eyes that are commonly seen in insect systems. Lateral

eyes (also referred to as compound eyes) are retinotopically organized whereas the

median eye system (ocelli) consists of simple lens. Ocelli have been found to contribute

to the processing of the compound eye images and functions primarily as an indicator to

changes of light (Mizunami et al., 1986; Hoyle, 1954). There is also evidence in flying

insects that ocelli act as horizon detectors (Taylor, 1981) .

The compound eye of cockroaches is composed of ommatidia which are units that

contain photoreceptor cells, pigment cells and support cells (Exner, 1891; Heimonen et

al., 2006). In Drosophila, the axons from the retina synapse directly onto an area of the

brain called the lobula which is beneath the axons of the photoreceptor cell. The output

neurons of the lobula project to the lateral protocerebrum near the central complex (Paulk

et al., 2009; Sanes & Zipursky, 2010; Strausfeld & Okumara, 2006). In addition, the first

order neurons from the ocelli feed in near the protocerebral bridge, but do not directly

enter the central complex (Hömberg, 2008). These inputs indirectly provide visual

information to the central complex concerning light levels, movement, and objects in the

visual field. Removal of the compound eyes results in the loss of the dark seeking

behavior (Okada & Toh, 1998). Anatomical and functional studies show that visual

information can influence the insect behavior(For further review, see: Srinivasan et al.,

1999).

Basic Visual Movement Detection

Moving stimuli can be used for navigation, foraging, and predator-prey interactions.

Many insect visual processing models are immensely complex and not the focus of this

project. In brief, a drift of retinal images in comparison to the surroundings such as when

a fly is flying toward an object causes changes in motor response(Gotz & Wandel, 1984).

Motion detection occurs in the optic lobe of the insect brain where cells act as elementary

motion detectors. Elementary motion detectors act through comparing the luminescence

in two adjacent ommatidia and multiplying them after one has been processed by a low

pass filter (one is likely to be processed first because of the delay in time depending on

how the stimulus is moving)( Borst & Bahde, 1988; Borst et al., 2010). The low pass

filter reduces the effect of frequencies above a specific value which is determined by the

processing cell. The output of the two ommatidia is eventually subtracted resulting in

downstream activation. The system is able to not only determine if the image is moving,

but also the direction of movement because of the lag processing (Srinivasan et al., 1999;

Borst & Egelhaaf, 1989). The resulting optomotor response is stabilizing and

compensatory which helps the animal orient its body so the visual field remains

stable(Pick, 1976; Tammero & Dickinson, 2002). This motion perception and “looming

stimuli” aid in the initiation of a landing sequence in some flying insects as well as

potentially contributing to escape and steering movements (Rosner & Homberg, 2013;

Srinivasan et al., 1999).

III. The Central Complex

Comparative Approach lends insight into function of Central Complex

Insects and other arthropods all display a stunningly vast array of behaviors.

While the focus of this study is on B. discoidalis, much of the existing literature comes from other insects such as Drosophila, desert locusts, and crickets. While there is variability in the structure of the central complex, the structure itself is highly conserved among insects(Strausfeld et al., 2002). Though considerable work has been done on visual processing in the central complex, it is important to note that even diplura, a

wingless and blind insect, has this structure indicating the central complex is essential to

survival rather than just important to visual processing(Böhm et al., 2012). The individual

nature of the motor and sensory needs for a species is reflected in the structure of the

central complex which appears to modulate if not initiate many of these behaviors. The

largest central complex is found in spiders, but forms of the central complex are also

found in crabs (Strausfeld et al. 2002). Regardless of this variation within the arthropod

phylum there are four similarities that are found across species (Loesel, 2011):

1. The fan-shaped body, the largest neuropil, is divided into horizontal layers that are then intersected by columnar cells 2. There are columnar cells that connect all the substructures 3. Some columnar fibers cross the midline to form a chiasm 4. The size and complexity of the central complex reflects the behavior of the animal

Overview of Central Complex Development

Most of the developmental work regarding the central complex has come from

studies in Drosophila and grasshoppers. However, this region of the insect brain is well

conserved and thus, the process has been shown to be similar across species though it

may occur at different life stages. For example, the central complex develops in

grasshoppers during embryogenesis whereas in Drosophila the central complex develops in the pupal stage (Boyan & Reichert, 2011; Williams & Boyan, 2008). The development of grasshoppers can be loosely divided into two main stages. In the first stage, a primary axon scaffold, a group of axons provide structural support for cells to grow, is established. At this stage, there is no modularity(Williams & Boyan 2008). This normally occurs in the first 40% of embryogenesis (Boyan & Williams, 2008). In the second phase of development, the modular neuropils and the precursor structures of the central

complex develop(Kaissling, 1997; Williams & Boyan, 2008). The first major neuronal

element of the central complex appears in the first instar of development. At this time

there is a group of cells known as the pars intercerebralis which eventually develop into the dip in the insect brain that separates the two protocerebral hemispheres(Strausfeld,

2012). The pars intercerebralis is a bilaterally symmetrical structure that contains the precursor cells for the W, X, Y, and Z cells which develop into the major group of axonal connections for the central complex(Williams & Boyan, 2008). The pars intercerebralis directs neural axon growth to and around the protocerebral bridge and into the other substructures of the central complex. These axon tracts are referred to as the W, X, Y and

Z respectively and are mirrored across the midline [W,X,Y,Z | Z,Y,X,W] (Boyan et al.,

2008; Williams & Boyan, 2008).The cell axons project from the protocerebral bridge to the fan-shaped body though most of them cross the midline rather than projecting straight down. This creates a chiasm that could be important for interhemisphere information sharing (Loesel, 2011). As the brain develops and the axons continue to grow, the connections become increasingly complicated. There are at least three other commissures that occur with the progeny of the W, X, Y, Z cells. The highly ordered columnar

organization of the central complex is visible mid-way through embryogenesis(Williams

& Boyan, 2008) .

The Central Complex’s influence on Spatial Orientation

Vision in insects is used primarily for navigation and object recognition (Giurfa &

Menzel, 1997). The compound eyes have been associated with steering during flight in both locusts and flies (Taylor, 1981;Gotz & Wandel ,1984). The locust is able to move its head to adjust for changes in the horizon when the compound eyes are present, but do so to a lesser extent when the compound eyes are surgically ablated (Taylor, 1981). The ability to judge distance and process images is important for flight control. In particular, changes in moving patterns will cause Drosophila to act on the movement of flight, but not the elevation of flight(Gotz & Wandel, 1984). Visual recognition of a target object by the fly can influence landing behavior (Borst & Bahde, 1988). In terrestrial insects such as desert ants, visual recognition of landmarks help them navigate back to their nest(Akesson & Wehner, 2002)(see The Central Complex and Memory for more details).

Other insects are able to migrate large distances consistently despite changing landmarks

(Heinze & Reppert, 2011; Sakura et al., 2008) ,using visual cues such as sun locations and polarized light patterns rather than object recognition.

The visual field has been shown to be at least partly represented in the columnar elements of the central complex(Liu et al., 2006; Strauss, 2002). Objects in the front of the visual field were found to be represented by the innermost columns whereas objects in the fly’s rear visual field caused an increase in activity in the lateral columns. In addition, the layers of the fan-shaped body encoded characteristics such as size and color

which was found to be important for visual object recognition (Strauss & Berg, 2010;

Triphan et al., 2010).

Recent studies in locusts and butterflies have shown activation of central complex

in response to polarized light that is found in the sky (Heinze & Reppert 2011; Heinze &

Hömberg, 2009). The e-vectors from polarized sunlight are detected by the

photoreceptors in the dorsal rim area of the eye (Heinze & Hömberg, 2009; Sakura et al.,

2008). This, then, is conducted to the central complex where the orientation of the e-

vector affects the activity of cells in the central complex. Many of these cells showed a

“polarization opponency” (Vitzthum et al., 2002) where one orientation of the e-vector

causes maximal activation and the opposite orientation causes minimal activation

(Vitzthum et al., 2002) which would allow for directional navigation.

Edge detection as well as spatial memory are encoded in the different layers of

the central complex (Heinze & Homberg, 2009). Drosophila with a defective

protocerebral bridge (tay bridge and ocelliless mutants) cannot accurately judge the

distance in a chasm resulting in the animal using abnormal behavioral sequences to cross

the gap. In addition, these animals are unable to properly orient their bodies while

crossing (Pick & Strauss, 2005).

The Central Complex Role in Changing Motor Behavior

Motor actions are often the only visible output the brain or any higher order

collection of neurons can make (Grillner, 2003). The central complex was initially linked

to behavior by Franz Huber. In the late 1950s and early 1960s he performed experiments

on short-horned grasshoppers by stimulating near the central complex with electrodes

(Strausfeld, 2012). In stimulating the central complex the grasshoppers produced sound

patterns through stridulation, a coordinated movement of the wings. Stridulation is used

by grasshoppers in order to mate and settle rivalries which are important behaviors for

reproduction. This was the first indication that the central complex was involved in the production of behaviors(Strausfeld, 2012). Excitation of the central complex through pressure injection of muscarinic agonists leads to stereotyped stridulatory behavior(Heinrich et al., 2001). This behavior appears to be elicited through the excitation of the command neurons in the central complex which in turn influence the descending neurons ultimately affecting the thoracic ganglia. This set of command neurons in the protocerebrum activate the thoracic pattern generators and cause a correct sequence of movements for the behavior to play out (Heinrich et al., 2001). In addition, studies in Drosophila (tay bridge mutant) with a deficient protocerebral bridge resulted in

lowered walking speed and activity that was rescued when protocerebral bridge function

was restored. Though the swing phase of walking was not changed, it was found that

swing speed was affected (Strauss & Heisenberg, 1993). Other mutations in the central

complex, lead to the fly only being capable of walking in circles. Drosophila with disrupted inter-hemispheric communication(sim mutants) were unable to participate in right/left “bargaining” and walked in circles though the local thoracic circuits were not impacted (Pielage et al., 2002; Strauss, 2002). It is clear that the central complex has some influence over both limb coordination and initiation of some behaviors important to survival.

The central complex has been directly linked to locomotor changes and behaviors using extracellular recording, stimulation and lesion techniques. Activation of neurons within the central complex is strongly correlated and in some cases predictive of changes

in stepping frequency (Bender et al., 2010). In this study, multi-channel recording wires

were inserted into the central complex of tethered cockroaches. Spontaneous walking

speed varied and activity in many of the central complex units altered their firing rate in

time with these locomotor changes. Many of these central complex units had a strong

correlation between an increase firing activity and walking speed. The firing rate changes

preceded locomotory changes. Furthermore, Guo and Ritzmann (2013) found units in the

central complex that had an increase in firing rate before a tethered turn was performed

and were biased to increases in ipsilateral turning speed(Guo & Ritzmann, 2013). Units

that were tuned to right turning were only found on the right side of the fan-shaped body

while units tuned to the left were found solely on the left side of the fan-shaped body.

Units tuned to forward walking speed independent of direction were located throughout

the fan-shaped body.

Lesions in the subregions of the central complex can lead to specific deficits in

motor response such as turning and tunneling. Lesions in the protocerebral bridge or the

ellipsoid body resulted in problems with turning and tunneling behaviors(Harley &

Ritzmann 2010). It has been established that cockroaches will turn away from the

direction of antennal contact with a surface (i.e. if the right antenna hits the wall, the

cockroach will turn left) in 98% of the cases of insects with intact central complexes.

However, cockroaches with lesioned central complexes had a reduced response wherein

there was either a delay in turning, failure to turn, or turning in the wrong direction.

Interestingly, these failures to turn were often only in one direction. Lesions in the lateral

accessory lobe and ellipsoid body resulted in execution deficits in both contralateral and

ipsilateral turns. Lesions in the lateral fan-shaped body led to more deficiencies in turning

than lesions in the medial regions of the fan-shaped body. The lateral fan-shaped body

lesions caused turning deficits only in one direction. This was the same for the

protocerebral bridge. A summary of the lesions and their effects can be viewed in Table

1. Cockroaches with lesions outside the central complex (i.e. the mushroom bodies)

showed no such deficits in turning(Harley & Ritzmann, 2010).

Table 1: Summary table of lesion experiments in the central complex. Turning deficits based on tactile information varied depending on which structures of the central complex were affected.(Harley & Ritzmann, 2010) Central Complex Lesions affects Lesion affects ipsilateral Substructure contralateral turning turning Medial Protocerebral bridge N/A N/A Lateral Protocerebral bridge Yes No Medial Fan-Shape Body N/A N/A Lateral Fan-Shape Body Yes No Ellipsoid Body Yes Yes Lateral Accessory Lobe Yes Yes

The Central Complex and Memory

While memory in insects is strongly associated with the mushroom bodies, another region of the insect brain, there is evidence that some memory processing occurs in central complex. The central complex has been implicated in visual memory in

Drosophila studies. The ellipsoid body in particular is important for maintaining movement toward a visually targeted object even if the object moves out of view.

Animals with central complex disruptions will “forget” their target and change direction

whereas those with normal central complex function will keep on the same flight

path(Strauss & Berg, 2010). The central complex also receives memory inputs from other

regions of the brain and is likely involved in memory circuitry. Damaging the central

complex causes deficits in memory access and consolidation though these processes may

not actually occur in the central complex(Strausfeld, 2012). The fan shaped body has

been shown to aid in object recognition in Drosophila through studies of the rutabaga

mutant. The fan-shaped body can help the individual avoid objects that it has negative

experiences with in order to enhance survival(Liu et al., 2006).

IV. Motor Behavior

Locomotion

Hexapods such as B. discoidalis walk in either a tripod, metachronal or an

“intermediate gait”. In a tripod gait, the ipsilateral front and rear legs swing forward with

the contralateral middle leg while the other three remain in stance ( Hughes, 1952;

Ritzmann & Buschges, 2007). Traditionally, this is considered to be the gait seen at

“faster” speeds. A metachronal gait generally occurs in terrestrial insects at low speed. In

the metachronal gait, the insect legs are lifted in a wave like manner from back to front.

An intermediate gait occurs when the animal walks in a combination of these gaits. In a

freely walking cockroach on a flat surface the animal tends to walk at one of two speeds

(Ritzmann & Büschges, 2007). Interestingly, recent studies of cockroaches tethered on an

oil plate show that even at low speeds cockroaches nearly always use a tripod gait(Bender

et al., 2011). In an arena with free walking cockroaches. There were two clusters of

speed observed. The first speed was an “ambling” pace (slower than~10cm/s) and was

often seen when the animals has antennal contact with the wall. The second cluster of

walking speed was a “trotting” gait (~30 cm/s) that was observed when the cockroach

lost antennal contact with the wall(Bender et al., 2011). This trot speed was about half the

speed seen in an escape response (~60cm/s)(Bender et al., 2011).

Each pair of legs has a different mechanical role in forward walking. By

measuring the ground reaction forces of the animals, Full and colleagues (1991)

determined that the prothoracic legs both aid in exploration and act as a brake whereas

the metathoracic legs are used to accelerate the animal. The mesothoracic legs can both

brake and accelerate during each leg cycle much in the way quadrupeds or bipeds

move(Full et al., 1991).

While straight walking is useful, survival of an insect is more dependent on its

ability to react to an environmental stimulus and execute a “transitional behavior”

(Watson et al., 2002). This signal to “turn” likely comes from the brain triggered by

some sensory cue captured by the eyes or antennae. This causes asymmetrical changes

mostly in the mesothoracic and prothoracic legs by affecting circuits within the thoracic

ganglia(Mu & Ritzmann, 2005;Hellekes et al., 2012). A single antenna touch can initiate

turning behavior. The turning movement was achieved through mediation of the inside

mesothoracic leg (ipsilateral to the direction of the turn) by changing the time of

extension of specific join segments.

In another study conducted by Gruhn curved walking was elicited in stick insects

using moving stripes as a visual stimulus(Gruhn, et al., 2009). The outer legs of the stick

insect (contralateral side to the direction of the turn) push forward into the turn whereas

the inner legs acted to lessen the vector angle by reducing stride length. The only

exception to this is the metathoracic leg which stops moving to perhaps act as a pivot

point. Furthermore, single and double leg preparations where the inter-leg coordination

was eliminated, was used to determine that turning is an active behavior where the

mechanism for movement is due to the presence of signals to the motoneurons rather than the turn being a result of passive changes in the body induced by the front leg (which initiates the turn sequence)(Gruhn et al., 2009).

V. Escape Behavior

Escape behavior is a vital survival response elicited by a variety of environmental

stimuli via different sensory channels in most species. Unlike the optomotor response that

clearly involves visual processing within the brain, most of the known escape circuitry

for cockroaches is located in the thoracic ganglia (Ritzmann and Eaton, 1997). However, intact brain circuitry has been shown to influence escape responses in cockroach

(Schaefer and Ritzmann, 2001). Generally, escape has two phases: (1) a quick turn away from the “danger” stimuli and (2) a more straight run that occurs in a more random direction (Camhi & Tom, 1978). When the escape response is activated by wind, the wind stimulus is detected by mechanoreceptive hairs on the cerci (Westin et al., 1977).

Activity in these receptors sums in the giant interneurons that project to the thoracic ganglia. These interneurons synapse with the type A thoracic interneurons which in turn activate local thoracic circuits(Ritzmann,& Pollack, 1988) and cause a contraversive turn i.e. away from the stimulus direction(Camhi & Winston, 1978; Comer et al., 1994). The type A thoracic interneurons (TIAs) characterized by Ritzmann and colleagues are multimodal (Ritzmann & Pollack, 1990) in that they are activated by wind or tactile stimulation. Likewise, escape response can be triggered by multiple modalities ( Schaefer et al., 1994).

Escape can be activated by different stimuli, such as wind or touch, perhaps by initially recruiting different components of the nervous system (Schaefer et al., 1994).

Anatomical and eletrophysiological recordings revealed that the mechanosensory organs of the antenna can affect the local thoracic circuits through descending motor interneurons (DMIs) to elicit escape(Burdohan & Comer, 1996). Although, wind evoked

escape responses are initially detected by the abdominal cerci and processed through the

giant interneurons rather than the antennae or other tactile sources, the motor turning

movements are the same(Schaefer et al., 1994). There are three types of escape turns seen

in cockroaches. Type I turns result in the animal moving forward and away from the

stimuli. Type II turns result in the animal making a large rotation away from the stimuli,

but does not move forward. A type III turn which is the least frequently used turn results

in the animal backing away from the source of the stimuli before making a type I or II

turn. These turns are seen in both wind and tactile induced escape (Nye & Ritzmann,

1992; Schaefer & Ritzmann, 2001).

Cockroaches stung by the wasp, Ampulex compressa, in the subesophageal

ganglion suppresses escape. Interestingly, brainless and sham surgery animals show

similar escape response. However, individuals that had wasp venom injected into the

subesophageal ganglia had a disruption in the connection with the thoracic interneurons

resulting in the lack of recruitment of the fast motoneurons which allow the insect to

escape (Libersat et al., 1999). This study found that the subesophageal ganglia had more

influence on escape than the brain itself did(Libersat et al., 1999).

Recent data on fly escape from visual threats demonstrates a motor planning

phase that leans the fly away from the threat prior to the actual escape jump (Card &

Dickinson, 2008). The authors speculated that the central complex may be involved in

this process. It is clear that higher order processing centers such as the central complex

and the subesophageal ganglion can influence reflex driven responses such as escape and

walking. However, a direct pathway from sensory structures and the thoracic motor

centers would come at a cost of increased latency potentially compromising the escape

response. Thus, it is important to establish whether the central complex is in a direct and

required link to the motor center.

VI. Procaine

Procaine is an anesthetic in the same class of drugs as Novocain. Procaine acts by

blocking voltage gated sodium channels and potassium channels in a dose dependent

manner. Much work has been done in vertebrate neurobiology regarding using

anesthetics to investigate the activity and necessity of different brain regions and

pathways. Within the arthropod phylum, a similar reaction to procaine has been

documented in bees. Devaud and colleagues used the patch clamp technique to show that

voltage dependent sodium and potassium channels were affected in a dose-dependent

manner (Devaud et al., 2007). The amplitude of the sodium currents in the bee brain was

reduced as the concentration of procaine was increased. Since honey bees and

cockroaches belong to class (Insecta) they are likely comparable. However, in the bee

brain the procaine effect lasted about 90 minutes. In our experiments extracellular data

shows that at our dose of procaine, the electrophysiological effects only last about 30

minutes from the injection time and behavior generally returns within 45 minutes post

injection time (T2-T3). Procaine has also been shown to reduce neural activity in other

species like the garter snake by inhibiting potassium and sodium channels and ultimately

reducing initiation of muscle contraction(Heistracher et al., 1969).

Significance of Project

In doing this project, we hope to add to the body of evidence that the central

complex is a sensory-motor integration center. We examined two behaviors in

conjunction with a reversible procaine block applied to the central complex. An

optomotor response was strongly altered after procaine injection into the central complex,

while controls in which saline was injected showed no such effect. In contrast, escape

speed was shown to not be modulated by the central complex.

Methods

Housing

All B. discoidalis were housed in a temperature controlled room at 27C with a 12

hour light/dark cycle. The colony was started from 250 cockroaches from Dr. Larry L.

Keely (Texas A&M University). Animals were raised in 5-gallon painter’s buckets with

bedding made of cardboard egg cartons with aspen shavings on the bottom. Water and

commercial chicken feed were added to the containers ad libitum. Only intact adult male

B.discodalis were used. Plastic tether with animal

Computer with stripe display

Air supported ball

Figure 3: Schematic of experimental set up (side-view). Data was collected of the ball’s rotation in two different directions; Front-back and right-left. The air supported ball allowed for natural walking of the cockroach. Data was indirectly correlated through measuring the ball’s position in the forward-backward and right-left direction.

Phase 1: Pretrial

Plastic flexible tethers approximately 1.5 inches long were attached to the B.

discoidalis pronotum using hot glue. The animals were mounted over an air supported ball facing a LCD computer monitor (Figure 3). Sensors taken from a computer mouse monitored the movement of the ball and hence the cockroaches intended movement in two orthogonal directions (left-right and front-back). Two trials were performed to assess the cockroach’s ability to turn in response to the moving stimuli. A custom linux program was used to generate a continuous pattern of moving black and white stripes with a width of 105 pixels and speed of 420 pixels/s. The screen was approximately 6 inches from the animal. If the animals showed no response to either stripe direction then

they were not used in further experiments. If the animal only responded in one direction

then this was noted and all subsequent trials used only that one direction. A starvation diet reduced the level of hemolymph in the animal’s body and made the subsequent surgery easier to perform. To generate this condition, cockroaches were housed in the

animal room for between 1-4 days without food or water. In some instances, cockroaches

were pretested 5 or 6 days before a trial. In this case food was provided for day 1 and 2.

No deleterious effect was noted in locomotory behavior due to lack of food and water.

Phase 2: Injections

Pretested animals were cold anesthetized by icing them for 15-30 minutes before

surgery. Injection pipettes were made using a pipette puller (Sutter Instruments). These

pulled pipettes were made from single barrel capillary tubes (World Precision

Instruments, OD/ID, 1.0/.58mm). The ends were broken using forceps to make a tip opening between 20-30μM. The tip openings were measured using a compound

microscope and none over 30μM were used. These pipettes were backfilled with either

the procaine or saline solution.10% and 20% procaine solutions were made. Both

procaine and saline solutions contained dextranflorescene (MW: 3000), a fluorescent dye,

as well as fast green (MW: 765) which allowed the solution to be easily visualized during

the injection process. The saline was a Blaberus saline that was standardly used for

physiology. Because B. discoidalis hemolymph makes large clots, it must be kept from

the brain during surgery to improve visibility and prevent the pipette from clogging. A

ligature was therefore placed around the animal’s neck in order to restrict hemolymph

through the cockroach’s neck. The animal was pinned on a piece of cork, wrapped in

foam and put into a tube that was placed under a dissection microscope. The head rested

on a neckplate and was waxed into place (Hypognathous position). The ventral surface of

the insect brain was exposed by cutting a hole in the cuticle between the eyes. The

surrounding trachea and fat were removed to expose the brain. The neurolemma was

generally left intact. The pipette was inserted into the brain and the solution was injected

using the PM 2000 (B) 4-Channel pressure injection system (Microdata Instrument Inc-

S. Plainfield, NJ). The injection was aimed at the center of the brain to increase the

likelihood of hitting the central complex. After the animal was injected it was taken out

of the restraints and the neck ligature was cut away to return blood flow to the brain.

Phase 3: Trial

The animal was given 10-15 minutes to recover before its righting behavior was

checked and any aberrant behavior was noted. The plastic tether was then hot glued to its

pronotum. The animal was then tethered to the air supported ball and Trial 1(T1, time

point, 0 minutes) was run. Each trial was 90 seconds and the visual stimuli, moving black

and white stripes, lasted 10 seconds and were started at 30 seconds into the trial. The

stripe direction was randomly selected. A trial was run every 15 minutes from trial 1 to

trial 5 for the total of 60 minutes on the ball and five trials. After the recording session,

cockroaches were then taken off the ball and the brains were prepared for histology.

Phase 4: Histology

If the animal had undergone an injection after being taken off the ball the

cockroach was heavily sedated with carbon-dioxide gas and decapitated. The brain was

removed and fixed overnight in a 4% paraformaldehyde solution at 2-4°C. The brain then

went through a wash and dehydration process to visualize the injection site (Table 2).

After the full dehydration process the brain was imaged using fluorescence microscopy in

a light microscope to see if the dye was near the central complex. If the dye point was located in X-Y vicinity of the central complex (in the center of the brain between the two hemispheres) then the brain was mounted in a DPX mounting solution (mix of distrene and xylene) and further imaged in the Z-plane using a confocal microscope to ensure that the dye was located in the central complex (Figure 4).

Table 2: Histological Process. The process took approximately 3 hours.

Treatment Time Repeated Paraformaldehyde 12 -18 Hours 1 Phosphate Buffer 15 minutes 2 Solution(PBS) 70% Ethanol in water 15 minutes 1 90% Ethanol in water 15 minutes 1 95% Ethanol in water 15 minutes 1 100% Ethanol 30 minutes 2 Ethanol - Methyl salicylate 20-30 minutes 1 solution (equal parts) Methyl Salicylate 20 -30 minutes 1

Mushroom Bodies

Central Complex

Figure 4: Confocal image of B. discoidalis brain with dye. Bright green is a result of the dextranflorescene with background green structures are identifiable. The red arrow indicates the bolus of dye which is located in the fan-shaped body (see Figure 2 for more details on central complex structure).

Electrophysiology

In a separate preparation, extracellular recordings were performed with the help of

Ms. Adrienn Varga on the central complex of B. discoidalis before and after injection

20% procaine and the 10% procaine. Injection pipettes were used to inject the procaine

concentration in 2 different insects. A 16 channel, 2 tine probe from Neurnexus was used

and was plugged into the headstage of a Neuralynx Cheetah (Bozeman, MT, USA) digital

interface. The sampling frequency was 30 KHz, the analog filter was set to 600 Hz (low

pass) and 6000 Hz (high pass). Sorting was done in Spike 2 version 7.10, K-means

clustering was followed by manual sorting with 20 msec ISI for separating units.

Histology was used to confirm the injection and recording sites.

Escape Behavior

Escape behavior was tested in 10 subjects by tapping the cockroach using a

mechanical probe on the abdomen. The probe had a set length that could be pushed back

and then when released would quickly tap the animals on the posterior part of the

abdomen to elicit escape responses. In these trials, optomotor responses were also

performed to compare the procaine effect to that behavior. In those cases, the stripes were

run at 30 seconds for a 10 second interval and the escape response was triggered at 60

seconds.

Data Processing

Data were collected using a mouse tracking software input into MATLAB using

simulink. Position of the cockroach in two different axes was calculated and the data

were plotted for each of the axes vs. time. The initial output values were of the position

of the ball in relation to the computer screen and this movement away from the center of

the screen was calculated into two separate measures.

(A) (B)

Figure 5: Example of raw output. (A) Forward-backward movement. The black line indicates where the 0 line is. Negative numbers indicate that the cockroach was walking forward. (B) Right-Left movement graph. The positive numbers indicate the cockroach was walking to the right and the negative numbers indicate that the cockroach is walking to the left. The first was the forward/backwards walking speed (cm/s, converted from

pixels/s) and the second was the angular velocity in the right or left direction (pixels/s

converted into radians/s) (Figure 5).

(A)

(B)

+ >

Figure 6: Example of data processing method. (a) The right-left data chart is filtered to represent only the direction of the stimulus. The motion stimulus was moving to the left and all the data points are in the negative range. (b) for the left direction visual processing threshold was set by taking 2* standard error of the below the mean position before the visual stimulus. If the mean of the position is below this value then a true value for a turn response is returned.

All stripes were run at 30 seconds in the 90 second trial. Stripe pattern was

randomly varied and either moved right or left. All the data points from 0-30 seconds

(right before the stripes were triggered) were filtered such that only the points in the

direction of the stripes were considered (positive - right turn, negative - left turn).

A right turn was classified by using the right-left positional data (see Figure 6,7):

2 ∗ .

A left turn was classified if the following statement was true:

2 ∗ .

This resulted in a yes or no operator that was then tabulated to get response averages per

trial. There were no turns that were in the opposite direction of the stripes. Significance

was determined using an unpaired t-test.

(A) (B)

Right

0 rad/s

Left

Figure 7: Example angular outputs. The moving stripes were played between the red stripes. (A) A right turn during the visual stimulus. The cockroach had a higher angular velocity during the time of the visual stimulus (B) A left turn due to the visual stimulus. The cockroach switched its walking direction when the stimulus was present.

Table 3: Summary of Experimental Groups. 15-20 cockroaches were in each experimental and baseline group.

Experimental Group Number of Cockroaches Control, No surgery 20 Sham Surgery 19 Saline Injection 15 10% Procaine Injection 16 20% Procaine Injection 15 Escape 20% Procaine Injection 10

Escape Response Analysis

Escape response was elicited with the use of a mechanical tapper that was set to

tap the abdomen on the animal. During escape trials, the visual stimuli started at 30

seconds and the escape was elicited at 60 sec. Escape speed was measured through

processing the forward/backward motion data and then taking the average speed during a

4 second period (58-62) wherein the stimulus was at 60 seconds. Sham surgery animals

were used as a control to demonstrate the surgery had no effect on the escape speed of the

animal.

Post Injection Behavior

Post injection behaviors were observed to ensure that the animals recovered from

the surgery. In the post-recovery period of the trial the cockroaches would run around the

bins often slamming into the side of the walls head first. Other behaviors that were also

commonly observed were animals running in circles for short periods of time. Some

animals would show little movement even when prodded. After 10 minutes, most

surgical subjects would walk for at least a short period of time. Sometimes locomotion was jerky or the stance posture was hyperextended. If normal stance or walking was not

seen after 10 minutes the animal was discarded because this was likely caused by damage

to the neck connectives. During this time period animals were more likely to fall over. In

addition, to these behaviors 48% of animals were unable to right themselves after

injection with 20% procaine in comparison to the 17% of animals who were unable to

right themselves after injection with saline.

Errors in Data Analysis

Turning behaviors were determined using a standardized metric by comparing the

cockroach’s forward walking behavior before the visual stimuli and the walking

movement during the stimuli. However, after the standarized turn test was performed, it

was clear upon visual inspection that there were some trials that were falsely labeled as a

turn (false positives) and some that were falsely indicated as a “not turn(false negatives)

(Figures 8,9). These errors could occur if the cockroach was turning before the stimulus

was played resulting in the animal continuing the behavior. Many methods to

automatically reduce the number of falsely identified trials, but in the end the most

effective was to manually correct the obvious errors. We generated figures that clearly

mark the false positives and negatives with different colors. The saline trials showed 8

visually identfied errors in turning (either false positives or negatives) over all 75 the

trials. The 20% procaine trials had 11 visually identified errors in turning(either false

positives or negatives) over all trials. When the false positive and false negatives were

manually corrected in the first trial for the saline and the 20% procaine the difference in

the optomotor response rate was still significantly different. There was only a slightly

stronger trend (p = .000219 corrected < .000229 uncorrected) in the manually corrected

data. Since the manually corrected data set was more accurate and did not affect the final

conclusion of the study, we felt justified in using the corrected values for further

analysis.

(A) (B)

Figure 8: Example of False Negatives. (A) the response between the red ines (when the visual stimuli was present. However, the response magnitude was similar to turns made by the insect in the pre-stimulus movement and since this method cannot take into consideration the fact that animal had stopped and was induced to walk in the direction of the stripes by the stimuli it failed to be scored as a turn. (B) The animal was moving more strongly toward the right in the time before the visual stimuli, however durng the stimulus the animal still maintained movement in the correct direction, just not at the same magnitude.

(A) (B)

Figure 9: Examples of False Positives. (A) The response during the visual stimuli is between the two red lines. The average response magnitude of the time during the stimuli is just slightly above the threshold required to classify a turn response. (B) The cockroach moves in both directions and it is unclear that it is a response to the visual stimuli rather than just random movement. However, this qualified as a response because the magnitude is greater toward the end of the visual stimuli to the right.

Results

Optomotor response was measured by demonstrating that the cockroach “turned” in the direction of the moving stripes. A baseline of optomotor response rate was established through the study of untreated naïve animals (Figure 10). Despite their untreated state, some animals failed to respond to the stimulus every trial. Thus, in examining the experimental conditions, it is important to keep in mind that the normal optomotor response under our experimental conditions is robust but imperfect in response rate.

Controls

Two types of control experiments were performed to ensure that the change in response rate seen in the procaine trials were due to the presence of the drug. A sham surgery control (Figure 11) was performed to ensure the deficits in optomotor response for the procaine groups were a result of the central complex manipulations and not the

surgery itself. A saline injection control was also done to demonstrate the results of the

procaine trials were not a result of brain damage from the pressure of injection of any

fluid (Figure 12). Saline injected animals showed a reduction locomotion for the

immediate 10 minutes post-injection likely due to the surgery itself, but did not show a

reduction in locomotion during any of the trials. Overall, there was no significant

difference among the no surgery optomotor trials and those of the two control trials

(ANOVA, df = 2 , p=.22) (Figure 15). The saline injection trials were the closest

procedure to the procaine injected trials but without the drug and thus were used for

direct statistical comparison with the procaine data.

Figure 7: Optomotor response for untreated cockroaches. Trials (n=20) were run over the course of an hour. 75% of the cockroaches showed an optomotor response in the first trial.

Figure 10: Optomotor response for untreated cockroaches. Trials (n=20) were run over the course of an hour. 75% of the cockroaches showed an optomotor response in the first trial.

Figure 11: Optomotor responses for cockroaches with sham surgery (n=19). Animals were stimulated five times over the course of an hour at the 30 second mark in the 90 second trial. At the first time point, there was a 58% optomotor response rate.

The Procaine Effect on Optomotor Response

Figure 13 shows the responses per trial for each cockroach injected with 20% procaine and Table 4 contains the statistical comparisons of these trials to the saline controls. Only 13% of the cockroaches injected with the 20% procaine in their central complex responded to the first visual stimulus compared to the 73% that responded in the saline trials. At the second time point, the response rate for the 20% procaine subjects more than doubled to 33%, however, it was still significantly lower than the response rate of the saline injected insects. Only the first two trials showed a significant difference in positive turn response to the visual cue. Beyond that recover there are no significant

differences in the turning response between the 20% procaine and saline responses. In

the procaine trials, 3 of the cockroaches did not walk in the time period the visual

stimulus was presented in the first trial. These were counted as “no response”. All those

cockroaches regained activity by the 15 minute mark(Trial 2). The remainder of the 20%

procaine subjects walked during the first trial, even if they did not turn.

Table 4: Turn response comparison between saline and 20% procaine trials. The procaine appears to have significantly altered turning behavior for the first two trials, but not the final three trials. P-values were calculated using a t-test.

Trial Time 20% Procaine Saline P-value Was effect (min) Response Rate Response Rate of drug significant? 1 0 13.3% 73.3% .00009 Yes

2 15 33.3% 73.3% .03 Yes

3 30 60.0% 86.7% .09 No

4 45 80.0% 73.3% 1 No

5 60 53.3% 60.0% .59 No

Figure 12: Optomotor Responses for cockroaches injected with saline. Trials (n=15) were run over the course of an hour. Time point 1 is at 0 minutes and trials were run every 15 minutes. The red box is an indication of a turn response and the dark blue boxes represent no turn. The light blue represents false positives and the orange boxes represent false negatives. Each animal was stimulated 5 times. Time point 1 is at 0 minutes and trials were run every 15 minutes ending at 60 minutes. The colors and time points are the same in all figures. There was a 73% response rate at the first time point (see Table 4).

Figure 13: Optomotor Responses for cockroaches injected with 20% procaine. Trials (n=15) were run over the course of an hour. The red box is an indication of a turn response and the dark blue boxes represent no turn. The light blue represents a false positives and the orange boxes represent a false negative. 13.3% of cockroaches responded at the first tme point.

Figure 14: Optomotor for cockroaches injected with 10% procaine. Only the first trial shows a significant difference from the saline injection response rate in comparison to schedule. At the first time point 37.5% of the cockroaches responded.

Table 5: Turn response comparison between saline and 10% procaine trials. The procaine appears to have significantly altered turning behavior for the first trials. P-values were calculated using a t-test.

Trial Time 10% Procaine Saline P-value Was effect (min) Response Rate Response Rate of drug significant? 1 0 37.5% 73.3% .04 Yes

2 15 50.0% 73.3% .42 No

3 30 62.5% 86.7% .10 No

4 45 75.0% 73.3% .78 No

5 60 43.8% 60.0% .24 No

100

90 80 optomotor 70 an

60 20% Procaine had

50 10% Procaine that

response 40 Saline 30 Sham 20 No Surgery cockroaches

10 % 0 0 15304560 Time Point (minutes)

Figure 15: Comparison of optomotor response in all experimental groups. Animals with 20% procaine in their brain had a significantly lower chance of turning in the first 3 trials in comparison to the control trials. There is no significant difference between the baseline animals (No surgery), a sham surgery, and the saline injection (ANOVA, df=3, p=.22)

Injections of 10% procaine also caused a significant difference (unpaired t-test,

T=-5.9, p = .04) in optomotor response rate of the first trial in comparison to the response rate of cockroaches injected with saline (Figure 14). However, in this case there was not a significant difference in response rate in the second trial or after that (Table 5).

Comparison of all experimental groups can be viewed in Figure 15. By the 30 minute mark there was no difference across response rates of all the groups.

Activity Levels

Failure to turn could occur in the procaine trials as a result of generally reduced activity levels. To examine this, overall activity levels for 20% procaine subjects are compared to the saline controls (Table 6). Activity was a calculation of the nonzero data points divided by all data points.

All trials were 90 seconds long and had 1800 data points total. The percent time standing

still was not significantly different between the 20% procaine and the saline trials. We

did note that there was often reduced activity immediately after surgery, but activity was

typically restored before the first trial was run.

Table 6: Average time spent Active during trial. P-values were calculated using an unpaired t-test. Activity was calculated by obtaining the total number of non-zero data points and dividing by the total number of points to calculate the percentage of points that were moving (n=15).

20% Saline P-Value Procaine Trial 1 58.4% 58.3% .98 Trial 2 54.4% 45.8% .31 Trial 3 50.5% 50.3% .98 Trial 4 53.8% 52.6% .86 Trial 5 55.4% 62.6% .36

Escape Response

We tested whether procaine injection into the central complex affected escape

responses. Escape response speed was averaged over a 4 seconds period that was

determined over the pretrials to be the average “period” of escape. This includes the

initial rapid movement and the secondary running period. There was no significant

difference in the average escape speed for the animals that underwent the sham surgery

and untreated animals (ANOVA, df =4, p=.94) Control trials of animals that had a sham

surgery were compared to the speed of escape of procaine animals tested over the course

of 45 minutes.

16 80 % of animals that responded to the motion

14 70

12 60

10 50 sham stimulus

8 40 20% 6 30 Procaine

4 20 Optomotor Response 2 10

Average forward speed during escape (cm/s) 0 0 0 153045 Time (min)

Figure 16: Average speed of escape for cockroaches under the influence of 20% procaine. There was no significant difference between the sham control and the procaine injected trials in all except at the 30 minute point (n=10). The right axis represents the percent of animals that had a successful optomotor response at the time point. The left axis indicates the speed of escape at the time point (gray bars). Standard error bars are shown.

Forward walking speed of the inject animals over the four trials did not vary significantly

(ANOVA, df= 3, p=.63). In the first two trials where the cockroach experienced the greatest of the procaine effect on optomotor response (Figure 16), there was no difference in escape speed relative to the control (Figure 16). However, in the third trial (30 minutes) (unpaired t-test, T= 1.8, p=.04) there was a significant slight decrease in speed.

This decrease in speed is not seen in the final trial (unpaired t-test, T= .3, p=.3).

The optomotor responses of animals in the escape response data set were comparable to those reported for the larger data set. The first trial of the escape response data set had a 10% response rate. This is comparable to the 13.3% seen larger 20% procaine data set. Similar trends were seen in the escape response data set where the majority of the cockroaches (70%) responded to the visual stimulus at the 30 minute

mark as compared to 60% for the larger data set. There was a slight decrease in the

optomotor response rate at the 45 minute mark for the escape experiment group at 50% as

compared to 53.3% for the larger data set. The decrease persisted in the final trials. Both

of these values are similar to the control group.

Extracellular Brain Recording

Extracellular recordings were used to confirm that neural activity within the

central complex was affected by procaine injections. After a successful recording was

obtained in the central complex, 20% procaine was injected into the brain. Rate of

spiking was plotted in Figure 17. Procaine was injected at 5 minutes after the data

acquisition commenced and the results show an immediate significant decrease in firing

rate (t-test, T = 12.5, p=0) from 5 minutes to 25 minutes (Figure 17). The firing rate

began to increase at the 20 minute mark (15 minutes post injection) and gradually

returned to a level that approached the original state about 25 minutes into the recording

(20 minutes after the injection). When 10% procaine was injected, the change in activity

was not as it was with the 20% procaine but there is still a reduction (Figure 18). These

recordings represent one trial each in one cockroach respectively and more trials need to

be done to demonstrate the neural effect of the drug throughout the central complex.

Nevertheless, it clearly shows a decrease in neural activity consistent with our behavioral

results.

8 (Hz) 6 rate 4 Firing 2

0 0 5 10 15 20 25 Time (min)

Figure 17: 20% Procaine influence on central complex spiking activity. Procaine is a reversible sodium channel blocker. When injected in the central complex cell firing rate is reduced (n=1), but returns after about 30 minutes. The red arrow indicates the injection time.

8 7 6 (Hz) 5

rate 4

Firing 2 1 0 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Time (min)

Figure 18: 10% Procaine influence on central complex spiking activity. When injected in the central complex cell firing rate is reduced (n=1). This represents one sorted unit. The red arrow indicates the injection time.

Discussion

Previous research found that localized electrolytic lesions affect decision making paradigms especially when it came to transitional behaviors such as turning. Concerns existed about whether there was damage done by the procedure itself despite the control lesions in the lateral regions of the brain showing an insignificant effect on turning

behavior(Harley& Ritzmann, 2010). In my study, procaine, a local anesthetic, was used

to temporarily silence localized regions in the brain such as the central complex. While

the degree of precise localization using procaine injection was not as high as it was in the

electrolytic lesions, the results showed that injections of procaine temporarily block

correct decision making by reducing optomotor responses while not influencing the

overall activity of the cockroach. The reversibility of the block supports the notion that the drug was responsible for the deficits in optomotor response. Damage that occurred as a result of the surgical procedure would have been permanent or at least would not have reversed over the course of the experiment. Two separate control groups were used to ensure that the result of the procaine injection was not a result of damage from the procedure itself. The saline injections and the sham surgery were found to not have significantly different response rates from the baseline trials (untreated group) over the course of each experiment (1 hour).This further lends support to the building array of experiments that the central complex is involved in the modulation of thoracic circuits and is a center of sensory information processing leading to motor changes (Mu &

Ritzmann, 2008).

Central Complex Influence on Optomotor Response

Effects of Procaine on Central Complex

Electrophysiological extracellular recordings were done in the central complex to confirm the effects of procaine on the firing activity. The 20% procaine clearly reduced the activity of neurons recorded near the injection site based on our preliminary experiment with 1 unit in 1 animal. This effect reversed with the activity level returning mostly to normal around the 30 minute mark. Similar findings were observed by another

student in our laboratory (Kathman personal communication). The recovery rate matches reasonably well with the behavioral data where recovery from the procaine occurred between 25 – 40 minutes post injection (includes recovery time) around the T2/T3 time point in the trial. This value is slightly different than what Devaud and colleagues found.

According to them procaine seems to affect cell spiking activity for up to 90 minutes(Devaud et al., 2007). However, Devaud and colleagues were injecting into the bee brain and a different structure, the mushroom bodies, which could account or the discrepancy (Devaud et al., 2007). Furthermore, they were not testing locomotor activity rather they were looking at learning. Although the spiking activity in Figure 17 began to return in 25 minutes, it is possible that a minimum threshold of descending activity may have to be met to elicit normal behavior. It is likely the return of normal behavior function precedes full recovery of brain activity.

While there was a reduction in central complex spiking activity for animals which had been injected with 10% procaine, it was not as strong as the 20% procaine (Figure

17, 18). This also resulted in less behavioral deficits with turning than the 20% procaine injections.

Effects of Procaine on Behavior

Although the 20% procaine clearly demonstrated that an intact and functioning central complex is necessary for optomotor responses, its dramatic effect is also a limitation. Future experiments would benefit from having the procaine generated more localized and subtle effects. Harley and Ritzmann (2010) found that lesions in all subregions of the central complex (ellipsoid body, protocerebral bridge, and fan-shaped body) affected turning. Not only did cockroaches fail to turn with the electrolytic lesions,

but some of them displayed delayed turning or would turn in the wrong direction (Harley

& Ritzmann, 2010). However, directional effects were isolated to the lateral regions of

the fan-shaped body.

In an attempt to achieve more subtle and localized effects, we injected a lower

concentration of procaine (10%) into the cockroach brain. The behavioral effects were

less global than 20% procaine where the optomotor response was severely reduced and

restricted only to the first time point. It is possible that the lower concentration of

procaine affected only a smaller region of the brain and so the deficits were more variable

from individual to individual in comparison to the 20% procaine (Figure 14). Another

potential explanation is that the lower concentration of procaine affected the same area

but did not effectively silence the cells and thus, the threshold activity required for the

optomotor response was still met. Unfortunately, our current histological techniques

cannot allow for definitive confirmation of the structures that were affected. While the

fluorescent dye was able to show the injection site of the drug it did not show the

migratory path of the procaine. Considering the different molecular weights of the

procaine versus the dye (much heavier), it is unlikely that they migrated at the same rate

or distance of the drug.

A detailed electrophysiological study in conjunction with a more precise

histological study would be needed to establish both the injection site and the effective

distance of the drug from that site. This could establish the extent of the physiological

impact of the drug. While we conducted a recording to establish that the drug did have an

electrophysiological effect, more recordings would be necessary to determine the

effectiveness of procaine and that was beyond the scope of this thesis.

Activity Levels

Drosophila with deficits in their protocerebral bridge or other genetic defects in the central complex have reduced number of walking bouts, walking speed and reduced response to optomotor stimuli (Poeck et al., 2008). Changes in arousal state have been linked to variable motor responses depending on the state of the animal (Niell & Stryker,

2010; Strausfeld, 1976). We found no significant difference in the activity level of the cockroaches in any of the trials of animals that showed behavioral deficits with injections

of the 20% procaine in comparison to those with saline injections (Table 6). The average

amount of time spent moving in all trials was consistently near 50%. Most animals still

walked even in the first trial which occurred 10-15 minutes after the injection. Reduced

arousal was seen for an extended period of time for up to 10 days in fully decapitated

cockroaches (Ridgel & Ritzmann, 2005). This could be potentially attributed to the

continued output of the subesophageal ganglion to the thoracic circuitry (Libersat et al.,

1999; Ridgel & Ritzmann, 2005; Schaefer & Ritzmann, 2001).

There was an imperfect response to the visual stimuli even in animals that did not

undergo any treatment. This is also seen in other studies with similar paradigms. Gruhn

and colleagues had a 75% a response rate of curved walking (turning) of stick insects to

the visual stimuli. However, in that case, 9% of the insects failed to respond to the visual

stimuli while the other 16% walked in the opposite direction of the stimuli(Gruhn et al.,

2006). Our data had a response rate typically had similar response rates of over 70% in

untreated animals as well as saline injected controls.

Escape Response

Escape response has been divided into two main events: a turn away from the

source of stimuli and a random fast run(Camhi and Tom, 1978). The cockroach escape

response has been heavily studied and indeed even the movement of the joints has been

documented by various forms of stimulation (i.e. wind, tactile). Direction was not

examined in this study since the stimuli was directed in the center of the body via the

tactile stimulus. The optomotor response is clearly reduced in the first and second trial,

but the escape speed remains the same in both the animals injected with procaine and

those that underwent the sham surgery Escape speed in untreated baseline animals was

measured to be ~8 cm/s (Figure 16). The procaine trials were no significantly different

from the control trials except in the third trial (30 minute point). We speculated that this

may be due to the adaptation of the escape system to the stimulus (Ritzmann,1984).

However, in the final trial at the 45 minutes, the escape speed is once again unaffected

leading us to conclude there may be an unidentified confounding variable in the escape

system itself perhaps due to the repeated activation of the system.

While there was some evidence that escape circuitry is influenced by brain

circuitry our work does not support that speed of response is influenced by the central

complex(Schaefer and Ritzmann., 2001). However, direction of turn or any response

time after stimuli was not examined in this project. A reasonable conclusion is that

descending activity influences the thoracic circuitry that directly controls cockroach escape turns, but that threatening stimuli directed at the abdominal region do not activate

circuits that must pass through the central complex prior to activation of the leg motor

neurons.

Conclusion

The central complex is a modular structure that is being investigated as a

premotor structure involved in planning, modulating, and even initiating movement

comparable to the basal ganglia(Strausfeld & Hirth 2013). 20% procaine was used to

manipulate the central complex, a region of the insect brain. In reducing the firing rate of

the central complex, deficits in optomotor response were seen. After about 30 minutes,

normal optomotor behavioral function was restored as the effect of the procaine

diminished. These behavioral abnormalities were not a result of the surgery or injection.

Average forward walking speed was not affected in tactile abdomen induced escape.

Injections of 10% procaine resulted in more variable behavioral optomotor response with

about half the animals still responding to the visual stimuli. Injection of procaine can be

used to manipulate circuits in the central complex and with some refinement of the

technique; it could be useful for dissecting out influences of various regions of the central

complex on specific behaviors. This work complements Harley’s results that showed that

electrolytic lesions in the central complex affect turning behavior(Harley & Ritzmann,

2010). It clearly supports the conclusion that the effects found by Harley were specific

for the central complex and not an effect of collateral damage within the brain. Such

effects would likely have persisted over time and the same effect would be seen in the

saline control trials. Through this work it is apparent that sensory information filtering in

through the central complex is able to influence locomotion likely through the thoracic

ganglion.

Future Work for Next Student

The procaine method could potentially be refined to the point where individual columns in the central complex would be able to be affected. However, we did not have as much success with this not because the tip size of the pipette was too small, but rather the pressure needed to inject into the brain would release a large amount of procaine in the brain. One of the challenges in this technique was controlling the amount of procaine released into the brain. We estimated volume initially by injecting with the same conditions into a dab of Vaseline and them measuring the diameter of the solution using a microscope. A high amount of variability was shown even if the pipette openings were the same diameter. The optimal setting so that the solution goes into the brain without damaging the tissue has yet to be determined.

Refinement of the technique could result in a mastery of local circuits that could aid in mapping circuitry and allowing for a workaround to using genetic techniques to get the level of specificity that is generally only available in systems such as Drosophila that have extensive genetic tools. The 20% procaine likely had a global effect and knocked out most if not all the central complex. There were four animals that did respond in the first trial. This was not unexpected as it is apparent that the different substructures of the brain can have profoundly different effects on the behavior of the animal. Furthermore, the dye and procaine are different molecular weights and thus, the dextran is able to provide us with an injection site, but little else. Future work should include a control of rhodamine red dye in order to examine with greater acuity the travel rate of the procaine.

There were some issues with histological observation as the dye, which was originally thought to be fixable, faded after a few weeks (in some cases within in week)

which made it impossible for confocal imaging resulting in loss of data. Rapid imaging

would improve the histological images.

Escape response has been characterized on a fairly detailed level in the

cockroach. We speculate that abdomen induced escape response mostly affects the local

thoracic circuits from the behavioral data. Joint analysis and electrophysiological

recordings would further confirm our results. Doing a more detailed study to see if the untreated animals had escape response speeds that varied over the course of an hour would be necessary to gain further insight into this topic as well.

Acknowledgements

I would like to thank all the members of the Ritzmann lab: Alan Pollack for initially teaching me a procedure and providing advice when I was troubleshooting as well as reviewing all the histological images; Nick Kathman and Peiyuan Guo for helping me with the procedure and answering questions at all hours when needed; Ada Varga for agreeing to do the electrophysiological recordings for me and reading a draft of this document. I would also like to thank Stephen Doyle who was able to assist me not only in general data collection, but especially on the escape response portion of this project.

Finally, I would like to thank Roy Ritzmann for all of his invaluable advice, patience, and support throughout the creation of this document. This work was supported by a grant from the National Science Foundation IOS-1120305.

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