Negotiation of Barriers by Intact and Brain-Lesioned

NEGOTIATION OF BARRIERS BY INTACT AND BRAIN-LESIONED

COCKROACHES

Cynthia Marie Harley

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Adviser: Dr. Roy Ritzmann

Department of Biology

CASE WESTERN RESERVE UNIVERSITY

January 2010 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

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candidate for the ______degree *.

(signed)______(chair of the committee)

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(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein.

To my husband Chris TABLE OF CONTENTS

Thesis Summary ...... 1

Chapter 1:

Introduction ...... 3

TRANSITIONAL BEHAVIORS ...... 5

ETHOGRAMS ...... 8

HEAD SENSORS ...... 9

Head sensor anatomy ...... 10

Antennae ...... 10

Maxillary Palps ...... 12

Vision ...... 13

Ocelli ...... 13

Compound Eyes ...... 14

PROCESSING SENSORY INFORMATION ...... 15

THE INSECT BRAIN ...... 16

Mushroom Bodies ...... 18

Antenno Cerebral Tract ...... 20

Central Complex ...... 21

Genetic Lesions ...... 24

Mechanical Lesions ...... 25

Recordings in the central complex ...... 25

Protocerebral Bridge ...... 28

Central body: the fanshaped body and ellipsoid body ...... 29

Lateral Accessory Lobe ...... 30

MY GOAL ...... 30

MOTIVATION ...... 31

FIGURES...... 33

Figure 1-1 Insect Nervous System ...... 34

Figure 1-2 Head Sensors ...... 35

Figure 1-3 Representation of the main regions within the protocerebrum of the

cockroach brain ...... 36

Figure 1-4 Schematic of the Central Complex ...... 37

Chapter 2:

Characterization of obstacle negotiation behaviors in the cockroach, Blaberus

discoidalis ...... 44

SUMMARY ...... 45

INTRODUCTION ...... 46

MATERIALS AND METHODS ...... 49

Animals ...... 49

Experimental Arena and Obstacles ...... 50

Lighting Conditions...... 51

Measurements ...... 52

Antennal Ablations ...... 53

Ethograms ...... 54

Climbing Strategies ...... 57

Shelf Height ...... 58

Eye Coverings ...... 59

RESULTS ...... 60

Block Obstacles ...... 60

Effect of Altered Antennal Length ...... 62

Shelf Obstacles ...... 64

Effects of Ambient Lighting ...... 66

Role of Vision in Dark Light Response ...... 68

Effects of Changing Shelf Height ...... 68

Body Posture Under Different Lighting Conditions ...... 69

Antennal Position Under Differing Light Conditions ...... 70

DISCUSSION ...... 70

Distance From Block Affects Climbing Success Rate ...... 72

Detecting Alternate Routes ...... 73

Ambient Lighting Influences Whether a Cockroach Climbs or Tunnels ...... 74

The Bias in Tunneling Was Only Present for Moderate Shelf Heights ...... 74

Ocelli Detect Light Levels and Influence the Light Bias on Shelf Behavior ...... 75

FIGURES...... 77

Figure 2-1: Block Climbing Behavior ...... 77

Figure 2-2: Distance from the Obstacle and Climbing Success ...... 78

iii

Supplimental Figure 2-1: Normalized Distance from obstacle and climbing success

...... 79

Figure 2-3: Strategies Used for Block Climbing ...... 80

Figure 2-4: Ethograms of Block Climbing ...... 81

Figure 2-5: Shelf Climbing and Tunneling is related to antennal contact ...... 82

Figure 2-6: The influence of shelf height and ambient lighting on climbing behavior

...... 84

Figure 2-7: Ocelli Determine if it is light or dark ...... 85

Figure 2-8: Antennae Sample the same space regardless of lighting ...... 86

Suppliment 2-2 Antennae sample the same space regardless of lighting ...... 87

Table 2-1: Dwell time after specific antennal contact patterns ...... 88

Chapter 3:

Discrete Lesions in the cockroach brain and their effect on the negotiation of barriers .. 95

SUMMARY ...... 96

INTRODUCTION ...... 97

MATERIALS AND METHODS ...... 101

Electrolytic Lesions ...... 102

Nonspecific damage during the lesion process ...... 103

Histology ...... 104

Behavioral Testing ...... 105

Lighting Conditions ...... 105

Experimental Arena and Obstacles ...... 106

Video recording and analysis ...... 110

Reflex Testing ...... 111

RESULTS ...... 112

Distance from the block and climbing success rate are decreased after lesions to

certain brain regions ...... 112

Head Contact with the Block ...... 115

Changes in block climbing strategy are present after the lesion ...... 115

Changes in Behavioral Sequences ...... 116

Changes in Turning Behavior ...... 117

Problems transitioning from a wall ...... 120

Changes in local reflexes ...... 120

DISCUSSION ...... 122

Protocerebral Bridge ...... 123

Fan-shaped Body ...... 124

Ellipsoid Body ...... 125

Lateral Accessory Lobe ...... 126

Control of Complex Behavior ...... 126

Reflexes ...... 127

Potential for Non-specific Effects ...... 128

FIGURES...... 130

Figure 3-1: Lesions are located in multiple brain regions ...... 130

Figure 3-2: A schematic of the obstacles used in this study ...... 131

Figure 3-3: Depiction of different turning behaviors ...... 132

Figure 3-4: Change in success of climbs is related to lesion location...... 133

Figure 3-5: After the lesion more individuals hit their head on the block in the light

and dark...... 134

Figure 3-6: Climb strategy changes in some individuals after the lesion...... 135

Figure 3-7: Behavioral variability changes after the lesion in individuals presented

with a shelf...... 136

Figure 3-8: Delayed turning increases after the brain lesion...... 137

Figure 3-9: Turning angles become more variable after lesions to certain brain

regions...... 138

Figure 3-10: Abnormalities in the FB and LAL relative to lesion location...... 139

Figure 3-11: Abnormalities in turning behavior increase in individuals with lesions to

the FB, EB, and LAL...... 140

Figure 3-12: Some individuals fail to reach the top of a wall...... 141

Figure 3-13: Changes in local reflexes associated with some lesion locations...... 142

Figure 3-14: Summary of Findings...... 143

Chapter 4: Discussion

DISCUSSION ...... 147

CHANGES IN BEHAVIORAL VARIABILITY ...... 149

THE CENTRAL COMPLEX AND THE CEREBELLUM: ...... 150

CAVEATS ...... 152

List of Tables

Table 2-1: Dwell time after specific antennal contact patterns ...... 88

List of Figures

Chapter 1: Introduction

Figure 1-1: The Insect Nervous System ...... 31

Figure 1-2: Head Sensors ...... 32

Figure 1-3: Representation of the main regions within the protocerebrum of the

cockroach brain ...... 33

Figure 1-4: Schematic of the central complex ...... 34

Chapter 2: Characterization of obstacle negotiation behaviors in the

cockroach, Blaberus discoidalis

Figure 2-1: Block Climbing Behavior ...... 77

Figure 2-2: Distance from the Obstacle and Climbing Success ...... 78

Supplimental Figure 2-1: Normalized Distance from obstacle and climbing success 79

Figure 2-3: Strategies Used for Block Climbing ...... 80

Figure 2-4: Ethograms of Block Climbing ...... 81

Figure 2-5: Shelf Climbing and Tunneling is related to antennal contact ...... 82

Figure 2-6: The influence of shelf height and ambient lighting on climbing behavior

...... 84

Figure 2-7: Ocelli Determine if it is light or dark ...... 85

Figure 2-8: Antennae Sample the same space regardless of lighting ...... 86

Suppliment 2-2 Antennae sample the same space regardless of lighting ...... 87

vii

Chapter 3: Discrete Lesions in the cockroach brain and their effect on the negotiation of barriers

Figure 3-1: Lesions are located in multiple brain regions ...... 130

Figure 3-2: A schematic of the obstacles used in this study ...... 131

Figure 3-3: Depiction of different turning behaviors ...... 132

Figure 3-4: Change in success of climbs is related to lesion location...... 133

Figure 3-5: After the lesion more individuals hit their head on the block in the light

and dark...... 134

Figure 3-6: Climb strategy changes in some individuals after the lesion...... 135

Figure 3-7: Behavioral variability changes after the lesion in individuals presented

with a shelf...... 136

Figure 3-8: Delayed turning increases after the brain lesion...... 137

Figure 3-9: Turning angles become more variable after lesions to certain brain

regions...... 138

Figure 3-10: Abnormalities in the FB and LAL relative to lesion location...... 139

Figure 3-11: Abnormalities in turning behavior increase in individuals with lesions to

the FB, EB, and LAL...... 140

Figure 3-12: Some individuals fail to reach the top of a wall...... 141

Figure 3-13: Changes in local reflexes associated with some lesion locations...... 142

Figure 3-14: Summary of Findings...... 143

viii

Negotiation of Barriers by Intact and Brain-Lesioned Cockroaches

Abstract

CYNTHIA MARIE HARLEY

I examined the role of specific regions of the cockroach central complex (CC) in controlling obstacle negotiation behaviors. The CC is a group of midline neuropils within the brains of all insects. They include the protocerebral bridge (PB), fan-shaped body (FB), ellipsoid body (EB), and lateral accessory lobes (LAL). It is thought to be involved in sensor-motor integration, which, contributes to the animals ability to negotiate obstacles. My goal was to examine the effects of lesions in specific regions of the CC on various obstacle avoidance behaviors. I first examined several behaviors in detail (Chapter 2) using the cockroach as a model organism. I found that a cockroach’s climbing success is associated with its distance from a block. In the absence of antennae, this distance shortens and the climbing strategy changes. The antennae are also used to determine whether the cockroach will climb over or tunnel under a shelf; a behavior which can be biased by ambient lighting. Because these behaviors are highly variable, it was necessary to establish their inherent variability before examining the effects of lesions. I, therefore, developed ethograms that divided complex behaviors into sequences of simpler elements and provided a quantitative measure of the probability of moving from one element to another at several choice points.

The role of specific brain regions could then be examined by creating electrolytic lesions within specific CC neuropils (Chapter 3). Lesions to the PB and EB only resulted in deficits in turning and shelf climbing, whereas, lesions to the FB and LAL resulted in deficits in those, as well

1 as, in climbing and local reflexes. Lesions to the lateral FB resulted in deficits in turning behavior, but those to the medial FB did not. Furthermore, lateral FB lesions only affected turns away from the lesion site. Individuals with lesions to the EB and LAL exhibited deficits in both directions, while lesions outside the CC and in the antennal commissural tracts rarely produced deficits.

These experiments support the hypothesis that the circuits within the CC and its related input and output regions play important roles in controlling transitions in locomotion.

Acknowledgements

I would like to acknowledge those who believed in me even when I did not believe in myself. I would especially like to thank Dr. Roy Ritzmann for unwavering faith, guidance, patience, advice, and for teaching me to be able to stand up and, without reservations, say that I do some amazing and interesting work. Additionally I would like to thank Dr. Mark Willis for being my behavior guru and Dr. Deb Wood for emotional support and valuable advice. The technical assistance of Al Pollack is greatly acknowledged. I also would like to acknowledge Nick Kathman for scientific discussions which improved this manuscript. And this could not have been done without the loving support received from my family and my husband.

The work presented here was made possible by support from the following grants (to RER): NSF IGERT Training Grant DGE 9972747, NSF Grant IOS-0845417, NSF Grant IOB- 0516587, Eglin AFB F08630-03-1-0003, AFOSR Grant FA9550-07-1-0149 Chapter 1: Introduction

Despite their small brains, insects, are able to carry out a multitude of complex motor tasks. These behaviors are not limited to uncoordinated trial-and-error acts, but rather, they are often targeted motions, which, in turn, suggest that it is necessary to coordinate sensory and motor information to guide the leg movements underlying complex maneuvering. Sensory information is necessary to determine and guide appropriate obstacle negotiation behaviors. For instance, to place their foot on top of a block, insects need both information about their distance from the obstacle and its height. This requires that somewhere within the nervous system sensory and motor information are integrated. It has been suggested, based largely on anatomy and gross lesions, that this integration occurs within a region of the brain known as the central complex (Huber, 1962; Strausfeld, 1999;

Wessnitzer and Webb, 2006; Williams, 1975). Lesions to this region of the brain have resulted in abnormalities in turning and stridulatory behavior (Huber, 1960; Ridgel et al.,

2007). Furthermore, recordings from this brain region have revealed cells which respond to antennal movement, visual information, and tactile stimulation and that these responses can be modulated by other sensory stimuli or the locomotor state of the animal (Heinze and

Homberg, 2007; Homberg, 1994; Homberg, 2004; Ritzmann et al., 2008). It does seem that this is a brain region which has been implicated in both sensory and motor tasks. . However, to properly assess the possibility that it is involved in the integration of this information, we need behavioral paradigms which require this integration. In this thesis I examined such behavioral paradigms and developed detailed ethograms of climbing. I then examined the effects of lesions within various brain regions including those that make up the central complex.

Transitional Behaviors

Whether you are a cockroach or a human your life is bound to be full of obstacles.

Getting around these obstacles depends on the ability to perceive them and to change actions to respond accordingly. In the case of the cockroach, these path altering actions have been termed ‘transitional behaviors’ (Watson et al., 2002). Examples of transitional behaviors include turning, climbing, and tunneling. These physical tasks often require changes in the walking pattern which may involve changes in body posture, gait, leg position, or ground reaction forces (Blaesing and Cruse, 2004; Jindrich and Full, 1999; Watson et al., 2002).

These complex behaviors are highly variable and transient. For instance, a previous study of climbing showed that there were multiple different climbing strategies which could be used to climb the same obstacle (Watson et al., 2002). These climbing strategies differed in the movement of the climbing limb as well as the anticipatory changes in body posture which occurred in relation to climbing. The intrinsic variability of these behaviors makes them difficult to study. Thus, such behaviors have not received as much attention as more stereotypical and repeatable movements such as straight-line walking or flying. Nevertheless, they may be the very behavioral elements for which a brain supervising lower control circuits is required.

To gain a better understanding of the complexity involved in transitional behaviors, it is important to first understand walking. Coordination of leg movements occurs through circuits in the thoracic ganglia. There is a thoracic ganglion for each pair of legs: prothoracic, mesothoracic, and metathoracic (figure 1-1). Pattern generators controlling an individual pair of legs communicate with those in the other thoracic ganglia to create these highly

5 coordinated walking patterns (Cruse, 1980). These pattern generators not only control when the leg moves, but can also control movement of the individual joints.

Information from sensors along the legs is used to modulate their movement through the use of local reflexes (for review see (Büschges and Gruhn, 2008)). These local reflexes change sign with changes in behavioral state. This has been studied previously in the femoral chordotonal organ (FCO), which measures the position of the femur-tibia joint

(Zill, 1985). In animals which are not walking, stretching the FCO results in excitation of extensor motoneurons and an inhibition of the flexor motoneurons in a manner known as a resistance reflex (Bässler, 1976). However, in an active animal, this same stimulus results in an ‘active reaction’ whereby stretching the FCO first leads to an excitation of the flexor and an inhibition of the extensor tibiae motoneurons (Bässler, 1988). Once the leg reaches a certain position, the flexor becomes inhibited and the extensor is excited. A similar activity- dependent reflex reversal is found in vertebrates (Pearson and Collins, 1993). It has been suggested that local reflexes such as these may be important to maintaining proper timing of joint movements during walking (Akay et al., 2001; Büschges and Gruhn, 2008).

Furthermore, modulation of reflexes such as these may be integral to establishing stable motor patterns for behaviors such as turning which requires different timing of leg movements than walking (Mu and Ritzmann, 2008b).

The distributed nature of walking control means that even when descending influences on the walking circuit are removed, cockroaches are still able to walk. However, the effect that this removal of descending influences has on walking gait is related to the location of the amount of descending information removed. Insects with lesions to the neck connectives exhibit abnormal posture and will take very few steps even after several days of

6 recovery (Graham, 1979a; Ridgel and Ritzmann, 2005). In contrast, when the suboesophageal ganglion (see fig 1-2) is left intact (connected to the thoracic ganglion but with connections to the brain ablated) the insects walk continuously for long periods of time

(Graham, 1979a; Ridgel and Ritzmann, 2005). However, these individuals fail to make adjustments in anticipation of turning and climbing and instead react to these objects in an uncoordinated manner (Ridgel et al., 2005; Ritzmann et al., 2005; Roeder, 1937). This is a sharp contrast to intact insects which when faced with a block make subtle changes in posture which allow them to retarget the motion of their front limb such that it reaches the top of the obstacle, often on the first attempt (Harley et al., 2009; Watson et al., 2002). Since the ability to make subtle changes in body position and limb movement is not present in insects with lesions to the neck connectives or circumoesophageal connectives, descending control must be necessary for control of complex leg movements (Mu and Ritzmann,

2008a).

The ability to physically perform these various behaviors is only one part of the puzzle. If these behaviors are targeted by information gained through evaluation of the barrier rather than performed through trial and error or brute force, then sensory information must be used to guide them. It would seem that this is the case. During climbing the insect must change its posture to raise its center of mass as well as make subtle changes in leg orientation to surmount the obstacle. The cockroach is able to move its foot from the substrate to the top of an obstacle without touching the front of the obstacle at all with its foot (Watson et al., 2002). This suggests that the limb is likely targeted toward the top of the block after some evaluation process. In addition to coordinating the physical movement, the proper behavior to respond to the obstacle must be implemented. Thus, the sensory information cannot solely make the cockroach aware of an obstacle in its path, it must also

7 be able to convey what the appropriate response to such an obstacle is -- should it climb, tunnel, turn, or find an alternate route? Furthermore, information about the insect’s internal state may influence the subsequent behavior. An insect which is attempting to escape will respond differently to an environment than one that is foraging (Harley et al., 2009). Thus the cockroach’s ability to evoke the appropriate response depends on its ability to assess and interpret information about the obstacle.

Ethograms

One of the barriers to understanding transitional behaviors is their inherent variability. Indeed, it may be characteristic of any brain-guided behavior that it is more variable than reflexive actions. A quantitative description of such complex behaviors is necessary if one is to experimentally manipulate the system to gain a better understanding of underlying mechanisms. Without such a framework, alterations could be attributed to the inherent variability of the system rather than to the experimental manipulation.

Both quantitative behavioral descriptions as well as an understanding of variability within the system can be represented by an ethogram. Ethograms quantitatively describe complex behaviors by separating one complex behavioral event into a series of simpler elements. These elements form a sequence detailing the probability of transitioning from one element to the next, thereby describing the original behavior while characterizing its variability (Lehner, 1996). This type of analysis has been used previously to describe many different behaviors; such as: courtship (Darrow and Harris, 2004; Pandav et al., 2007), agonistic encounters (Adamo and Hoy, 1995; Karavanich and Atema, 1998; Nilsen et al.,

2004), exploratory behavior (Clark et al., 2005), and predatory behavior (MacNulty et al.,

2007). Combining ethograms with other techniques has allowed researchers to determine brain structures and pathways involved in specific behaviors (Diamond et al., 2008; Ewert,

1987), to establish whether a single sensory modality or a combination of multiple modalities is used for a particular behavior (Goyret et al., 2007; Raguso and Willis, 2002), to characterize deficits in genetically modified organisms (Crawley, 1999; Pick and Strauss,

2005), and to create computer models for testing neurobiological hypotheses (Blaesing,

2006). Here, we use this technique to examine the role of sensors in obstacle detection behaviors as well as to assess changes in behavior that are related to lesions to various regions within the brain.

Head Sensors

Coordination of directed behaviors such as climbing requires sensory information.

This sensory information is needed not only for the cockroach to perceive the obstacle, but also for it to be able to respond properly by lifting its leg high enough to climb or turning the appropriate direction far enough to negotiate a wall. Thus, the sensors located on its head are an important piece of understanding the puzzle of how any animal negotiates barriers in its natural environment. What information is important to the cockroach when it is responding to an obstacle? How is that information obtained? Where is it processed? To answer these questions, we must develop not only an understanding of the head sensors themselves, but we must also develop a paradigm which will enable us to test for changes in the insect’s ability to obtain or respond to sensory information.

Cockroaches will turn away from antennal contact with a wall. In fact, their turning behavior can be induced by contacting a single antenna (Okada and Toh, 2006). It is likely

that turning could also be induced by visual stimuli as is the case in stick insects (Dürr and

Ebeling, 2005). Indeed cockroaches have a number of sensors which would be capable of

directing such behaviors. These are the compound eyes, simple eyes, antennae, and maxillary palps. Furthermore, it has been suggested that limb contact with a wall is enough to induce turning behavior (Ridgel et al., 2007). To develop an understanding of how these sensors can be used to evoke these behaviors we must first understand what they are capable of

sensing.

Head Sensor Anatomy

There are a remarkable number of sensory structures located on the heads of insects.

A description of all types is beyond the scope of this thesis. Rather, I will focus upon the

structures that will become important in later chapters; i.e., mechanosensors of the antennae

and maxillary palps and those associated with vision.

Antennae

The antennal flagellum is composed of approximately 150 segments (Toh, 1977).

Groups of two to three marginal sensilla, singly innervated campaniform sensilla, are located

on the proximal edges of every other flagellar segment (Campbell, 1972; Schafer and

Sanchez, 1973; Toh, 1977). Campaniform sensilla are oval-shaped domes within the cuticle of the cockroach. They are stimulated when their short axis is compressed making them directionally sensitive to cuticular stress (Bell and Adiyodi, 1982; Zill et al., 2004). On the leg, they are located near the joints enabling them to process information about posture and

locomotor control (Pringle, 1938b; Pringle, 1940; Zill, 1990). It is likely that here they are used to sense strain within the antenna and thus its movement and the location of object contact along the flagellum. In addition to these campaniform sensilla, there are also a number of hair-shaped sensilla along the flagellum capable of sensing mechanosensory and chemosensory information (Bell and Adiyodi, 1982).

The base of the antenna is made up of two segments, the scape and the pedicel that are thought to communicate the position and posture of the antennae (figure 1-2). The scape is a ball-like joint that articulates with the head capsule. The pedicel is the next most distal segment and is connected to the scape by a hinge-like joint, that, in cockroaches, can only be moved in the vertical plane (Okada and Toh, 2000). These two segments have five different types of mechanoreceptors. The antennal chordotonal organ is sensitive to both vibration and tension (Ikeda et al., 2004; Toh, 1985). In addition to the flagellar sensors, there are hairplates located along the scape and pedicel (Okada and Toh, 2000). These are located such that they are stimulated by movement of these joints. On the head-scape joint, they are located at ventral, dorsal, and lateral positions along the scape. Additional hairplates are also located along the hinge-like scape-pedicel as well as along the dorsal and ventral areas of the pedicel. This suggests that these hair plates are located for proprioceptive purposes. If the hairplates are shaved the cockroach contacts objects with its antennae less often and shows deficits in orientation behavior (Okada and Toh, 2000; Okada and Toh, 2001; Okada and

Toh, 2004). These deficits in object localization are likely because when an object is contacted with the antenna, its location is related to where that antenna is in space at the time of contact. In addition to these hair plates there are campaniform sensilla which form a ring around the distal edge of the pedicel and others at various locations along the scape and pedicel. In addition to these cuticular receptors there are chordotonal organs. One of these is

11 termed the Johnston’s organ and stretches around the pedicel-flagellum (PF) junction responding to flagellar movements in all directions (Staudacher et al., 2005). Another group of chordotonal organs at the PF joint respond to movements of the flagella in specific directions. Others at the scape-pedicel joint that respond to downward flexion of the pedicel

(Petryszak, 1975; Staudacher et al., 2005; Toh, 1981).

Maxillary Palps

While there is an extensive literature on the involvement of maxillary palps in chemosensory tasks (for examples see (Blaney and Duckett, 1975; de Bruyne et al., 1999)), they appear to be capable of additional mechanosensory function (Klein, 1981). Unlike the antennae, these structures are made up of all the same segments and joints as are found in the leg, with the absence of the complex tarsal (foot) segments. This complex joint structure confers remarkable precision onto their movements. The passive movement and, to a lesser extent, active movement of the palp is sensed by an array of campaniform sensilla located along each of the 5 joints (Pringle, 1938a). Certainly, these sensors should enable them to determine their location in space; however, it is also possible that these structures could be used to sense obstacles in the environment. Their mechanosensory capabilities have not previously been documented. Here, we were unable to determine whether or not the maxillary palps can sense obstacles. However, we can not negate the possibility that they can be used to compensate for loss of a sensor or decrease in sensory information.

While, in this thesis, I have concentrated on the function of antennae and palps in mechanosensory applications, it is important to mention that they are also covered in chemoreceptors and hygroreceptors (Bell and Adiyodi, 1982). However, while these sensors probably have profound effects on brain-guided orientation behavior, discussion of them is beyond the scope of this project.

Vision

The other sensors that will become important to behaviors presented in this thesis are visual structures. As with many insects, cockroaches have two pairs of eyes: one simple

(ocelli) and one compound.

Ocelli:

Ocelli have lower spatial resolution than compound eyes, but have a higher transmission speed and greater sensitivity to changes in light level (Goodman, 1981;

Laughlin, 1981; Mizunami, 1994). Ocelli are most known for their function as horizon detectors in locusts and dragonflies (Reichert et al., 1985; Schuppe and Hengstenberg, 1993;

Stange, 1981; Taylor, 1981). Most insects which use their ocelli for horizon detection have two front facing ocelli and a third which is located on the dorsal surface of the head

(Warrant et al., 2006; Wilson, 1975). It has been suggested that this configuration is necessary to detect the horizon (Homberg, personal communication). However, an exception has been noted in dragonflies which have a median ocellus with an unusually thick curved lens (Stange et al., 2002). Physiological evidence suggests that the structure of the eye

in combination with the neural architecture of second- and third-order neurons allows

processing of horizon information (Berry et al., 2006). In contrast, while the ocelli of most

diptera look upward they do not have this specialized lens and are, therefore, not known to be used for horizon detection. What may their ocelli be used for? It has been shown that houseflies are able to use their ocelli to orient to edges (Wehrhahn and Reichardt, 1973).

However, how or whether this ability is used in their natural environment is unknown.

Cockroaches differ from flies, locusts, and dragonflies in that they only have two ocelli, both of which face forward. Anatomical studies suggest that the receptors of what once was the dorsal ocellus is divided between the two forward facing ocelli (Mizunami,

1995a). It is possible that the lack of a dorsal ocellus inhibits the ability to detect horizon.

Thus, cockroach ocelli may have a different function than those previously mentioned; they are anatomically distinct from those of other insects in that they possess a larger lens and more photoreceptors (Mizunami, 1996; Weber and Renner, 1976). This configuration likely does not allow for horizon detection which begs the question: what is the function of ocelli in the cockroach. While it is possible that cockroach ocelli may enable them to orient to objects, it is thought that compound eyes are responsible for most of an insect’s object perception leaving the function of these simple eyes in cockroaches an enigma.

Compound Eyes:

In addition to ocelli, cockroaches possess superposition compound eyes which are

adapted to low-light conditions. This sensitivity comes at the cost of visual acuity (Land,

1981; Wolken and Gupta, 1961). Even so, nocturnal insects are able to visually navigate

around stationary objects (Varju and Reichardt, 1967; Wehner, 1981), pursue moving

objects (Wehner, 1981), and estimate object distance (Collett, 1978; Wallace, 1958; Wallace,

1959) at very low light levels. There is even evidence that visual information can be used to

guide antennae toward objects within the visual field (Honegger and Campan, 1981; Ye et

al., 2003), suggesting that the visual and mechanosensory systems could act alone or in

concert. Indeed, recent evidence has suggested that ocellar information may modulate

processing of certain primary sensory inputs (Willis et al., 2009). Furthermore, projections

from ocelli go to other sensory areas of the brain as well as to the thoracic ganglia

(Mizunami, 1995a; Mizunami, 1995b; Mizunami, 1995c).

Processing of Sensory information

For obstacle negotiation behaviors to occur, the sensors need to assess the obstacle and

guide changes in motor behavior. This complex sensory motor integration suggests that

higher-order processing is needed to create different movements such as the asymmetric middle leg motions associated with turning and postural adjustments associated with raising the body to climb. What is the involvement of different brain regions in these behaviors?

Are the same brain regions involved in all complex behaviors or are there different regions involved in each behavior? To begin to answer these questions, one must first understand the different regions of the insect brain and their basic function, as we currently understand it.

The insect brain

While control of the basic walking program lies in pattern generators in the thoracic ganglia (for review see (Cruse, 1990)), more complex movements rely on descending signals from the brain. The walking pattern is set by a combination of joint-specific pattern generators and inter- and intra- joint reflexes (Büschges and Gruhn, 2008). However these leg movements can be modulated to create more complex behaviors. For example, one of the movement patterns associated with turning involves the middle leg femur-tibia joint.

During straight walking, these joints extend during stance to pushing the insect’s body forward. However, during turning, the legs on the inside of the turn often extend during late swing and flex during stance creating a pulling motion in the direction of the turn (Mu and

Ritzmann, 2005). Changes in walking behavior have been associated with changes in activity of local circuits in the thoracic ganglia that ultimately control leg movement (Akay et al.,

2007; Mu and Ritzmann, 2008a).

Research in both vertebrates and arthropods has shown that descending information from the brain is necessary to modulate, coordinate, and adapt motor patterns to produce effective walking (Grillner and Wallen, 2002; Kien and Altman, 1992). While insects which have had this connection severed (either by decapitation or lesion of the neck connectives) are still able to take a few steps, they often do not walk well (Graham, 1979b; Roeder, 1937) and exhibit postural abnormalities (Graham, 1979a; Ridgel et al., 2005; Roeder, 1937).

Furthermore, they fail to adjust their behavior in the presence of an obstacle or to show goal-directed behaviors (Ridgel et al., 2005; Ritzmann et al., 2005).

Excitatory signals from the suboesophageal ganglion (SOG) are active during leg movement, flight, and rest (Altman and Kien, 1987; Gorelkin et al., 2001; Roeder, 1967).

Lesions anterior to this brain region, that disconnect the brain from the SOG, result in individuals that walk continuously, in a regular gait, for long periods of time (Ridgel et al.,

2005; Roeder et al., 1960). Thus, it is likely that while signals from the SOG are necessary to maintain normal walking, they also need to be modulated by higher areas of the brain.

Furthermore, while these individuals walk normally, they are unable to change their walking motor program and thus are unable to avoid obstacles using the kinds of behaviors seen in intact insects (Ridgel et al., 2005; Roeder et al., 1960).

Beyond its involvement in adjusting motor programs, the brain is also necessary for processing multi-modal sensory information. Indeed much of its volume is involved in primary processing of visual and antennal stimuli. This information then must be integrated with motor information to evoke the proper obstacle negotiation behavior. Evidence has shown that the brain is crucial for initiating behavior patterns, indicating that interactions between sensory processing and command formation occur somewhere within the brain

(Huber, 1960). However, where in the brain this occurs is still a matter of debate.

Furthermore, the brain regions involved may be specific to particular behaviors or a unique set of conditions and the control of these behaviors could be distributed throughout multiple different brain regions.

Several brain regions within the protocerebrum (see figure 1-3) have been implicated in integration of sensory and motor commands. The protocerebrum of the insect can be divided into several sub-regions. The most notable are the mushroom bodies (MB), antenno- cerebral tracts (ACTs), lateral accessory lobe (LAL), and the central complex (CC). Each of these regions has a different known function.

Mushroom Bodies

The MBs can be divided into four main sub-regions: the alpha lobes, beta lobes,

pedunculus, and calyces (Mizunami et al., 1998a). As the MBs are not a main focus of this

paper, this is only a brief overview of their function. The reader is directed to the following reviews for more detailed accounts, (Mizunami et al., 1998b; Mizunami et al., 1998c; Okada et al., 1999). The MBs receive a variety of sensory inputs. Many of these inputs are carried from the antennal lobes via the antennal cerebral tracts and are thus olfactory and mechanosensory (Li and Strausfeld, 1999; Malun et al., 1993; Okada et al., 1999; Zeiner and

Tichy, 2000). Other inputs originate in the lateral protocerebrum, circumoesophageal connectives, and visual regions of the brain (Nishino et al., 2005). Output connections from the MBs project to other areas of the protocerebrum (Li and Strausfeld, 1999). It is thought that this output from the MBs (transmitted via protocerebral circuits) is sent to premotor centers which supply the thoracic motor centers (Strausfeld et al., 1998). The involvement of the MBs in locomotor planning is supported by evidence that their activity occurs 100-

2000ms prior to the onset of locomotion (Okada et al., 2003). Furthermore, when this region of the brain is stimulated, locomotion stops (Huber, 1960). This has lead to the idea that the MBs are involved in integration of sensory and motor signals and thus in hierarchical processing for behavioral control (Okada et al., 1999). In Drosophila that have been conditioned to choose a flight direction, genetic mutations that affect the MBs have resulted in an inability to resolve conflicting situations (Tang and Guo, 2001), suggesting that sensory motor integration within the MBs may even operate at the level of behavioral decisions.

The most prominent function of these structures is their strong role in place memory and experience-dependent behavior modification (Erber et al., 1980; Kwon et al.,

2004; Lent et al., 2007; Mizunami et al., 1998c). While this appears to be the case in a number of species, it should be noted that differences in the role of these structures in various behaviors may exist between species or even between developmental stages of the same species. The MBs show a notable increase in volume, in some insects, around the time that adult foraging behavior occurs (Fahrbach et al., 1997; Withers et al., 1995). This increase in volume has an experiential component as these structures are reduced in individuals that have undergone sensory deprivation (Fahrbach et al., 1998). Chemical or genetic lesions to these structures result in a reduction in olfactory learning behavior in Drosophila (de Belle and

Heisenberg, 1994; de Belle and Heisenberg, 1996). In fact, simply cooling either the calyx or

α-lobe of the MBs after the initial stimulus disrupts learning behavior in bees (Erber et al.,

1980). Physical lesions to the pedunculus and β-lobe resulted in a reduction in visual place memory, which was only significant with bilateral lesions. This suggests that a single MB may be enough for memory formation (Mizunami et al., 1998d). However, this ability does have its limits; when the brain is divided into two hemispheres, learning in the MB on one side does not translate to learning on the other side (Lent et al., 2007). The involvement of MBs in memory may be limited to specific aspects of place memory. One study in Drosophila suggests that MBs are important to memory retrieval but not acquisition or storage (Dubnau et al., 2001).

Antenno-Cerebral Tract

The antenno-cerebral tract (ACT) is composed of several tracts of axons which carry sensory information from the antennal lobes to other areas of the protocerebrum. These tracts are most well characterized for carrying olfactory information (Homberg et al., 1988;

Strausfeld, 2009). However, it is possible that they also carry mechanosensory information.

Neurons from the antenna project to the antennal lobe. Once the antennal nerve reaches the antennal lobe it divides into one tract that projects to the antenna mechanosensory and motor center (AMMC) and another that terminates in the antennal lobe and contains mainly olfactory neurons (Han et al., 2005). Processes which then project from the AMMC to the antennal lobe have been identified in locusts and crickets (Schafer and Rehder, 1989;

Vitzthum et al., 1996). Thus, it is possible that the ACTs carry not only olfactory information from the antennal lobe, but also mechanosensory information. This antennal information travels via the ACTs to areas of the brain such as the MBs and the CC

(Homberg et al., 1988).

Central Complex

The central complex (CC) is thought to have a role in sensori-motor integration

(Williams, 1975). The CC is composed of several interconnected neuropils: the fan-shaped body (FB) (also called the upper division of the central body), ellipsoid body (EB) (also referred to as the lower division of the central body), protocerebral bridge (PB), and paired nodules. The FB and EB receive afferent fibers and connect to the lateral accessory lobe

(LAL) which has descending inputs to the ventral body, a premotor area of the brain (Okada et al., 2003; Strausfeld, 1999). Connections from motor reporter units that arise from the

thoracic ganglia and provide information regarding ongoing motor activity are also present

in the CC (Homberg, 1987; Homberg, 1994).

The CC is found in virtually all insects and some crustacea. Highly similar neuropils

have also been found in chilopods (centipedes), chelicerates (spiders), diplopods (millipedes),

annelids and even onychophors (velvet worms) (Homberg, 2008; Loesel et al., 2002;

Strausfeld, 2009). However, this highly conserved structure is not without variations between organisms. Comparative studies of these variations give us insight into the putative function of this structure.

This structure is thought to be involved in asymmetric body movements because it is

greatly reduced in insects such as water striders, which row using their right and left legs at

the same phase as well as in lepidopterans which use their legs for grasping but not for

walking (Flogel, 1878; Strausfeld, 1998b; Strausfeld, 1999). In water striders, this reduction of the CC even includes the lack of an ellipsoid body and a vestigial protocerebral bridge

(Strausfeld, 1999). In a related insect, Notonecta glauca (the common back swimmer), a

reduced CC is also present. Here, in addition to a smaller CC, the protocerebral bridge is

split into two halves with most terminals restricted to a single half (Strausfeld, 1999). These

insects do not walk well and instead use symmetrical movements of the legs to swim (Flogel,

1878; Strausfeld, 1999). Thus, it seems common for insects that use symmetrical leg

movements or that do not use their legs for walking to have a reduction in the CC

(Strausfeld, 1999). In contrast, this structure is enlarged in insects that manipulate objects

with their front legs, cell-building social insects, and insects such as cockroaches which make

asymmetrical turning movements (Loesel et al., 2002; Strausfeld, 1999).

Studies on the development of the CC in beetles have shown that its size increases with each successive instar, or developmental stage, coinciding with limb development

(Breidbach, 1989; Breidbach et al., 1992). Flies have an additional modification of the CC.

Instead of forming an arch as it does in most other insects, their ellipsoid body forms a complete torus (Strausfeld, 1999). Functionally, there is not a known reason for this; however, the more an insect uses vision during flying, the more closely its ellipsoid body resembles a torus suggesting possible functional differences in this structure between insects which use visual information for navigation and those that do not (Strausfeld, 1998b;

Strausfeld, 1999). It does seem that anatomical differences in this structure support the notion that it is involved in sensory-motor integration, specifically the creation of asymmetric movements.

Thus, while this structure is present in a wide range of species, differences in its anatomy are present and associated with differences in locomotor strategy. The cross-species evidence that this structure is involved in asymmetric movements and sensory motor integration is further supported by connections within the structure that are common between a number of species. As stated above, the CC is composed of several distinct neuropils—the protocerebral bridge (PB), fan-shaped body (FB), ellipsoid body (EB) and paired nodules (NO) (figure 1-3). Neurons in these structures have complex arborizations.

In fact, the fan-shaped body got its name because its extreme dendritic arborization results in it having a fan-like appearance (Strausfeld, 1999). The PB, FB, and EB all have a highly patterned columnar organization (Homberg, 1987; Williams, 1975).These columns allow for the modules to interconnect with others within the same region as well as different regions creating a cross-talk which is similar to that typical of higher order processing in the vertebrate brain (for examples see (Bower and Parsons, 2003; Diamond et al., 2008; Tanifuji

22 et al., 2006; Tsunoda et al., 2001)). The PB is composed of cells arranged into 16 columns. In flies the structure is divided such that 8 columns are on the left (L1-L8) and 8 on the right

(R1-R8) (Muller et al., 1997; Strausfeld, 1999; Williams, 1975). Both the EB and FB are composed of 16 columns (figure 1-4) (Williams, 1975). These columns consist of compact dendritic trees which make them readily visible in histological sections. . The columns of the

FB and PB are interconnected such that the most lateral column on the left side of the PB connects to the most lateral column on the left side of the FB. There are also connections that cross the midline, such that the most lateral column on the left of the FB also receives inputs from the most lateral column on the right of the PB (Heinze and Homberg, 2008;

Strausfeld, 1999; Williams, 1975). The EB also receives connections from the PB. These are arranged such that columns R1-R8 of the PB connect with even numbered columns of the

EB and L1-L8 connect with odd numbered columns (Heinze and Homberg, 2008; Muller et al., 1997; Strausfeld, 1999; Williams, 1975). The EB is underneath and slightly in front of the

FB (figure 1-3) and receives connections from it in many, but potentially not all insects

(Heinze and Homberg, 2008; Strausfeld, 1998a). The pairing of these columns is highly ordered in the manner in which it connects to other CC structures ((Strausfeld, 1999), see figure 1-4). In addition to their ordered connections with one another, these neuropils receive ordered arborizations from afferents (Strausfeld, 1999). The PB, FB and EB send projections to the LAL such that each lobe receives connections from both the left and right sides of each of the neuropils (Heinze and Homberg, 2008). Each nodulus can be divided into a mantel that receives connections from the ipsilateral side of the FB and a core that receives connections from the contralateral side (Strausfeld, 1999). The elaborate and ordered crossover between these modules could allow for integration of information from both sides of the brain. The connections between this structure and the ventral body (a

premotor area of the brain) suggest that this integration of information is used to coordinate

motor behavior.

This anatomical and evolutionary evidence for the role of the CC in locomotion can

be further supported by data obtained from lesion studies.

Genetic Lesions

Genetic lesions of the CC have been instrumental in our understanding of its role in

behavior. In flies, genetic mutations which affected the interconnection of different regions

within the CC resulted in a decrease in locomotor activity (Martin et al., 1999). While these

individuals were still able to maintain a tripod gait typical of insects; all of the 15 different

mutant lines of Drosophila with structural deficits in the CC exhibited impairments in walking behavior (Ilius et al., 1994; Strauss and Heisenberg, 1993). These impairments in walking

behavior can be organized into seven different categories: (1) the temporal pattern of swing

phases, (2) the spatial placement of legs, (3) the proper initiation and maintenance of step

length, (4) the across-body symmetry of step length, (5) the range of stepping frequencies,

(6) the swing phase duration, and (7) the swing speed of legs (Strauss, 2002; Strauss and

Heisenberg, 1993).. Furthermore, there is a high degree of correlation between the degree of

the motor deficit and the severity of the structural deficit (Strauss and Heisenberg, 1993).

Despite these deficits in the ability to modify or initiate walking behavior, these insects were still able to walk using a tripod gait pattern, likely because the control of rhythmic stepping resides in the thoracic ganglia (Strauss, 2002). Interestingly, mosaic flies with a unilaterally

mutant thorax are still able to walk in straight lines as long as their CC (specifically the fan-

shaped body and ellipsoid body) is intact. If it is not, they walk in circles, suggesting that this

24 combination of structures is able to compensate for deficits in performance of a particular side of the body (Strauss and Heisenberg, 1993; Strauss and Trinath, 1996).

Mechanical Lesions

In crickets, large lesions of the CC led to inhibition of walking and stridulation behavior, while electrical stimulation of this area resulted in forward walking as well as turning behavior (Huber, 1960). Further supporting the involvement of the CC in turning, large-scale frontal lesions which encompassed the entire CC resulted in insects that failed to turn when presented with a wall (Ridgel et al., 2007). In these studies, no lesions outside of the CC resulted in turning abnormalities, suggesting that this structure is necessary for

“normal” turning behavior. Sagittal lesions of the CC resulted problems in turn direction.

For example, some of these insects turned into the wall instead of away from it. These results were also heterogeneous in that some generated profound deficits, while other individuals performed the task normally (Ridgel et al., 2007). The heterogeneity shown with these large-scale lesions suggests that lesions within the same brain region (such as the CC) will have different effects. Thus, the role of CC regions in supervising locomotion, may be highly specific. Indeed, they may even be task specific, with different obstacles requiring different sensory information and motor behavior and, therefore, involving different regions of the CC or even specific areas within a single neuropil.

In addition to obstacle negotiation behavior, the CC is also involved in stridulatory behavior in the cricket and other insects. Male crickets sing by raising their wings and scraping them together rhythmically (Huber, 1962). If the CC is cut in half, male crickets are no longer able to chirp (Huber, 1962). Stimulation of the CC resulted in irregular

movements of the forewings with associated changes in chirping sounds (Huber, 1962).

While the mushroom bodies were found to be involved in initiation, continuation, and song

choice, the CC was found to be involved in modulating the temporal pattern of the chirps

(Huber, 1962). Thus it is possible that the CC is involved in translating information from the

MBs into motor commands that control stridulation motor circuits located in the thoracic

ganglia (Huber, 1962). Injecting muscarinic agonists into the CC of grasshoppers evokes

stridualtion, while blocking the production and release of nitric oxide in the CC prevents it

(Weinrich et al., 2008),suggesting that release of neurotransmitters in this brain region

controls onset and ending of stridulatory behavior. As stridulatory behavior involves a motor

behavior, the scraping of the wings, it is possible that a similar transmitter release would

affect walking behavior. Indeed, some recent evidence suggests that this may be the role of octopamine in the central brain (Haspel et al., 2003; Rosenberg et al., 2007).

Recordings in the Central Complex

Recordings within the central complex (CC) have revealed its multisensory nature in bees, locusts and cockroaches (Homberg, 1985; Milde, 1988; Ritzmann et al., 2008). The CC has a number of cells with complex dendritic architecture. One subtype of these cells, the giant fan-shaped neuron (GFS), has been shown to respond to visual and tactile stimulation

(Homberg, 1994; Williams, 1975). Multi-unit recordings in resting cockroaches also suggest that the CC is sensitive to multimodal stimuli (Ritzmann et al., 2008). A large number of units were recorded in the EB and FB that respond to lateral and medial deflection of the antennae at their base. Most units that were responsive to antennal stimulation were also light sensitive, responding either phasically or tonically, to persistent changes in ambient

lighting (Ritzmann et al., 2008). The responses of virtually all of these units were velocity

dependent and about one-third of them were biased toward a specific direction or to

movement of one of the two antennae. An examination of responses from several units at

once revealed detail of the stimulus, suggesting that direction of antennal movement may be

described by activity within a population of cells (Ritzmann et al., 2008). Recordings in the

CC of locusts identified cells in the PB and EB that were responsive to orientations of

polarized light (Heinze and Homberg, 2007; Vitzthum et al., 2002). Different columns within

the PB were sensitive to different orientations of polarized light resulting in an organization

that creates a topographic map (Heinze and Homberg, 2007).

The activity of some of cells in the CC has been shown to be associated with the

behavioral state. In crickets, cells which arborize near the CC and respond to multimodal

stimuli have activity patterns which are dependent on the locomotor state of the insect

(Staudacher and Schildberger, 1998). These multimodal descending neurons in crickets

respond to chirping or visual stimuli; however this response was only present when the insect was walking (Staudacher and Schildberger, 1998). Similar neurons which change activity with onset of flight have been found in both the CC and lateral accessory lobes of locusts (Homberg, 1994). This suggests that care must be used in interpreting studies of restrained insects. Thus, despite our knowledge of the anatomy of CC and response patterns of many component neurons, its potential involvement in locomotion, the function of the individual regions of the CC are less well understood.

Protocerebral Bridge

The protocerebral bridge (PB) is thought to be involved in the integration of visual

information from both hemispheres of the brain and has fibers which run from right to left

within the structure (Bausenwein et al., 1994; Strausfeld, 1999). However, it is not solely a

visual structure. In fact, it is fully developed in blind cave beetles (Ghaffar et al., 1984). In

addition, it is known to receive projections from the antenno-cerebral tracts suggesting it

also has a role in mechanosensory and/or olfactory information processing. Genetic lesions

of the PB resulted in Drosophila which failed to walk in a straight line and had bouts where their walking was uncoordinated (Strauss et al., 1992). This lack of coordination results from their lack of ability to match swing speed with stepping frequency (Poeck et al., 2008).

However, as the walking rhythm is a result of circuits in the thoracic ganglia, the movement

of the legs relative to one another remains unchanged with these individuals continuing to

use a tripod or tetrapod gait (Poeck et al., 2008; Strauss, 2002). When these individuals try to

turn, they fail to place their legs appropriately and end up tripping over their own feet

(Strauss et al., 1992). Because turning requires that insects adjust step length, this region has

been suggested to have an involvement in turning behavior (Strauss and Heisenberg, 1993).

While this structure is important to the ability to change stride length, it is discontinuous in

insects that use their front legs for grasping suggesting that there is some task specificity to it

(Strausfeld, 1999). Perhaps sensory information from this structure is used to modulate coordination of walking.

The central body: The fan-shaped body and ellipsoid body

Much of the literature about the fan-shaped body (FB) and ellipsoid body (EB) refers to these structures together as the central body. These two structures do have a number of similarities. For instance they both receive extensive connections from the PB in a manner which is highly patterned (see figure 1-4) (Strausfeld, 1999). In addition, these structures are supplied by afferents from sensory neuropils thought to be involved in higher-order sensory processing (Strausfeld, 1999). Most cells recorded from in this region respond to multimodal stimuli (Homberg, 1994; Ritzmann et al., 2008). This suggests, at the very least, a role in the integration of sensory information. Genetic lesions to the FB and EB result in flies that display altered orientation behavior and quickly lose their bearings when flying toward a target (Strauss, 2002). While the EB has been shown to have cells sensitive to polarized light orientation, the FB does not have any such cells (Vitzthum et al., 2002). It has been suggested that the EB has a role in sky compass orientation while the FB does not (Heinze and Homberg, 2009). Furthermore, this region has an involvement in spatial orientation memory (Neuser et al., 2008). The FB has neurons which are involved visual pattern memory, specifically visual contour and elevation (Li et al., 2009). While it would seem that these two structures are involved in orientation to visual stimuli, their behavioral function in less visual insects is currently unknown.

The lateral accessory lobe

The lateral accessory lobes (LALs) are composed of a series of neurons that connect to the ventral nerve cord (Homberg, 1994; Strausfeld, 1999). The LALs are prominently connected to the CC in a number of different insects (flies (Strausfeld 1976; Hanesch 1989),

bees (Homberg, 1987), beetles (Wegerhoff et al., 1996), moths (Homberg et al., 1988) and

locusts (Homberg, 1994; Williams, 1975). These structures are located on each side of the

CC and carry ascending and descending information to and from the CC. While the LAL is

not often considered part of the CC, we place it in this category because of its

interconnection with the CC. This region has been shown to have involvement in motor

behavior, specifically the control of flight in tethered locusts (Homberg, 1994).

In moths, cells in the LAL also respond to pheromone (Kanzaki et al., 1991; Kanzaki

and Shibuya, 1986). The most well known of these is the “flip-flop” cell in Bombyx mori.

These cells, when activated by pheromone show two distinct patterns thought to be linked

to the zig-zagging motion typical of pheromone tracking behavior (Kanzaki et al., 1992;

Olberg, 1983a). It is thought that this zig-zagging behavior may be controlled through reciprocal inhibition of connections between the two lateral accessory lobes (Kanzaki et al.,

1992; Mishima and Kanzaki, 1999; Olberg, 1983a). During this behavior, turns are made in the direction of the antenna receiving the higher concentration of pheromone (Olberg,

1983b). Thus, this region receives known sensory stimuli and has been found to be involved in direction and motor control of locomotion.

My goal:

The goal of this thesis is to describe complex transitional behaviors in cockroaches in a quantitative manner that allows for experimental manipulation (chapter 2). I then follow up on the mechanical and genetic lesions that are described above to examine the behavioral effects of discrete electrolytic lesions in specific regions of the brain, with particular focus

30 upon the CC (chapter 3). I tested each lesioned cockroach on a range of behaviors, each of which had been previously described with varying degrees of detail and most of which had been examined after more gross lesions and brain deficits. The results begin to demonstrate how various individual neuropils within the CC influence these behaviors and in one case, the FB, shows effects that are restricted to a specific region of that structure.

These experiments support the hypothesis that the circuits within the CC and its related input and output regions play important roles in controlling transitions in locomotion. More refined behavioral experiments should now be performed to refine this understanding, ultimately combining with recent advances in recording and genetic manipulation within these brain regions. With a combination of such techniques, our understanding of the behavioral role of the central complex should be forthcoming.

Motivation:

Through increasing our understanding of sensory-motor integration in the insect brain we can better understand how information is processed in general to guide complex behaviors. While it is apparent that insects use brain circuitry to perform these complex behaviors, we only have a very elementary understanding of their brains, especially when compared with our knowledge of mammalian brains. Here, we examine where in the brain, control of specific behaviors resides. Similar lesion studies have contributed to our understanding of the function of mammalian brains and thus provides a good starting point for the understanding of insect brains (for example see (Drew et al., 2008)). When combined

31 with recording studies, we can gain understanding of how circuits confer control on the system.

This process can contribute to future comparative studies between the brains of vertebrates and invertebrates. Is it the case that a multi-legged creature requires the type of control embodied by a cerebellum-like organ to sequence behavioral subroutines or optimize sensation, or can a totally different system exist? Comparisons between locomotion in cats and insects have been made on the level of lower circuits and leg mechanics have revealed many similarities (Pearson, 1993; Ritzmann et al., 2004), suggesting that at least analogous brain structures should exist. Regardless of the level of similarity between vertebrates and invertebrates, this type of study will enable us to understand more about how brains process complex information about the environment.

Since my goal is to examine the role of brain circuits in controlling complex behaviors, it became necessary to first quantify the variability that is inherent to those behaviors. Absent the kind of quantitative descriptions found in Chapter 2, I would not have been able to positively conclude that changes in behavior were due to lesions. Armed with the ethograms of climbing and tunneling behaviors and the quantitative data on pre-lesion animals described in Chapter 3, I could now examine the effects of lesions with greater confidence.

Figure 1-1: Insect Nervous System. The main parts of the insect nervous system. Between each set of legs there is a thoracic ganglia. The influence of descending inputs on locomotor behavior can be removed by severing the neck connectives (between the suboesophageal ganglion and the prothoracic ganglion) or the connection between the brain and the suboesophageal ganglion. Figure from (Mu, 2008)

Figure 1-2: Head Sensors. Figure from (Bell and Ayodi, 1982). The scape (Sc), pedicel (Pd), and Flagellum (Fl) are all visible as are the Ocelli (Oc), and compound eye (Co).

A D

B E

C F

Figure 1-2: Representation of the main regions within the protocerebrum of the cockroach brain. A-F are cartoon representations of sections of the insect brain. Each section is 60 microns deeper than the previous section. A is the shallowest section F is the deepest. While other sections were deeper and shallower than these, only sections containing regions of interest were drawn. G is a color representation of the sections superimposed on each other, with an accompanying legend. This is an adaptation of a figure in (Mizunami,1998). The proto-, deuto-, and trito cerebra are fused into a single mass (Strausfeld, 1999). The deuto- and trito- cerebra receive sensory afferents from gustatory and chemo receptors, provide motor axons to muscles, and are anatomically distinct from the protocerebrum (Strausfeld, 1999). The protocerebrum is the most frontal of these neuromeres. Here, the antennal lobes, which are located within the deutocerebrum are also depicted.

Figure 1-3: Schematic of the central complex adapted from (Strausfeld, 1999) (right) and (Wessnitzer and Webb, 2006) (left). The schematic shows the highly patterned connections between the PB and the EB (left) and FB (right). It also shows a schematic of the patterned output to the LAL.

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Chapter 2:

Characterization of obstacle negotiation behaviors in the cockroach, Blaberus discoidalis

This material published previously in Journal of Experimental Biology: Harley, C.M, English, B.A., Ritzmann R.E. (2009)

Summary:

Within natural environments, animals must be able to respond to a wide range of obstacles in their path. Such responses require sensory information to facilitate appropriate and effective motor behaviors. The objective of this study was to characterize sensors involved in the complex control of obstacle negotiation behaviors in the cockroach, Blaberus discoidalis. Previous studies suggest that antennae are involved in obstacle detection and negotiation behaviors (Camhi and Johnson, 1999; Comer et al., 1989; Watson et al., 2002). During climbing attempts, cockroaches swing their front leg, which then either successfully reaches the top of the block or misses. The success of these climbing attempts was dependent on their distance from the obstacle. Cockroaches with shortened antennae were closer to the obstacle prior to climbing than controls, suggesting that distance was related to antennal length. Removing the antennal flagellum resulted in delays in obstacle detection and changes in climbing strategy, from targeted limb movements to less directed attempts. A more complex scenario, a shelf which the cockroach could either climb over or tunnel under, allowed us to further examine the role of sensory involvement in path selection. Ultimately, antennae contacting the top of the shelf led to climbing while contact on the underside led to tunneling However, in the light, cockroaches were biased toward tunneling. A bias which was absent in the dark. Selective covering of visual structures suggested that this context was determined by the ocelli.

Keywords: ethogram, tactile orientation, antennae, ocelli, vision, climb, tunnel, Blaberus discoidalis, cockroach, dark, light, ethogram, bilateral antennectomy, short antennae, antennal lesion

Introduction:

To properly negotiate obstacles in their path, animals typically need to alter their behavior. For instance, an animal that is walking in a straight line and encounters an obstacle may respond to it by initiating any of a number of behaviors, such as climbing, tunneling, jumping, escaping, or turning. However, the animal may first have to evaluate the object to determine the appropriate response. These objects may be predators (Comer et al., 2003), prey items (Catania and Kaas, 1997; Dehnhardt et al., 2001), tall obstacles

(Watson et al., 2002), or walls (Camhi and Johnson, 1999; Cowan et al., 2006; Wiesel and Hubel, 1963). To respond appropriately, the animal must detect and extract specific properties of the objects it encounters. While this is often thought of as a visual process, many insects and vertebrates use mechanosensory information for navigation (Patla et al.,

1999). Vertebrate examples include rats (Brecht, 2006; Mehta et al., 2007; Mitchinson et al., 2007; Towal and Hartmann, 2008), shrews (Anjum et al., 2006; Catania, 2000), harbor seals (Dehnhardt et al., 1998) and star-nosed moles (Catania, 1999; Catania and

Kaas, 1997), which use their whiskers (or appendages around the nose in the case of the star-nosed mole) to sense objects in their environment. Even humans, despite our visual nature, are able to use mechanosensory information to respond to objects within our environment (Roland, 1992). While some invertebrates can use mechanosensors on their front limbs to sense obstacles (Blaesing and Cruse, 2004b; Pick and Strauss, 2005), they can also gain mechanosensory information from the antennae (Camhi and Johnson, 1999;

Dürr and Krause, 2001; Horseman et al., 1997; Pelletier and McLoed, 1994; Zeil et al.,

1985).

Insect antennae are covered with a variety of mechanosensors. For instance, the basal segments of the antennae possess hair plates, campaniform sensilla, and chordotonal organs. The hair plates encode the position of the antenna in both the horizontal and vertical planes (Okada and Toh, 2001). This position information is an important element in obstacle localization. Without it, tactile orientation to obstacles is impaired (Okada and

Toh, 2000). Campaniform sensilla are located both at the base of the antenna and along the flagellar segments (Campbell, 1972; Schafer and Sanchez, 1973). It is thought that these sensors detect where contact was made along the flagellum and are involved in wall-following behavior (Camhi and Johnson, 1999). The combined information detected by each of these sensors should enable the cockroach to identify an object’s location relative to itself. However, it is likely that the antennae are able to sense much more about obstacles than just their position.

Previous behavioral observations suggest that antennae are likely candidates to guide obstacle negotiation behaviors in cockroaches (Camhi and Johnson, 1999; Okada and

Toh, 2000; Okada and Toh, 2006; Staudacher, et al, 2005, Ye et al., 2003). Information about an obstacle is obtained prior to limb contact, encoded within the nervous system, and then is used to guide the new behavior. For example, cockroaches are able to climb over an obstacle using a single front limb movement without that limb ever touching the front of the obstacle (Watson et al., 2002). If the height of the barrier is altered, the cockroach changes its rearing height appropriately (Watson et al., 2002). Another insect which uses this strategy, the potato beetle, fails to rear when their antennae are removed

47 suggesting that the antennae provide critical cues for directing this behavior (Pelletier and

McLoed, 1994).

While antennae may provide critical information, they are not the only sensors available to the cockroach. As with most insects, cockroaches also have two pairs of eyes: one simple (ocelli) and one compound. Evidence suggests that visual information can be used to guide antennae toward objects within the visual field (Honegger and Campan,

1981; Ye et al., 2003), suggesting that the visual and mechanosensory systems could act alone or in concert. Indeed, recent evidence has suggested that ocellar information may modulate processing of certain primary sensory inputs (Willis et al., 2008).

No matter which sensory system is employed, an appropriate response to some objects will require a more thorough evaluation of the object. Unlike simple reflex events, the process of this initial evaluation can be somewhat variable. Thus, the essential properties of the obstacle that are extracted and how they guide these behaviors are currently unknown. A quantitative description of such complex behaviors is necessary if one is to experimentally manipulate the system to gain a better understanding of underlying mechanisms. Without such a framework, alterations could be attributed to the inherent variability of the system rather than to the experimental manipulation.

48 elements. These elements form a sequence detailing the probability of transitioning from one element to the next, thereby describing the original behavior while characterizing its variability (Lehner, 1996).

Previous behavioral studies investigated the role of mechanosensory information in gap crossing behaviors (Blaesing and Cruse, 2004a; Hutson and Masterton, 1986). In stick insects, changes in step type and velocity occur with gaps of different sizes. These alterations are influenced by whether or not the gap was detected by the antennae or by the front legs (Blaesing and Cruse, 2004a). Similarly, in rats a gap was crossed only after the rat’s whiskers contacted the other side of the gap (Hutson and Masterton, 1986).

In this paper, we investigated the role of cockroach antennae in negotiating vertical obstacles. Previous studies suggested that cockroaches use their antennae to guide movements associated with block climbing (Watson et al., 2002). Here we examined specific behavioral elements to determine exactly which features of the obstacle are important in guiding appropriate motor responses and how those features are detected.

We found that antennae played a major role in guiding the cockroach over both blocks and shelves, but this occurred in a context determined by visual information.

Materials and Methods:

Animals:

Blaberus discoidalis cockroaches were raised following methods described in Ridgel,

Alexander, and Ritzmann, (2007). Both male (average length 47.4 mm +/- 2.02 mm, average weight 2.33 g +/- .21 g, N = 38 measured) and female (average length 52.48 mm

+/- 2.06 mm, average weight 4.41 g +/- .54 g, N= 35 measured) adult insects were used.

No significant behavioral differences were found in this study between males and females. There was no statistical evidence suggesting that the day on which an individual was tested influenced its behavior (Generalized Estimating Equation (GEE), (Hanley et al., 2003; Hardon and Hilbe, 2003)).

Experimental Arena and Obstacles:

At the beginning of each trial the cockroach was placed in a plastic release cage

(measuring 5cm wide x 5 cm high x 9cm long) which was then set in the arena

(measuring 5cm wide x 10cm high x 58 cm long). All trials were recorded using two digital video cameras (Photrontm, San Diego, California, USA), one on either side of the

arena operating at 60fps. This frame rate allowed us to obtain enough temporal resolution

to track antennal movements which for empty arenas and those containing a block were

respectively on average 5.9 cycles/sec (+/- 1.5, N= 7) and 4.9 cycles/sec (+/- 1.2, N=7) in

the light and 3.3 cycles/sec (+/- 1.6, N=7), 3.5 cycles/sec (+/- 1.7) in the dark. To avoid

possible chemical communication, the experimental arena was cleaned with ethanol at

least 30 minutes before the start of any trials. The obstacle within the arena was chosen at random prior to the start of the experiment through the use of a random number generator. This obstacle could either be an acrylic block (50 mm wide x 11.7 mm high) or a glass shelf which was made of 1 mm thick glass and measured 50 mm wide and

50 reached a height of 11.4 mm. For tests involving ”naïve” individuals, each insect performed one trial such that no insect was influenced by experience. In tests involving surgical modification (shams, antennal shortening, antennectomy, or eye coverings), each individual performed up to 4 trials. The release cage was opened at the beginning of the trial. If the cockroach failed to leave the release cage within 5 minutes or failed to complete a trial before 10 minutes had passed, it was removed. This removal was permanent for non-modified individuals. For modified individuals, if the individual became inactive after more than one trial, it was then removed from the arena. In the event that modified individuals only performed one trial, they were later returned to the arena. If they became inactive again before performing a second trial, they were removed permanently.

Lighting Conditions:

Tests were performed under two lighting conditions; referred to simply as “light” and

“dark”. Tests under light conditions were performed during the last three hours of the cockroach’s light cycle (12 hours light/12 hours dark); for the dark condition, tests were performed during the first four hours of the dark cycle. This timing was chosen because cockroaches are most active during the end of their light phase and beginning of their dark phase (Gunn, 1940; Tobler and Neuner-Jehle, 1992). Animals were entrained to a

12-hour light/dark cycle at 27º C for a minimum of 48 hours prior to testing. On the day of the experiment, they were removed from the environmental chamber and placed in the experimental room for one hour prior to the start of the experiment in order to allow them to adapt to experimental room conditions. The experimental arena within this room was

51 illuminated to 350 lux incident light (2800 lux reflected light) (Gossen Luna-Pro light meter, Nürnberg, Germany) by fluorescent lights and two infrared strobe lights that were synchronized to the cameras (Infrared Strobe II, AOS Technologies AG, Baden,

Switzerland). This lighting condition approximated an overcast day. The addition of infrared lights did not alter light levels over that of the fluorescent sources. At the start of the cockroach’s subjective night, the room was made dark (0.17 lux, the lowest non-zero light level detected by our light meter), approximating light levels during a quarter moon

(Falkenberg and Clarke, 1999). The cockroaches were given one hour to adapt to the dark before testing under these conditions began. Under these lower light conditions, the infrared strobes added (non-visible) light to the arena causing measurable light levels to reach 7-11 lux. Under either lighting condition, the cameras were capable of recording detailed images as the insects moved. Individual insects were only tested under one of the two lighting conditions.

Measurements:

The horizontal distance between the cockroach and the block was measured using the

Winanalyze motion analysis software package (Mikromak, Berlin, Germany). This distance was measured as a horizontal straight line from the front of the pronotum to the plane of the block at the beginning of each climbing attempt (swing phase). For modified individuals, this measurement was only taken after the first climbing attempt to assess the onset of climbing behavior. Here, climbing attempts were defined as pronounced vertical movements, of one or both front legs, directed toward the top of the block. These movements were easily distinguishable from walking movements. In the case of elevator-

52 type climbing movements (see below), measurements were only taken once at the beginning of the first swing, because, by definition, a single elevator movement uses at least two swing movements. The Winanalyze software package was also used to calculate dwell time, the time between contact of the obstacle with the cockroach’s antennae and the onset of climbing or tunneling.

This same software was used to measure antennal angles. The angle between the tip of the antenna, the pronotum, and the most posterior portion of the abdomen of the cockroach was measured to approximate the position of the antennal tip relative to the body axis. As contact with an obstacle would change the antennal trajectory, we only measured prior to antennal contact with an obstacle (if one was present). The antennal angle was then organized into 5 degree bins for each trial. The percent of time spent in each bin was then averaged for the treatment group. The proportion of each trial represented by a given bin was calculated. These proportions were then added together and are represented by the distance from the origin (Figure. 8). This gives an approximation of the amount of time spent in a given region of space. For this circular data, means were calculated for raw data using Oriana 3.0 software (Kovach Computing

Services, Anglesey, Wales). An ANOVA was then used to compare means from individuals of different groups.

Antennal Ablations:

For antennal ablations, the flagellum of each antenna was cut, either to 10 mm (between one half and one third of the original length) or removed entirely (leaving the scape

53 intact) under cold anesthesia. Animals with these ablations will be referred to respectively as having “short antennae” or “bilateral antennectomies”. The tip of the cut end of the antenna was coated with VetBond (3M, St. Paul, Minnesota, US) to prevent loss of hemolymph. After ablation surgeries, individuals were given at least 20 hours to recover before behavioral testing. Sham animals for this procedure were anesthetized and handled but nothing was done to their antennae. These individuals were given at least one hour to recover prior to the start of testing.

Ethograms:

Ethograms were created by separating the behavior into smaller defined elements which do not overlap temporally (Figures 2-1B, 2-4, 2-5 B, E). These were defined as follows:

Approach: The cockroach left the release cage thus approaching the obstacle. All

sequences started with this behavioral element.

Return: The cockroach turned (more than a quarter turn) away from the obstacle;

starting to return to the beginning of the arena (release cage). It then had to turn

around again to re-approach the obstacle.

Antennal Contact: Any part of the antenna(e) contacted the obstacle. Contact may

have continued beyond this point. Contact for the shelf obstacle could be further

separated into three distinct patterns: over/over (both antennae contacted the top

of the shelf, under/under (both antennae contacted the underside of the shelf),

54 over/under (one antenna contacted the top of the shelf and one contacted the underside). The over/under contact pattern also included trials where one antenna contacted the top and bottom of the shelf before the second antenna made initial contact. Both antennae always contacted the obstacle prior to climbing or tunneling. Antennae did occasionally touch the walls of the arena; however, this contact only involved the tip of the antenna and did not result in noticeable changes in behavior, and thus, was not included in the ethogram.

Body Contact: The cockroach’s leg, body, or head contacted the obstacle. This was only noted in animals that had received bilateral antennectomies. In the other individuals, it was unusual for the body to contact the block prior to the subsequent behavior, and this contact never preceded antennal contact.

Climb: This was defined as a vertical movement, of one or both front legs, directed towards the top of the obstacle (Figures 2-1 A1, A2, A3, 2-5A1). This behavior often involved postural changes. However, as this could occur during one or across multiple elements in the sequence, they were left out of the ethogram for simplicity. The end point of the vertical swing movement was characterized by the location of the foot which either may have (success) or may not have (miss) reached the top of the block. If a swing resulted in a miss, then the insect would swing again; subsequent swings could then either miss again or could be successful.

Tunnel: This behavior only occurred with the shelf and was defined by the

cockroach’s tarsus passing under the shelf (Figure. 2-5A2). To be counted as a

tunnel, the entire tarsus had to break the front plane of the shelf.

End: Sequences ended when the individual’s second foot reached the top of the

obstacle or, in the case of shelf tunneling, when the individual’s thorax passed

under the shelf.

These elements are either physical movement of the insect or the actions of the antennae.

These items were combined in the attempt to understand how antennal contact with an obstacle is involved in these behaviors. The timing of transitions between elements in the behavioral sequence was determined by examining the video records with the Photron

Fastcam Viewertm (San Diego, California, USA) software. We combined data from all

individuals to create a first-order transitional probability matrix (Lehner, 1996). This

matrix recorded the number of times one behavioral element followed another. As this

was a first-order matrix, we only considered the immediate transition from one behavior to the next. The matrix was then used to create a sequence of elements which represents the entire behavior. While additional transitions were possible, they were not exhibited in

any trials by any individuals and thus were not included in the ethogram. While many of

the elements within this ethogram could be further divided into even smaller elements,

we felt that additional detail was not essential for this study. Further work is necessary to

understand the active tactile sampling strategy of the antennae. In the results section, we

use insets of these ethograms to highlight the behavioral element discussed in each figure.

Climbing Strategies:

Climbing swings or attempts could be separated into multiple strategies, which were defined as follows:

Controlled Rear: During or before the climbing swing, the cockroach raised the

front of its body, changing the body-substrate angle (Cruse, 1976; Dürr and

Brenninkmeyer, 2001; Pelletier and McLoed, 1994; Watson et al., 2002;

Yamauchi et al., 1993). It then swung its leg toward the top of the block.

Elevator: The front leg swung and either failed to contact the block or contacted

the face of the block; it then swung higher toward the top of the block (Cruse,

1980; Pearson and Franklin, 1984; Watson et al., 2002).

Brute Force: The cockroach pushed its head and body into the obstacle until that

force resulted in its body pushing up and over the obstacle.

T1 on top:The cockroach used a high limb trajectory such that its foot contacted

the block while its body remained horizontal. It then used this front leg to pull its

body up and over the block (provided that the leg reached the block) (Watson et

al., 2002).

All 6: The cockroach simultaneously extended all six legs, elevating the whole

body during or prior to the climbing swing (described previously as “elevate” in

(Watson et al., 2002).

Jump: The cockroach extended both hind legs in a jumping movement which

propelled the body both upward and forward in order to climb the block (Watson

et al., 2002). This often involved simultaneous climbing trajectories from both

front legs.

It should be noted that some of these strategies such as “controlled rear” and “all 6” require coordination of multiple limbs to move the whole body, whereas, others such as

“T1 on top” and “elevator” only require the coordinated movement of a single limb.

Thus, these strategies present distinct motor control issues which should be further analyzed in the future. In some trials cockroaches made multiple attempts to climb prior to successfully reaching the top of the block. The climbing strategy was recorded for each attempt regardless of success. Each attempt began when the leg started to move, whether it was picked up from the substrate or resumed movement while in the air. The leg would then swing, either landing on top of the block or missing the block and continuing to extend toward the substrate. The cockroach’s leg would then stop moving forward, defining the end of the attempt.

Shelf Height

To examine the effect of changing shelf height on climbing and tunneling behavior, we set shelves with their tops at each of the following heights (mm): 8.9, 10.3, 10.8, 11, 11.7,

12.3, 12.9, 14. These heights were chosen to test four that were higher and four lower than the 11.4mm height used in the original shelf experiments. Before the experiment began, a random sequence of shelves was determined and assigned to each individual.

Unlike the other shelf and block experiments, here animals performed more than one trial with each encountering at least 3 (up to all 8) shelf heights. No individuals faced the same shelf height more than once. Prior to the start of each trial the appropriate shelf was placed in the arena. The cockroach was then placed in a release cage which was then set in the arena (measuring 5cm wide x 58 cm long). Statistics for this experiment were performed using the generalized estimating equation (GEE) protocol in SAS (Cary, North

Carolina, USA). This statistical method allowed us to account for multiple uses of the same individual (Hanley et al., 2003).

Eye coverings:

For eye coverings, dental wax was melted and mixed with carbon powder to create an opaque black wax (Roberts, 1965). This was used to cover the ocelli, compound eyes, or both. Shams were cold anesthetized and both their simple and compound eyes were covered with wax which had not been mixed with carbon and thus was still translucent.

The melted wax was poured into thin flexible sheets which were attached to the eyes using moderate heat. These individuals were anesthetized with cold and were given one day to recover prior to behavioral experiments. To prevent these individuals from removing the wax, they were placed on corks and restrained with pins placed through the

59 pronotum and wings. Special care was taken to make sure that the pins did not damage the legs. Before the beginning of their trials these subjects were freed and allowed to walk within a container for a few minutes. Upon completion of the trials, they were examined post mortem to ensure that their eyes were still covered. Due to the proximity of the eyes and antennal joints, before the start of behavioral trials all individuals were examined to insure full mobility of antennal joints.

Results

Block Obstacles:

To diagnose changes in block climbing behavior associated with experimental treatments, we first had to determine the inherent variability of the behavior in naïve individuals. To accomplish this task, we broke the entire behavioral sequence into simpler elements by creating an ethogram (Figure 2-1B). Each number on the ethogram along with its arrow represents the frequency of transitions from one state to another. The weight of the arrow is also indicative of this transition frequency. All sequences started with the cockroach approaching the block (Approach). The insect could then return to the beginning of the arena (Return) without having contacted the block with their antennae or after they contacted the block with their antennae (Antennal Contact) (Figure 2-1A1). They then proceeded into climbing behavior (Climb) which often involved changes in body- substrate angle called rearing (Figure 2-1A2) (see (Watson et al. 2002)). Rearing either occurred before or during the leg movements associated with the actual climb. The climb began with the cockroach swinging one or both of its front legs to either reach the top of

60 the block (Success) (Figure. 2-1A3) or failure to reach the top (Miss). If a swing resulted in a miss, then the insect would swing again; subsequent swings could either miss again or be successful. Here, under light conditions, our 58 individuals made 88 climbing attempts (58 successes, 30 misses). Once their tarsi had successfully reached the top of the block, the middle and hind legs would extend to push the insect’s body upward, thereby surmounting the block and ending the sequence.

Naïve cockroaches missed the top of the block 45% of the time (Figure 2-1B). Because success of climbing attempts depended on the cockroach using information about the obstacle’s height and distance to target its limb trajectory, it was possible that there was an optimal distance from the block where climbing attempts were most successful. . To examine this possibility, we measured the distance between the cockroach and the block during climbing attempts. The majority of climbing trajectories (58 out of 88 attempts) occurred at distances less than 11 mm and most of these (50) were successful.

Conversely, 22 of the 30 misses occurred at distances greater than 11 mm (Figure. 2-2A).

Even when the distance from the block is normalized to account for insects of varying body length, we notice the same separation between successes and misses (supplemental

Figure 2-1). Together, these observations suggested that distance from the block is important to climbing success and thus would be an important parameter for the cockroach to sense.

Effect of Altered Antenna length

If antennae do indeed detect the distance from the block, then shortening the antennae should influence the distance at which climbing commences. To investigate this possibility, we measured how far cockroaches with sham lesions, short antennae, and bilateral antennectomies were from the block when they made their first climbing attempt

(Figure. 2-2B). Shams were, on average, the farthest from the block when they attempted to climb (7.5 mm). Cockroaches with short antennae were significantly closer at 5.9 mm

(p<0.001, GEE). Individuals with no antennal flagellum were the closest to the block at

3.2 mm (p<0.001, GEE).

As distance from the block was related to climbing success and antennal length affected that distance, we would expect changes in rate of climbing success in individuals with modified antennae. Definite changes in the frequency of misses were present (Figures 2-

2D and 2-4), but the relationship of miss frequency compared to shams was different between the two antennal treatments. Insects with bilateral antennectomies missed significantly more than shams on their first attempt (p<0.01, Tukey means comparison).

However, animals with short antennae missed significantly less often than shams

(p<0.05). Why might success increase with shorter antennae? Successful climbing was related to the cockroach’s distance from the block, with most successful attempts occurring within 6 mm of the block (Figure 2-2A). With their average distance of 5.9mm from the block, cockroaches with shortened antennae attempted to climb in the optimal range more often than sham animals. Interestingly, for bilateral antennectomies, the

62 mean distance for successful first attempts was 2.4 mm while it was 3.8 mm for misses, suggesting that an additional factor was altered in these trials.

The increase in misses in cockroaches with bilateral antennectomies could be attributed to a total change in climbing strategy. There are multiple strategies a cockroach can employ in order to climb over a block. However, rearing was the most commonly used by intact cockroaches (57%, Figure. 2-3) (Watson et al., 2002). In this strategy, the cockroach raised the front of its body so that a typical front leg swing would place the front foot on top of the block (Ritzmann et al., 2004). This targeted limb motion and compensatory change in body posture appeared to be guided by the height and position of the obstacle. If the necessary information was acquired by the antennae, cockroaches without antennae might be compelled to switch to a completely different climbing strategy. Cockroaches with short antennae still predominantly used rearing to surmount the block (57%, Figure. 2-3). However, cockroaches with bilateral antennectomies changed to elevator leg movements (47%), with only 22% attempting controlled rearing.

In the elevator strategy, the cockroach would swing its leg toward the top of the block; the leg would miss (often touching the block face) and then was swung higher searching for the top of the block. Clearly a strategy that relies on trial and error, would result in increased misses. Bilateral antennectomies also showed an increase in brute-force climbing behavior (25%), which occurs when the cockroach pushes its body into the block such that it slides up the object’s face to the top. Both of these strategies occurred close to the block (Figure 2-2B) and relied on trial and error suggesting that these individuals used the strategy available to them with their decreased sensory information.

If spatial information provided by the antennae was critical to normal barrier responses, shortening or removing the antennae could also alter other aspects of those behaviors.

These exploratory behaviors are variable by nature. However, by comparing the ethograms associated with the different treatments, we were still able to assess differences (Figure. 2-4). For bilateral antennectomies, we had to modify the antennal contact portion of the sequence to body, head, or limb contact. Body contact (Body

Contact) was not observed prior to antennal contact (Antennal Contact) in shams or insects with short antennae.

In addition to the aforementioned changes in success and climbing strategy, the ethograms also revealed altered frequency in returns after antennal contact (Figure. 2-4, dotted circle). There was a general trend whereby animals with short antennae and bilateral antennectomies returned after antennal contact at a much lower frequency than their sham counterparts (p<0.001, p<0.01, respectively, χ2 ). This decrease in variability,

suggested that by modifying the antennae, we decreased the availability of stimuli to

which the animals reacted. Alternatively, this could suggest a change in the behavioral

state of the animal in the arena.

Shelf Obstacles:

A shelf obstacle created a more complex paradigm whereby the cockroach had two

different paths available to it. Now they could either climb over or tunnel under the

object (pictured in Figure. 2-5A). To understand what factors were involved in path

64 selection, we constructed another set of ethograms. The shelf ethogram was similar to that which was developed for block climbing in that it starts with an approach

(Approach) which can lead to the cockroach turning around (Return) before or after it contacted the shelf with its antennae (Antennal Contact). Initial contact was always with the antennae and could be classified in one of the following three ways: (1) both antennae contacting the top of the shelf (Over/Over), (2) both contacting the underside

(Under/Under), or (3) one contacting the top of the shelf while the other contacts the underside (Over/Under). Subsequent contacts involved transitions from one of these contact states to another. These could occur multiple times in one sequence before the cockroach proceeded to the final behavior; either climbing over (Climb) or tunneling under (Tunnel) the shelf which marked the end (End) of the sequence (Figure. 2-5B).

Both antennae always contacted the shelf prior to climbing or tunneling behavior.

The frequency of approach and return for shelf climbing was similar to block climbing

(Figure 2-5B). Differences occurred after antennal contact, when the insect proceeded along one of the two paths: climbing or tunneling.

Two critical instances were examined: initial antennal contact and ultimate contact just prior to climbing or tunneling. “Initial contact” refers to the state of contact when the antennae first touched the shelf. “Ultimate contact” refers to the situation just prior to climbing or tunneling actions. Whether the cockroach climbed over or tunneled under the shelf was highly correlated with the manner in which its antennae contacted the block at both of these time points. The cockroach initially contacted the underside of the shelf

65 with both antennae in 36 trials. Of these instances, 31 resulted in tunneling behavior

(Figure. 2-5c). Trials where initial contact had both antennae over the shelf were evenly split between climbing and tunneling (4 of 7). Cockroaches with one antenna initially on either side of the shelf moved both antennae to one side and proceeded accordingly

(Figure. 2-5 B, C). The ultimate antennal contact pattern perfectly predicted whether the cockroach climbed or tunneled (Figure. 2-5 D). That is, climbing always occurred immediately after both antennae were placed above the shelf (14/14), whereas, tunneling always occurred after both antennae were placed underneath the shelf (41/41). Thus, the animal appeared to resolve the initial over/under antennal pattern, but then acted according to the ultimate pattern.

Effects of Ambient Lighting:

In our light condition, we noted that nearly three quarters of trials resulted in tunneling

(Figure.2-5C and D and 2-6A, p < 0.01, χ2 test). Because cockroaches are nocturnal

animals, we suspected that the bright ambient light conditions may have affected the

relative probability of climbing or tunneling by causing the subjects to seek out shelter

(Kelly and Mote, 1990), thereby biasing them toward tunneling (Halloy et al., 2007;

Jeanson and Deneubourg, 2007). We, therefore, repeated the observations under dark

conditions. Although the data in the dark still appeared to have a slight bias toward

tunneling (Figure. 2-6A), the difference between climbing and tunneling was, in fact, no

longer significant (p > 0.5, χ2 test).

Similar to the light, in the dark initial antennal contact revealed that 35 out of 39 trials in which the cockroach contacted the underside of the shelf with both antennae resulted in tunneling and 12 out of 13 trials in which the cockroach had both antennae on top of the shelf resulted in climbing (Figure. 2-5 E, F). In the dark, the pattern seen in the behavioral outcome of the ultimate antennal contact also reflects whether the cockroach climbed or tunneled. That is, all 37 individuals that had both antennae over the shelf right before responding chose to climb, whereas all 48 individuals with both antennae under the shelf tunneled (Figure. 2-5 G). Under both lighting conditions, there were a few individuals that responded to the shelf before both antennae were on one side. In this situation, the cockroach in the light tunneled and both individuals in the dark climbed

(Figure 2-5D,G).

Biases related to lighting conditions may result in changes in the delay between antennal contact and subsequent behaviors. For this reason, we determined the amount of time the individuals dwell within the antennal contact states before moving on to climbing or tunneling (Table 2-1). Overall this dwell time was significantly (p < 0.05, Tukey means comparison) longer in the dark than it was in the light. However, other trends were noted.

The shortest dwell time in the light occurred when both antennae were under the shelf, leading to tunneling (Table 2-1), this was significantly shorter than the same situation in the dark (p < 0.05, Tukey means comparison). Interestingly, in the dark, the shortest dwell time occurred in the opposite situation when both antennae were above the shelf.

Role of Vision in Light/Dark response:

Cockroaches have two pairs of eyes which are both capable of sensing light. To assess the involvement of compound eyes versus ocelli in the light vs. dark behavioral bias, we covered the eyes with carbonized wax or, in the case of shams, non-carbonized wax

(Figure. 2-7). Shams and individuals with covered compound eyes still showed a significant bias toward tunneling in the light (p<0.05, ANOVA, Tukey means comparison). This bias was absent in individuals in which the ocelli or both compound eyes and ocelli were covered (p<0.77 and p<0.9 respectively, ANOVA, Tukey means comparison). Indeed, individuals with both ocelli covered showed no difference in the light than shams or normal animals in the dark. Climbing prevalence in the light for shams was significantly different than that of ocellar coverings and combination compound eye and ocellar coverings (p<0.01 and p<0.001 respectively, GEE).

The Effects of Changing Shelf Height:

Shelf height could affect the probability of climbing or tunneling. At low shelf heights, the behavioral outcome was biased toward climbing, whereas at high shelf heights, tunneling was more prevalent (Figure. 2-6B). At a shelf height of 8.9 mm, 17/24 insects climbed in the light and 17/22 climbed in the dark. In contrast, at a shelf height of 12.9 mm. 4/24 insects climbed in the light and 7/27 climbed in the dark. It was important to note that even at these extreme test heights, both climbing and tunneling behaviors did occur. Therefore, the shelves were never placed at a height where only one outcome was physically possible. Despite their similarity at high and low shelf heights, between 10.8 and 11.8 mm heights, the climbing and tunneling behavior revealed significant

68 differences between the two lighting conditions (p<0.001, GEE). These differences are consistent with the data reported in Figure 2-6A for a shelf height of 11.4mm.

Body Posture Under Differing Light Conditions:

The differences in shelf-directed behavior between the two lighting conditions could possibly have been explained by changes in posture. If, in the light condition, cockroaches maintained a lower posture as they walked (i.e., held their body closer to the ground), they would be predisposed to contact both antennae under mid-range shelves, leading to greater incidence of tunneling behaviors. Conversely, a higher posture in the dark would result in greater incidence of antennal conditions leading to climbing. If this were the case, the pattern of antennal contact with the shelf could be a consequence of altered posture rather than a causal step in the choice of climbing or tunneling. To assess this possibility, we measured the height of cockroach over the floor in an empty arena under both lighting conditions as the cockroach walked from the beginning of the arena to where the obstacle would be. We then calculated the average of these values. In the light, pronotum height was 9.35 mm (9 trials from 9 individuals (3 males, 6 females)) whereas in the dark the value was 9.0 mm (8 trials from 8 individuals (3 males, 5 females)). These were not significantly different (p = 0.39, two sample t-test).

Furthermore, despite size differences, we found no significant difference between the pronotum height or climbing probability of males and females.

Differences in Antennal Position Under Differing Light Conditions:

Differences in antennal movements between the two lighting conditions could also have resulted in changes in antennal contact leading to behavioral differences. For this reason we digitized the angle between the antennal tip, antennal base, and most posterior portion of the abdomen for the antenna closest to the camera. Because it was possible that objects in the visual field could change antennal trajectory (for example see (Ye et al.,

2003)), we examined antennal movements when there was an obstacle (Figure 2-8c,d) in the arena as well as when the arena was empty (Figure 2-8 a,b). We found no differences in mean antennal direction between the two lighting conditions (Figure 2-8). Nor did we find any differences in antenna direction with or without an object in the arena. As the distribution of the data is skewed, it seems as though the mean is not located near the most prominent antennal angles. This skewing is better illustrated in supplemental figure

2-2.

Discussion

When an animal encounters an obstacle, it often must modify its behavior in order to negotiate the obstacle. This often requires the animal to redirect its leg movements so that it can climb, tunnel, turn, or step over the obstacle. These tasks are dependent upon sensory information for the animal to appropriately adjust its course. For instance, cats walking in cluttered environments are able to avoid stepping on objects to a high degree of accuracy, a feat which is dependent upon visual information to plan targeted limb trajectories (Sherk and Fowler, 2001). Such targeted limb trajectories are not limited to mammals; cockroaches are able to swing their foot from the ground to the top of an

70 obstacle in preparation for a climb (Watson, et al., 2002). In naïve insects, these swings were successful at reaching the top of the block the majority of the time (Figure 2-1B).

Furthermore, the majority of attempts occurred at a distance of less that 11mm from the block where climbing swings were successful, suggesting that distance from the block was a factor in the initiation of climbing behavior (Figure 2-2A).

Here, our data suggest that while other sensors are available to the cockroach, they rely heavily upon their antennae to guide climbing and tunneling behaviors. We confirmed this notion by altering the antennae and demonstrating a series of predictable alterations in the behavior of our experimental groups. Previous studies suggested antennal involvement in navigation through wall-following (Camhi and Johnson, 1999; Cowan et al., 2006), anemotaxis (Linsenmair, 1973; Rust and Bell, 1976), and escape (Comer et al.,

1994). In other studies, antennae were shown to take part in active searching (Okada and

Toh, 2000) where movement of antennae can be guided by visual stimuli (Honegger and

Campan, 1981; Ye et al., 2003) leading to object tracking (Honegger and Campan, 1981).

These antennal-related mechanisms can lead to orientation toward obstacles (Blaesing and Cruse, 2004; Durr, 2000; Durr and Krause, 2001; Okada and Toh, 2000; Staudacher et al., 2005; Zeil et al., 1985) or postural adjustments associated with obstacle contact

(Dürr and Brenninkmeyer, 2001; Pelletier and McLoed, 1994; Watson et al., 2002). Here, we were able to show that antennae clearly played a role in directing obstacle climbing and tunneling behaviors. We were also able to identify some of the properties that the cockroach extracts from antennal information in navigating these barriers.

Distance from a block affects climbing success rate

What factors were involved in whether or not a swing was successful? For a swing to be accurately targeted toward the top of a block, the cockroach must be able to establish both the height of the obstacle and its own distance from the obstacle (Figure 2-1B).

Previously, it was shown that cockroaches adjust their body position accordingly for obstacles of different heights (Watson et al., 2002). Here, the impact of antennal length on both distance from the block as well as climbing strategy suggests the possibility that an active sensing mechanism is involved. This notion is further supported by a previous study which showed that once initial contact with an obstacle occurs, the movement of the antennae changes to continually contact the obstacle (Okada and Toh, 2006). Is this evaluation closed loop or open loop? It is possible that this evaluation could be a simple closed-loop scenario whereby the cockroach raises its body until a specific antennal angle is obtained, at which point it begins a climbing swing. Alternatively, the cockroach could calculate the height of the block from antennal information prior to climbing and move accordingly. Of course, in either case, control could reside in other sensory modalities such as vision. However, if that were the case, we would not have expected to see a change in climb strategy in individuals without antennae (Figure. 2-3). Instead, the increase in elevator and brute force strategies after antennectomy suggested that cockroaches without antennae were unable to obtain the sensory information necessary to employ their usual controlled rearing strategy. Interestingly, the elevator strategy has been shown to be a prominent climbing strategy in intact locusts and stick insects (Cruse,

1980; Pearson and Franklin, 1984), suggesting differences in obstacle-sensing behavior between cockroaches and these insects.

In contrast to obstacle height, sensing distance appears to employ a more passive antennal function. While it was possible that this sensory task could be the result of active sensory discrimination, it was more likely related to the mechanical properties of the antenna.

Decreasing antennal length resulted in a decrease in the distance at which the cockroach first attempted to climb (Figure. 2-2B). Thus, the cockroach appears to simply rely upon contact with an appropriate length antenna to establish proximity to the obstacle leading to a climb. Previous studies suggested that this is the case when insects are maintaining distance from a wall (Camhi and Johnson, 1999; Cowan et al., 2006; Durr et al., 2003;

Durr and Matheson, 2003).

Detecting alternate routes

To create a more complex situation, we presented the cockroach with a shelf which allowed it to take one of two paths; it could climb over or tunnel under the shelf (Figure

2-5). As with block climbing, several sensory modalities were available to the cockroach to establish an appropriate path over or under the shelf. However, again antennal contact appeared to play a dominant role. This was made evident by the strong relationship between the form of antennal contact immediately preceding climbing or tunneling and the pathway that was actually taken (Figure 2-5). A similar critical point is seen in stick insects as they cross gaps. Once the far side of the gap is touched by either a front leg or the antennae, leg movements are re-directed from walking patterns so that the stick insect can successfully span the gap and reach the other side (Blaesing and Cruse, 2004b).

Ambient lighting influences whether a cockroach climbs or tunnels

While antennal contact clearly affected the path that the cockroach took over or under a shelf, it was not the only factor involved (Figure 2-5). Cockroaches were biased toward tunneling in the light and in the dark this bias was absent (Figure. 2-6A), suggesting that the light created a context around this behavior. Previous work demonstrated that cockroaches tend to seek out shelter from light when placed in an arena (Halloy et al.,

2007; Jeanson and Deneubourg, 2007; Kelly and Mote, 1990; Meyer et al., 1981; Okada and Toh, 1998). Other insects have also been found to change their behavior under different lighting conditions. For instance, tropical katydids change mate-attracting strategies under certain lighting conditions. During the new moon they call to attract mates, whereas during the full moon they use tremulations more often than calling, a method which does not transmit the signal as far but reduces the predation risk present under relatively bright lighting conditions (Belwood and Morris, 1987; Lang et al., 2006).

Similarly, here, the cockroaches may be predisposed to find shelter from predators in the light while exhibiting normal foraging behavior in the dark. All of these studies point to the context-dependent nature of complex behaviors, which should be considered in neuroethological studies.

The bias toward tunneling in the light was only present for moderate shelf heights

Similar to antennal effects, the effect of ambient lighting was not absolute. A bias toward tunneling in the light was not found at all shelf heights (Figure. 2-6A). At low shelf heights climbing was the predominant behavior regardless of lighting conditions, while at higher elevations tunneling prevailed. A window existed between 10.8 and 11.8 mm,

74 within which a consistent difference in the proportion of the behaviors between the two lighting conditions was revealed.

A similar contextual bias is present in leeches, which are biased to crawl in waters under

10mm in depth and swim at greater depths (Esch et al., 2002). In the leech, each of these behaviors has a unique pattern of cellular activity. While a large population of cells is responsible for the decision to swim or crawl, manipulation of a single cell can bias the system to perform one behavior or the other (Briggman et al., 2005). It is possible that a similar population of cells exists in the cockroach brain, whose activity controls whether the cockroach climbs or tunnels and that ambient lighting and antennal inputs change their activity. Indeed, populations of antennal sensitive cells that were also sensitive to ambient light have recently been described in the cockroach central complex (Ritzmann et al., 2008). Similar light-related changes in crayfish behavior have been found to be associated with changes in neural activity (Liden and Herberholz, 2008).

Ocelli detect light levels and influence the light-based bias on shelf behavior

For these behaviors to differ in light and dark, light levels must somehow be detected.

While the compound eyes have been implicated in controlling the shade response (Okada and Toh, 1998), our data showed that the ocelli were solely responsible for light-related biases in shelf behavior (Figure. 2-7). Cockroach ocelli are large in comparison to those of other insects and have a unique anatomy (Mizunami, 1995a). Neurons related to the ocellar system have been shown to project to a number of brain regions including the central complex (Goodman, 1976; Mizunami, 1995b). While ocelli in other insects have

75 been implicated in flight control (Reichert et al., 1985; Schuppe and Hengstenberg, 1993;

Stange, 1981; Taylor, 1981), no behavioral function in cockroaches had previously been demonstrated.

A1 — Approach B

A2 — Climb

A3 — Success

Figure 2-1 Block Climbing Behavior: Approaching the block (A1), swinging the leg to climb (A2), climbing (A3). (B) Ethogram of block climbing in the light. Arrows represent a direct transition from one behavior to the next. The number on the arrow and its thickness represent the frequency of that transition. This was calculated by dividing the number of times a specific transition was made by the total number of transitions exiting a specific element. All behavioral sequences begin with the cockroach approaching the block (Approach). It can then turn around and walk away from the obstacle (Return) before or after antennal contact (Antennal Contact). The cockroaches would then enter a climbing sequence (Climb) which could either be successful, with their foot reaching the top of the obstacle (Success) or not be successful (Miss). In the event that the cockroach missed, it would then produce another climbing motion, which again could either be successful or not. The end of the behavioral sequence occurred when the cockroach climbed the block. The beginning and end of the sequence must be “Approach” and “End” respectively, for this reason these elements are represented in bold. This sequence represents the responses of 58 individuals (one trial per individual).

A 15 Success B a Miss 12 k (mm)

10 b 10 8

6 c 5 4

Percent of Attempts 2 Mean Distance from Bloc from Mean Distance 0 0 0 5 10 15 20 Sham Short Bilateral Horizontal Distance from Block (mm) C 100 c 80 N = 16 n = 40 a 60 N = 14 n = 35 b 40 N = 15 n = 57 20

0 Percent First Attempts which Miss Attempts First Percent Sham Short Bilateral

Figure 2-2 Distance from obstacle and climbing success. (A) Success is dependent on distance from the block. The horizontal straight line distance was measured between the cockroach and the obstacle at the beginning of each climbing attempt. Climbing attempts were categorized as success or miss. The data from 88 steps by 58 individuals are represented as a histogram. The distance from the obstacle for successful attempts is significantly less than that for unsuccessful attempts (p<0.0001, GEE). Statistics were performed on raw data. (B) Shortening antennae results in cockroaches getting closer to the block before climbing. Average distance from the block on first attempts in sham (white), short (gray) and bilateral (black). Each individual performed up to 4 trials. The horizontal distance was measured in the same manner as (A). The number of trials (n), and number of individuals (N) is as follows: sham (n = 36, N = 14), short (n = 42, N = 15), bilateral (n = 31, N = 14). Individuals with bilateral antennectomies were significantly closer than individuals with short antennae and shams (p<0.001, GEE). Individuals with short antennae were significantly closer to the block than shams (p<0.001, GEE). (C) Shortening antennae changes success for first attempts. Black bars indicate bilateral antennectomies, gray bars indicate shortened antennae, and white bars indicate shams. The first climbing attempt made in each trial of each individual is counted as either a success or a miss. We calculate the percent of first attempts which are misses for a given individual. No significant differences existed between light and dark. Individuals with shortened antennae miss less than shams (p<0.05 Tukey means comparison) and individuals with bilateral antennectomies (p<0.001, Tukey means comparison). Individuals with bilateral antennectomies miss more often than shams in (p<0.01, Tukey means comparison). Letters in (B) and (C) indicate the independence of statistical comparisons whereby if two conditions were not significantly different they would share the same letter. 78

Supplement

Success 12 Miss

Percent of attempts Percent 2

0 0 1020304050 Distance from Block (Percent Body Length)

Supplemental Figure 2-1: Normalized distance from obstacle and climbing success. (A) Success is dependent on distance from the block. The horizontal straight line distance was measured between the cockroach and the obstacle at the beginning of each climbing attempt. This was then divided by body length to normalize for individual size. Climbing attempts were categorized as success (foot reaches the top of the obstacle) or miss (foot does not reach the top of the obstacle). The data from 88 steps by 58 individuals are represented as a histogram. The distance from the obstacle for successful attempts is significantly less than that for unsuccessful attempts (p<0.0001, GEE). Statistics were performed on raw data.

100 Sham 90 Short 80 Bilateral 70 60 50 40 30 20

Percent of Attempts 10 0 Jump Brute Elevator All 6 T1 on Controlled Force top Rear

Figure 2-3 Strategies used for block climbing in Sham (white), Short (gray), and Bilateral antennectomies (black). There is no significant difference between the distribution of climb strategies in individuals with short antennae and shams, but there is a significant difference between both of these groups and bilateral antennectomies (p<0.001, Pearson’s χ2 test). Each animal performed up to 4 trials, which may have contained more than one climbing attempt. The number of trials (n), number of measured climbing attempts, and number of individuals (N) are as follows: sham (n=36, attempts=56, N=14), short (n=42, attempts=46, N=15), bilateral (n=31, attempts=48, N=14).Despite that by definition they contain more than one climbing swing, elevator strategies were only counted once.

ShortShort Sham Bilateral B AntennaeAntennae Antennectomy A B C

.23

Figure 2-4 Ethograms of Block Climbing in Sham (A), Shortened Antennae (B), and Bilateral Antennectomy (C) cockroaches under light conditions. Arrows represent a direct transition from one behavior to the next (behaviors described in Figure 1). The number on the arrow and its thickness represent the frequency of that transition. Dashed squares and circles represent regions which will be focused on within the chapter. Each individual performed up to 4 trials; number of trials (n) and number of individuals (N) is as follows: sham (n=36, N=14), short (n=42, N=15), bilateral (n=31, N=15).

Light Dark E B

Light Dark C100 F Initial Antennal Contact 100 Initial Antennal Contact Climb Climb Tunnel Tunnel 80 80

60 60

40 40

20 20

0 0 Over/Over Over/Under Over/Under Under/Under Over/Over Over/Under Over/Under Under/Under to to to to Over/Over Under/Under Over/Over Under/Under D G 100 Ultimate Antennal Contact 100 Ultimate Antennal Contact Climb Climb Percent of Trials 80 Tunnel 80 Tunnel Percent of Trials

60 60

40 40

20 20

0 0 Over/Over Over/Under Under/Under Over/Over Over/Under Under/Under 82

Figure 2-5 Shelf climbing and tunneling is related to antennal contact. (A) pictures of climbing (A1) and tunneling (A2) behavior. (B, E) Ethograms of shelf behavior in the light (B) and dark (E). Arrows represent a direct transition from one behavior to the next. The number on the arrow and its thickness represent the frequency of that transition. Dotted lines were used when 2 or fewer individuals performed a specific transition. Antennal position relative to the shelf was determined as being both over the shelf (over/over),both under the shelf (under/under), or if one antenna contacted the top of the shelf and the other contacted the underside the pattern was recorded as (over/under). Either the first (C,F) Initial Antennal Contact) or ultimate antennal contact with the shelf prior to climbing or tunneling ((D,G) Ultimate Antennal Contact) was recorded. For over/under initial antennal contacts, the following contact pattern was also recorded. This situation always resolved such that both antennae were on one side. Usually the two antennae contacted the shelf around the same time. In cases when one antenna contacted the shelf first on one side then the opposite before the second antenna could contact the shelf, that contact was scored the same as if one antenna contacted the top of the shelf and the other contacted the underside. Data for light (B, C, and D) represents 58 seq uences from 58 individuals (14 climbs and 42 tunnels). Data for dark (E , F, G) rep resents 86 sequences f rom 86 individuals.

climb

A 100 tunnel*

80 N. S. 60

40 Percent of Trials 20

0 Light Dark B Light 100 Dark n = 21 Light (ethogram) 80 Dark (ethogram)

n = 24 60 n = 24 n = 22 n = 27n = 27

40 n = 28 n = 27

n = 26

Percent Climbs 20 n = 27 n = 27 n = 24 n = 26 n = 26 n = 24 0 n = 23 9 1011121314 Shelf Height (mm)

Figure 2-6 The influence of shelf height and ambient lighting on climbing behavior. (A) Climbing and tunneling in insects presented with a shelf under different ambient lighting conditions. Naïve cockroaches were placed in the arena with an obstacle they could climb over or tunnel under. The light condition represents 58 trials (14 climbs and 42 tunnels). The dark condition represents 61 trials (26 climbs, 35 tunnels). The error bars in this and subsequent figures represent the +/- standard deviation (calculated using methods for binomial data). In the light, the climbing and tunneling percentages are significantly different (p<0.01, χ2 test), in the dark, this difference is not significant (p>0.5, χ2 test). (B) Prevalence of climbing with different shelf heights. The empty circles represent the light condition, while the filled circles represent the same behavior under dark conditions. Individuals faced multiple shelf heights, but were not exposed to a single height more than once. All responses from all individuals were averaged to create a percent response. When examined as a whole distribution, the light and dark distributions are significantly different at (p<0.05, GEE). The effect of shelf height on climbing proportion is significant (p<0.001, GEE). When examined individually, points at 10.8, 11, and 11.7mm differed significantly from their counterparts in the opposite lighting condition (p<0.001, GEE). Square symbols represent data from the shelf height used to create the ethogram (fig 5 A and D) and represents 58 naïve individuals for the light and 86 naïve individuals for the dark.

Compound eye coverings were not significan not were eyecoverings Compound the individual’s res Figure 2-7 were significantly higher than those ofthe Shams ocelli (D)this was not significant. Light values for dark(p<0.05,ANOVA,Tukey means comparison). Foroce compound eye covering (C) showed a higher percentage of climbs inthe light than the our ethogramnaïveindividual datafor sh andthe on B,C, Drepresent Squares translucent wax A)sham or carbonize experiments were performed in boththe

Ocelli determineifitislightordark Average Percent Climbing Trials 100

C 100 20 40 60 80 20 40 60 80 0 0 ponse. This was averaged acro Compound Eye Covering Dark n 12 N = n N =9 Sham Eye Covering = 48 =38

p<0.05 p<0.05 d wax B) ocelliC) compound eye, D) s inlight (gray,n=56) and dark am light (gray) and dark (black)re dark (black) and light (gray) conditions. Eyes were either covered wit N = 12 N = n Light Light = 48 =48 n N =11 tly different from shams(p<0.24, GEE). GEE). shams(p<0.24, from different tly

= 43 ss individuals for the average percentof climbs. All (A) (p<0.01, GEEand p<0. . The trials. The for eachindividual were then averagedobtain to the Ocelli (B)and compound eye and ocelli (D)coverings B D lli (B)orcoverings ofboth the compound eyes and Compound Eye and Ocelli Covering EyeCompound Ocelli and Dark n = 44 N = 11 n = 40 N = 10 (black,86). Bothshamsn= and (A) Ocelli covering Ocelli sponses. Filled circles inA represen compound eyeandocelli covering N. S. N.S. 001, GEE, respectively). 001, GEE, Light n = 40 n = 10 N = n =48 12 N =

85 t h . A Light Dark 90 B 90

1.0 120 60 1.0 120 60

150 30 150 30 0.5 0.5

0.0 0.0

180 0 180 0 0.00 0.00

Empty Arena

0.5 0.5 210 330 210 330

1.0 240 300 1.0 240 300 270 270 C 90 D 90 120 60 1.0 120 60 1.0

150 30 150 30 0.5 0.5 Block

0.0 0.0 180 0 0 180 0 0.00 0.0

0.5 0.5 210 330 210 330

1.0 1.0 240 300 240 300 270 270

Figure 2-8 Antennae sample the same space regardless of lighting. Naïve cockroaches were placed in the empty arena (A,C) or in the arena when a block obstacle was present (B, D). Vertical movements of the antenna ipsilateral to the camera (occurring prior to antennal contact with the block when it was present in the arena) were digitized. This is presented above in black where the angle is the angle between the antennal tip, antennal base, and the most posterior point on the abdomen (to approximate the body axis). To calculate distance from the origin, the antennal movements were divided into 5 degree bins. The proportion of each trial represented by a given bin was calculated. These proportions were then added together for the individuals with a given treatment to approximate the amount of time spent in a given region of space. The circular means are represented by the dotted white line and standard deviations by the gray triangle. No significant differences were found between the means or variances of treatment groups (ANOVA). Means and standard deviations are as follows: empty arena light mean = 179.6 degrees, s.d = 46.3 degrees, block light: mean = 172.7 degrees, s.d.= 38.9 degrees, empty arena dark: mean = 173.1 degrees, s.d = 26.8 degrees, block dark: mean = 175.3 degrees, s.d = 26.1 degrees. 86

Supplement

A Light B Dark

0.15 0.15

0.10 0.10

0.05 0.05 Empty Arena

0.00 0.00

Average proportion of trial spent at angle at spent trial of proportion Average 0 60 120 180 240 300 360

Average proportion of trial spent at angle at spent trial of proportion Average 0 60 120 180 240 300 360 Antennal Angle Antennal Angle C D 0.15 0.15

0.10 0.10 Block

0.05 0.05

0.00 0.00 Average proportion of trial of trial spent at Average proportion angle

Average proportion of trial spent at angle at spent of trial proportion Average 0 60 120 180 240 300 360 0 60 120 180 240 300 360 Antennal Angle Antennal Angle Supplemental Figure 2-2 Antennae sample the same space regardless of lighting. Naïve cockroaches were placed in the empty arena (A,C) or in the arena when a block obstacle was present (B, D). Vertical movements of the antenna ipsilateral to the camera (occurring prior to antennal contact with the block when it was present in the arena) were digitized. To calculate y the antennal movements were divided into 5 degree bins (x). The proportion of each trial represented by a given bin was calculated. These proportions were then added together. This gives an approximation of the amount of time spent in a given region of space. No significant differences were found between the means or variances of treatment groups (ANOVA). Means and standard deviations are as follows: empty arena light mean = 179.6 degrees, s.d = 46.3 degrees, block light: mean = 172.7 degrees, s.d.= 38.9 degrees, empty arena dark: mean = 173.1 degrees, s.d = 26.8 degrees, block dark: mean = 175.3 degrees, s.d = 26.1 degrees. Certain means appear to not be where one would expect because of the skewness of this data (illustrated here). The circular means are represented by the dotted line.

Both Over Over /Under Both Under All

Mean n Mean n Mean n Mean N Light 2.96 ± 1.8 6 2.56 ± 1.89 15 1.80 ±1.98a 33 2.14 ± 1.95b 54 Dark 1.79 ± 1.61 8 4.07 ± 3.8 27 3.37 ± 3.67a 25 3.48 ± 3.55b 60 Table 2-1 Dwell time after specific antennal contact patterns. This is a measure of the time between initial antennal co ntact and the initiation of climbing or tunneling behavior. Note that the mean dwell time (seconds) of all trials in the dark is significantly (p<.05, Tukey means comparison) higher than that of individuals in light. Furthermore the dwell time for individuals who contacted the shelf with both antennae under is significantly (p<0.05, Tukey means comparison) lower for individuals in the light than it is in the dark.

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Chapter 3:

Discrete Lesions within the Cockroach Brain and their Effect on Negotiation of Barriers

Summary

An animal’s ability to respond to obstacles in its path is necessary for it to successfully function within its natural environment. Responding to these obstacles involves transitioning from walking to other behaviors, many of which are more involved than a simple reflex. For these behaviors to be successful, insects must not only have enough information about the obstacle, but they must also interpret the information properly and be able to evoke a new motor pattern. A deficit in any one of these areas would result in problems with the subsequent behavior. For the sensory information to guide these complex motor behaviors it must first be integrated with motor information. This may occur within the central complex (CC), a region of the brain which receives information from both motor reporters and sensory modalities (Strausfeld, 1999a). Large scale lesions within the CC result in turning deficits such as, failure to turn and turning in the wrong direction (Ridgel et al., 2007) as well as inability to negotiate block obstacles (Alexander, 2004). However, these effects were heterogeneous with some lesions having dramatic effects while others had none; suggesting ‘hot spots’ within the CC which are critical to certain behaviors. Here, we use more discrete electrolytic lesions to examine the role of certain sub-regions of the CC in various obstacle negotiation behaviors. We found that cockroaches with lesions to the PB and EB exhibit abnormalities in turning and shelf behaviors. While, individuals with lesions to the FB and LAL, exhibit abnormalities on those behaviors as well as block climbing, wall climbing, and in local reflexes. The turning deficit is limited to individuals with lesions to the lateral FB. Those with lesions to the center of the FB are able to turn normally. Furthermore, abnormalities are only in the direction opposite to the lesion location. In contrast, individuals with lesions to the EB and LAL exhibited turning abnormalities in both directions.

Introduction

Cockroaches are agile insects that excel at responding to obstacles in their natural environment. When a cockroach climbs over a substantial obstacle at slow speeds, it first evaluates the obstacle. The distance from the obstacle, its height, and whether or not alternate routes exist are all factors in how the insect responds (Blaesing and Cruse, 2004;

Harley et al., 2009; Pick and Strauss, 2005; Watson et al., 2002b). In initiating climbing behavior, the cockroach makes postural changes by raising the front of its body such that small changes in front leg movement will place it on top of the obstacle (Watson et al.,

2002b). All of these changes require neural adjustments at least for the initiation of newly directed movement. Sensory information from the environment must be integrated with proprioceptive information about body position, then used to make motor changes resulting in targeted movements.

Changes in movement direction can be collectively referred to as “transitional behaviors”. Transitional behaviors can be thought of as changes from straight walking to another behavior as a result of changes in body posture, ground reaction forces, changes in gait, or leg position (Dürr and Ebeling, 2005; Jindrich and Full, 1999; Mu and Ritzmann,

2005; Watson et al., 2002a). In some cases, transitions occur as a consequence of simply bumping into or stepping onto an object. For example, a small block placed in front of a cockroach only generates postural changes after the cockroach steps on it (Watson et al.,

2002b). It does so as a consequence of the front leg pushing down after reaching the top of block with normal swing movement. However, taller blocks require an initial postural adjustment before the swing of the front leg will reach the top of the object. The height of the postural adjustment is correlated to the height of the block. Thus, the cockroach appears

to evaluate the object and then actively adjust its posture through descending commands

interacting with thoracic circuits controlling the legs. The altered posture then affects leg

reflexes that generate additional motor adjustments to complete the climb (Watson et al.,

2002a).

Control of the basic walking motor program is present in the thoracic ganglion (for

reviews see (Bässler and Büschges, 1998; Büschges and Gruhn, 2008; Cruse, 1990)), while

sensory information from the legs is involved in controlling the timing and magnitude of the

motor activity (Büschges, 2005; Grillner, 2003). The walking pattern is modulated by basic

reflexes whereby changes in signals from the sensors along the leg can induce changes in the

walking motor program (Akay et al., 2001; Bucher et al., 2003; Hess and Buschges, 1999).

These reflexes do not just control the movement of the whole leg, but also affect the timing

of movements of individual joints (Büschges, 1995). However, the reflexes which govern the

movement of these joints relative to each other can be modulated by descending activity

(Mu and Ritzmann, 2008a; Mu and Ritzmann, 2008b) and may even reverse as occurs when

stick insects walk backward (Akay et al., 2007).

While it would seem that much of the control needed for locomotion is present in

the thoracic gangliaof arthropodsor the spinal cord of vertebrates, research in both v of

these groups has shown that descending information from the brain is necessary to

modulate, coordinate, and adapt motor patterns to produce effective walking (Grillner and

Wallen, 2002; Kien and Altman, 1992). While insects which have had this connection

severed are still able to walk, they often do not walk well (Graham, 1979b; Roeder, 1937), and they fail to adjust their behavior in the presence of an obstacle or to show goal-directed behaviors (Ridgel and Ritzmann 2005, Ritzmann et al. 2005). When the circumoesophageal

98 connectives are severed, bilaterally isolating the brain from the suboesophageal ganglion and all thoracic ganglia, individuals walk in a highly regular gait for long periods of time, but show no ability to modify their behavior in the presence of obstacles (Graham, 1979a;

Ritzmann et al., 2005; Roeder, 1937; Roeder et al., 1960).

Which regions of the insect brain contribute to descending motor control? The central complex (CC) is a region within the insect brain thought to have a role in the modification of motor programs and sensory motor integration. Indeed, it has been suggested that this highly ordered brain region may “supervise” locomotion (Strausfeld,

1999b). The CC is composed of several interconnected neuropils: the fan-shaped body (FB), ellipsoid body (EB), protocerebral bridge (PB), and paired nodules that are located in the protocerebrum of virtually all insects (Strausfeld, 1999b). Nevertheless, little is known about how the CC might exert its influence on motor patterns. It has been suggested that this structure translates processed sensory information from the mushroom bodies or other loci into commands which excite the circuits of the thoracic ganglia (Huber, 1962). Recordings of activity in the PB revealed neurons that are responsive to specific orientations of polarized light and are arranged in a manner consistent with a topographic map of the sky

(Heinze and Homberg, 2008; Heinze and Homberg, 2009). Multi-unit recordings in the FB and EB described numerous multi-sensory units that are responsive to antennal movement as well as changes in ambient light (Ritzmann et al., 2008). However, these structures are not solely sensory, the FB and EB receive connections from motor reporters arising from the thoracic ganglia (Homberg, 1994), and stimulation in the CC can produce leg movements or calling song in crickets (Heinrich et al., 2001; Huber, 1960; Pollack et al., 2006; Weinrich et al., 2008). Furthermore, the CC and the lateral accessory lobe (LAL), which receives

99 projections from the CC, send axons to the ventral body (a premotor area of the brain)

(Homberg, 1994; Okada et al., 2003; Strausfeld, 1999b).

Manipulation of the CC does have motor consequences. Lesions to the CC structure led to inhibition of walking and acoustic behavior in crickets (Huber, 1960; Huber, 1962). In

Drosophila, CC structural mutants show defects in walking speed, leg coordination, and directional control of walking and flight (Ilius et al., 1994; Strauss, 2002; Strauss and

Heisenberg, 1993). Indeed, the central complex appears to play a key role in motor planning

(Heinze and Homberg, 2009). However, not much is known about the role of individual CC structures in obstacle negotiation. In cockroaches, large-scale lesions that disrupted the entire CC revealed heterogeneous results associated with deficits in turning behavior. Some individuals failed to turn, turned the wrong direction, or showed delayed turning, while others with damage within the CC exhibited normal turning behavior (Ridgel et al., 2007) .

The heterogeneous consequences of these large-scale lesions suggest a compartmentalization within the CC, with various regions playing different roles in controlling individual behaviors. This arrangement may even be task specific, as different obstacles will require different sensory information and motor behavior and may, thus, be susceptible to damage in different brain regions.

Here we report on the behavioral effects of discrete electrolytic lesions within the

CC and surrounding areas of the cockroach brain. Lesions were generated in various regions of the CC and then the subjects’ ability to negotiate a range of barrier types was compared to pre-test conditions. While previous studies typically examined only a single behavior, here we present brain-lesioned cockroaches with several different types of obstacles, each requiring different sensory cues and motor adjustments. This strategy allowed us to compare the

100 potential role of different regions within the CC in various obstacle negotiation behaviors.

Behavioral responses to our experimental obstacles have been examined previously in both intact and cockroaches with more profound lesions. Shelf and block climbing are complex behaviors for which a previous study used ethograms to describe sequences of sub-elements

(Harley et al., 2009). That study also demonstrated significant differences in climbing behavior under light and dark conditions. Therefore, we also tested behaviors under these conditions. Our results begin to show how the various CC neuropils, or regions within individual neuropils, influence transitional movements as the cockroach negotiates objects in its environment.

Materials and Methods

We examined the effect of discrete electrolytic brain lesions in 47 Blaberus discoidalis individuals. These lesions were located in six different regions of the brain; four of which are elements of the CC. After the lesion, the behavior of these individuals was tested on four different obstacles. After behavioral testing, local reflexes were assessed in select individuals.

Not all lesioned individuals were tested on all obstacles; some were only tested on a subset.

The number of individuals with lesions to each location is as follows for each region (except where noted): mushroom bodies = 4 individuals, antennocerebral tract = 7, protocerebral bridge = 5, fan-shaped body = 11 (of those 4 were located medially and 7 were located laterally within the structure), ellipsoid body = 4, lateral accessory lobe = 7, and 9 lesions are located outside all of these structures in a category called ‘other’ (Figure 3-1).

101

Electrolytic lesions

Cockroaches were first anesthetized using cold (-16°F). Once they stopped moving,

a ligature was placed around the neck to reduce blood flow to the brain. Because the

hemolymph of Blaberus becomes opaque when it is exposed to air, the neck ligature allowed for better visualization of the brain during the lesion procedure. Most individuals moved normally after the ligature was removed. If an individual dragged its maxillary palps on the ground or exhibited an inability to support its head with its neck muscles, it was discarded as these may be symptoms of damage to the neck connectives. Once the ligature was in place, the cockroach was placed into a plastic tube and its head was opened as described in

Ritzmann, Ridgel, and Pollack (2008). A small slice was made in the sheath surrounding the brain. We found that a damaged 16-channel silicon-based probe (NeuroNexus Technologies,

Ann Arbor, MI) provided a sharp and effective knife for this purpose. Once this was done a ground wire was placed into the top of the head capsule. Then an eligoy steel probe (Micro

Probes, Inc., Gaithersburg, MD), which had first been dipped in calligraphy ink (Windsor and Newton, Piscataway,NJ) to coat the tip, was placed into the brain. Current was injected

through the probe into the brain. The amount of current was kept constant by taking into

account the resistance of the probe. The resulting lesion size was related to current duration.

The majority of the lesions used a 30-second duration which resulted in a lesions of 100-

150µm in diameter. For scale, the fan-shaped body of Blaberus discoidalis is 400µm from edge- to-edge. The calligraphy ink would deposit on the brain tissue as the probe was inserted.

This allowed us to better visualize the lesion site histologically.

It should be noted that the brain structures and lesions are three dimensional. Here, we did not analyze the effects of the depth of the lesion. Because of the large size of our

102 lesions, it is likely that most of them went all the way through a given structure; however, additional examination is required to examine whether or not different lesion depths have different effects on behaviors.

Once the lesion was made, the probe and ground wire were removed from the cockroach. Dental wax was used to re-affix the removed head cuticle. Special care was taken to prevent the wax from contacting the antennae or eyes of the cockroach. Once the head cuticle was back in place, the ligature around the neck was removed, the cockroach was freed from the tube, and allowed to recover overnight (at least 12 hours) before further testing.

Non-specific Damage During the Lesion Process

We cannot state for sure that the only damage from this process is located within the region identified as the lesion. Inserting the probe could cause damage to any structure which it passed through. To assess the extent of damage, we inserted the probe into 5 individuals without running the lesioning current. The calligraphy ink was used to determine the probe’s location within the brain. In 4/5 individuals, ink from the probe was found in the FB, in the other it was found in the mushroom body. These individuals did show some deficits; however, those were similar to individuals with lesions to regions which the probe passed through. Those individuals where the probe contacted the FB exhibited delayed turning in 20.8% of trials and failure to turn in 8.3% as compared to 55.9% and 7.1% respectively with the electrolytic lesions. Neither of these behaviors were present in the individual where the probe contacted the mushroom body. During block climbing behaviors the number of climbing attempts under 5mm from the block was the same in these individuals as it was in those which had electrolytic lesions to the FB. However these attempts had a higher success rate than that of individuals with the electrolytic lesion (72%

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vs. 64%). Since both the increase in delayed turning and the success of climbing attempts

within 5mm of the block, were less than the value seen in lesioned individuals, we conclude that while the insertion of the probe probably did cause damage, it was less extensive than that caused by the electrical current.

While it could be suggested that the rest of the surgical procedure may also lead to deficits, we did not observe any differences between intact controls, shams and those individuals with lesions outside the of the CC neuropils, LAL and ACTs. Each of these insects would have gone through the same procedures as those that experienced lesions in the CC. Furthermore, a previous study found that just opening the insect’s head (as was done in our procedure) did not increase abnormalities in turning behavior over that which had been seen in prior to the lesions (Ridgel et al., 2007).

Histology

After behavioral testing was completed, brains were carefully removed and placed into AAF fixative (5% Acetic acid, 85% Alcohol and 10% Formaldehyde) for a period of three days. After this time the brains were dehydrated in a graded ethanol series and embedded in Paraplast Plus (McCormick Scientific, St. Louis MO). Tissue blocks were sectioned at 12µm and stained lightly with 1% toludine blue which allowed for identification of brain structures. The calligraphy ink that transferred from the probe would still be high contrast in comparison to the light stain. Lesions were then able to be identified under a conventional light microscope. This identification was made by locating a hole in the tissue of approximately 100µm2 that spanned 8 or more sections and was aided by additional

contrast from the calligraphy ink (when present).

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Diagrams of these lesion locations are illustrated in figure 3-1. For clarity we have

separated the lesions into those within the CC (1b) and those outside of it (1c). In subsequent figures, the brain lesion locations are color coded reflecting the behavioral change relative to the pre-lesion trials. In these data, the proportion of trials where a specific behavior was exhibited was calculated for each individual pre-lesion and post-lesion. The difference between post-and pre-lesion behaviors was calculated. Once determined it was used to color code the lesion sites on the diagrams. This was done by calculating the standard deviation for the pre-lesion behavior. The behavior could increase by one standard deviation (orange), two standard deviations (red), it could decrease by one or two standard deviations (green and blue, respectively), or it could stay the same (black).

Behavioral Testing

Lighting conditions:

Behavioral tests were performed under two lighting conditions: referred to

simply as ‘light’ and ‘dark’. In the light condition, the experimental arena was

illuminated to 350 lx incident light (2800 lx reflected light) (Gossen Luna-Pro light

meter, Nürnberg, Germany) by fluorescent lights and two infrared (IR) strobe lights,

which were synchronized to video cameras (Infrared Strobe II, AOS technologies

AG, Baden, Switzerland). This lighting condition approximated an overcast day. The

addition of IR lights did not alter light levels over that of the fluorescent sources.

During tests in the ‘dark’ condition the room was only illuminated to 0.17 lx ( the

lowest non-zero light level detected by our light meter), approximating light levels

105 during a quarter moon(Falkenberg and Clarke, 1999). The cockroaches were given one hour to adapt to the dark before testing under these conditions began. Under these lower light conditions, the IR strobes raised measurable light levels in the arena to 7–11 lx. However, this extra IR light was probably not visible to the cockroaches. Under either lighting condition, the cameras were capable of recording detailed images as the insects moved.

Animals were entrained to a 12 h:12 h light:dark cycle at 27°C for a minimum of 48 h prior to testing. On the day of the experiment, they were removed from the environmental chamber and placed in the experimental room for one hour prior to the start of the experiment in order to allow them to adapt to experimental room conditions. Tests under light conditions were performed during the last three hours of the cockroach’s light cycle (12 h:12 h light:dark); for the dark condition, tests were performed during the first four hours of the dark cycle. This timing was chosen because cockroaches are most active during the end of their light phase and the beginning of their dark phase (Gunn, 1940; Tobler and Neuner-Jehle, 1992).

Experimental arena and obstacles:

Cockroaches faced up to four different obstacles which were put in random order at the beginning of the experimental period (Figure 3-2). To accurately assess abnormalities, individuals performed multiple trials on each obstacle during both pre-lesion tests and post-lesion tests. At the beginning of each trial the cockroach was placed in a release cage measuring 5cm wide x 5cm high x 9cm long. The release cage was opened allowing the cockroach access to the arena. Once the trial was

106 finished, the cockroach was returned to the release cage. The obstacles were as follows.

Block :

The block was a simple obstacle which the cockroach would climb over

(Figure 3-2A). It measured 50mm wide and 11.7 mm high. While it may take the cockroach multiple climbing attempts for its foot to reach the block, this eventually occurred in all trials. Climbing attempts could be termed ‘successful’ where the front foot reaches the top of the block, or ‘miss’ where the pronounced movement of the climbing leg fails to reach to the top of the block (Harley et al., 2009). Trials on this obstacle were determined as being completed when both of the cockroach’s feet reached the top of the block. Each cockroach did this task a minimum of twice and a maximum of four times under each lighting condition.

Climbing attempts were easily identifiable pronounced movements of the front leg which were directed toward the top of the block. The grand mean was calculated for each lesion group. Pre-lesion trials were compared to post-lesion trials using raw data in a 2x2 contingency table. The same method was used to compare all other elements of the block climbing behavior which were tested. Climbing strategies have been described and classified previously in Harley et al. (2009).

Distance from the block was measured in the horizontal plane from the edge of the block to the front of the pronotum (the cuticular shield on the dorsal surface of the cockroach’s thorax). The statistical significance of the distance from the block during the first climbing attempt was determined using an ANOVA (Tukey means comparison).

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Shelf:

The shelf provided an obstacle which the cockroach could climb over or

tunnel under (Figure 3-2B). It is a glass shelf which measures 1 mm-thick and

measured 50 mm wide and 11.4mm high. Trials on this obstacle were completed

when both of the cockroach’s feet reached the top of the obstacle (climb) or when

the individual’s thorax passed under the shelf (tunneling) (Harley et al., 2009). Each

cockroach did this task a minimum of three and up to four times for each lighting

condition.

Analysis for the shelf experiment was performed using methods previously

described in Harley et al. (2009). Whether or not an increase in behavior or a

decrease in behavior was statistically significant was determined by a χ2 test on a 2x2

contingency table which compared pre-lesion and post-lesion behaviors. This

allowed us to place behaviors in the categories of increase, decrease, or new. The

significance of new behaviors was not tested as they had never occurred in pre-lesion

trials during this or previous studies (Harley et al., 2009). Whether or not an

individual exhibited a significant change in the behavior was determined by a change

of two standard deviations from the pre-lesion values for the population.

Turn:

The turning arena was a U-shaped arena similar to that used by Ridgel et al.

(2007) (Figure 3-2C). This arena exploits the fact that cockroaches will turn away from antennal contact with a wall to induce turning movements. The outer walls of the U were 18cm each, with the inner walls placed such that a channel 6.5cm wide

108 was present allowing for the cockroach to move freely. This particular arena can be put over a piece of glass with a mirror located underneath it at 45 degrees. This allowed for a view of the cockroach’s underside which enabled us to measure the turn angle. Furthermore, filming through the mirror meant that we did not have to move the cameras for the other obstacles. Cockroaches did at least three right turns and three left turns. However, in cases where it was deemed necessary, more turns were recorded to insure accuracy of behavior classification.

Analysis of turn angle was performed by marking the cockroach’s position when its antenna first touched the wall. Then the body axis (a line from the cockroach’s head to its posterior) was compared to the original position to obtain an angle. This angle was only assessed at each middle leg step following antennal contact. This choice allowed us to avoid discrepancies in the data due to walking speed. Middle legs were used because timing of their movements was consistent during turns. Front legs, on the other hand, can engage in searching behaviors or occasionally are used to attempt to climb or dig under the walls of the arena. During these behaviors, middle legs would not move, making it easy to exclude them from our analysis. In pre-lesion tests, nearly all individuals reached a 30-degree turn in 8 middle leg steps. Thus, a behavior was labeled “failure to turn” if the cockroach did not turn at least 30 degrees within 8 steps of antennal contact. While cockroaches usually turn away from antennal contact with a wall, occasionally, they would turn the opposite direction. We defined “turning in the wrong direction” as a turn of 30 degrees or greater toward the wall (Figure 3-3).

As “failure to turn” and “turning in the wrong direction” behaviors were relatively rare, we had to use a different method of color coding the lesions. This

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involved using the number of occurrences of the behavioral event for each individual

(during pre-lesion tests) instead of the proportion. Whether or not a statistically

significant change in a behavior occurred was determined by using a 2x2 contingency

table (χ2) which compared pre-lesion and post-lesion occurrences of the behaviors

using the raw data. Whether or not the mean direction was significantly different

was determined through the use of the Watson-Wheeler F-Test in the Oriana

software package (Kovach Computing Services, Angsley, Wales).

Climbing wall:

This arena is a large Styrofoam block (19cm high x 19 cm long) (Figure 3-

2D). The cockroach would climb up the wall (vertical surface) and then when it

reached the top, it would transition to walking on the horizontal surface. With this

obstacle, we focused particularly upon cockroach’s ability to transition from the

vertical to horizontal surfaces. Plexiglas walls (placed 6 cm apart) were used to limit

lateral movement and keep the cockroach in focus during filming. Cockroaches

surmounted this wall three times in each lighting condition.

Video recording of behaviors and analysis:

Each behavior was recorded using two high-speed digital video cameras

(PhotronTM, San Diego, CA) (or one in the case of the turning behaviors). One of these

cameras was located on the side of the arena, the other was located above the arena for all

obstacles except the turn. Turning trials were recorded using the side view camera, which

took video of a mirror mounted at 45 degrees under the turning surface. All videos were

taken at 60 frames per second. Video records were saved directly to a PC computer hard

110 drive for subsequent analysis. Videos were analyzed with Winanalyze motion analysis software (Mikromak, Berlin). This software enabled us to determine the cockroach’s distance from the block in addition to the changes in body angle (turning) or posture.

Reflex Testing

Twenty-one insects were tested for reflex effects related to the lesion. Nine of these had previously undergone behavioral testing. The remaining 12 insects had brain lesions and never underwent behavioral testing. Myograms (EMGs) were taken by implanting 64µm copper wires insulated with polyurethane coating to the tip (MWS Wire Industries, Westlake

Villiage, CA) in the main coxal depressor muscle of the T2 (second thoracic) leg. Signals were amplified using an AC amplifier and recorded digitally with Axoscope data acquisition software and a Digidata 1322A interface (Axon Instruments, Sunnyvale, CA). The main coxal depressor muscle is innervated by a single slow excitatory motor neuron (Ds) one fast depressor motor neuron (Df) and three inhibitors (Pearson and Iles, 1970). Once the wires were imbedded and secured with cyanoacrylate, the cockroach was restrained exposing its dorsal side. The second thoracic leg was secured such that the coxa-trochanter (CTr) and femur-tibia (FTi) joints were fixed at the 90 degree position. A small incision was then made into the distal femur. The cuticle and muscle were removed to expose the femoral chordotonal organ (FCO) apodeme. Blaberus saline was used to keep the FCO moist. The apodeme was grabbed using a pair of forceps mounted to a speaker (Pasco Scientific). Its most distal end was cut. A function generator was then used to create a ramp (500ms) and hold (5sec) stimulus through the speaker. This moved the FCO 0.4mm which was equivalent to a 45 degree movement of the joint. Each animal was tested 10 times, with at least a minute between tests. When possible, we recorded from both the left and right T2 leg.

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Analysis for the reflex experiment was performed using methods described in Mu and Ritzmann (2008). Normalized histograms of spikes were created by dividing the recordings (10 seconds pre-stimulus, 0.5 sec ramp up, 5 second hold, 0.5 sec ramp down, and 10 seconds post stimulus) into 104 bins. The results of each trial were added to create a global histogram. Statistical comparisons were done by comparing the 500ms before the ramp stimulus and the 500ms during the ramp stimulus. Any statistical significance of the change in the number of spikes was determined through the use of an ANOVA.

Results

47 cockroaches received electrolytic brain lesions and were subsequently examined for behavioral deficits. The behaviors that were examined were chosen because details of several had been described in detail previously (i.e., block and shelf climbing (Harley et al., 2009), turning (Jindrich and Full, 1998; Mu and Ritzmann, 2005) and for most, the effects of more gross lesions had been described previously (i.e., effect of circumoesophageal connective lesion on block climbing and transition from a taller wall (Ritzmann et al., 2005) and effect of gross lesions within the CC on turning (Ridgel et al., 2007).) In all cases, behavioral conditions after the lesion were compared to observations on the same individuals prior to generating the lesions.

Distance from the block and climbing success rate are decreased after lesions to certain brain regions

Prior to examining the effects of lesions of particular brain regions on climbing, we first analyzed properties of the entire lesion population (Figure 3-4a,b). We noted that initial

112 climbing movements in pre-lesion trials occurred at an average distance of 8.3mm from the block. Post-lesion trials occur closer to the block at an average distance of 5.3mm. While in pre-lesion trials, only 24% of climbing attempts occur at distances of less than 5mm of the block, post-lesion this increases to 53%. To examine if this change in distance is uniformly distributed among individuals with all brain lesions, we looked at the number of attempts occurring at distances of under 5mm from the block for lesions within each brain region studied. There was a significant increase in these attempts at 5mm for individuals with lesions to the ACT (p<0.001), MB (p<0.05), FB (p<0.005), and LAL (p<0.001 ANOVA)

(Figure 3-4c). Lesions to other areas of the brain did not result in a significant change (figure

3-4c).

These climbing attempts can either be successful, where the foot reaches the top of the block, or a miss, where the foot fails to reach the top of the block. Individuals at smaller distances from the block are more likely to be successful than those that are further away

(Figure 3-4a). A similar trend was noted previously in naïve individuals (Harley et al., 2009).

Indeed, in that study, surgically shortening the antennae increased the number of close-up attempts leading to an increased the success rate. After the brain lesion, this was not the case. Successful attempts and misses were equally likely to occur at each distance from the block (58% of lesioned animals) This success rate was almost half that for pre-lesion trials

(96%) (Figure 3-4b). Thus, unlike antenna shortening, brain lesion results in more attempts closer to the block, but without any increase in success rate.

This observation led us to assess whether or not this decrease in success was associated with lesions to particular regions of the brain. Here, we used the average number of climbing attempts per lesion as a measure of climbing success. Once an individual has a

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successful climbing attempt, the trial ends. Therefore, while successful individuals may have

a single attempt, individuals which are unsuccessful will have multiple attempts per trial. By

investigating the change in the number of successful attempts from pre-lesion to post-lesion,

we are able to account for individual differences. Here, a positive number means that there is

an increase in the number of attempts per trial, i.e., a decrease in climbing success (Figure 3-

4d). We found a significant change in individuals with lesions in the lateral accessory lobe

(LAL) (p<0.005, χ2). A small increase in attempts per trial (1 standard deviation of the pre-

test population (s.d.= 0.45)) is present in individuals with lesions to the ACT. However, that

was not statistically significant.

While under normal circumstances individuals are quite successful at climbing when

they are closer to the block, here we noticed that this was not the case. To further focus on

this issue, we looked at attempts occurring at less than 5mm from the block (a region where

96% of pre-lesion climbing attempts were successful). We also assessed if this decrease in success is related to the region of the brain lesion. While attempts per trial can be used as a measure of success when looking at the whole population, here it may not be. An individual that made no attempts at a distance of less than 5mm in the pre-lesion trials may make a single attempt at that distance in the post-lesion trials. This would lead to an increase in the

number of attempts per trial under 5mm, but not necessarily a change in success rate.

Therefore, we calculated the change in success rate of these attempts. If an individual was

just as successful at distances of less than 5mm as they were before, this number would be 0.

A negative value denotes individuals which are less successful in the post-lesion trials than

they were previously and positive number indicates individuals that are more successful post-

lesion. This decrease in successful attempts close to the block was only significantly different

from pre-lesion trials in individuals with lesions to the FB (p<0.005), and LAL (p<0.001),

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two of the groups which showed an increase in the number of attempts close to the block.

(Figure 3-1c) Because we had a large number of lesions in the FB, we could further

discriminate lesions in the medial and lateral regions of this neuropils. Here, both regions

showed a significant decrease in successful attempts (Figure 3-4e).

Head Contact with the Block

After the lesion, many of individuals completely failed to respond to antennal contact with the block resulting in them hitting their head on the obstacle. This behavior suggests that the subjects have difficulty detecting the block, which could result from

antennal or visual deficits. We, therefore, tested the cockroaches under both light and dark

conditions. The incidence of head contact increased in individuals with lesions to the FB

(p<0.001) and LAL (p<0.005), regardless of ambient light conditions. Similar delayed

responses have been described previously as an effect of CC lesions in Drosophila

(Heisenberg 1994). Interestingly, individuals with lesions in the PB or ACT only showed a

significant increase in head contact when tested in the light (p<0.05) (Figure 3-5).

Changes in block climbing strategy are present after the lesion

There are multiple strategies that a cockroach can employ when attempting to climb

a block (Harley et al., 2009; Watson et al., 2002b). The most common strategy used by intact

cockroaches is controlled rearing (70% of pre-test attempts) (Figure 3-6a). During this type

of climbing, the cockroach raises the front of its body so that when it swings its front legs,

their tarsi will land on the top of the block. In post-lesion tests, this climbing strategy was

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only used in 33% of attempts. This decrease was significant in individuals with lesions to the

ACT (p<0.05), FB (p<0.05), and LAL (p<0.001) (Figure 3-6d). After the lesion, controlled

rearing gave way to a large increase in elevator and brute force strategies (Figure 3-6a).

During the elevator strategy, the foot misses, often reaching the face of the block. It then swings progressively higher until it reaches the top of the block. This strategy was used in only 11.6% of pre-lesion tests but increased to 21.5% of post-lesion tests. However, an increase in elevator strategy was only statistically significant in individuals with lesions to the

LAL (p<0.01) (Figure 3-6c). The brute force strategy occurs when the cockroach pushes forward after contacting the block until it body slides up the face and over the block. While this strategy was hardly present in trials occurring before the lesion (0.5%), after the lesion its use increased to 27.3% of attempts. Similar increases were found in cockroaches that had had their antennae removed (Harley et al., 2009). This may suggest an inability to receive antennal information or to respond to that information. This increase was statistically significant in individuals with lesions to the ACT (p<0.01), MB (p<0.05), FB (p<0.01) and

LAL (p<0.01) (Figure 3-6b).

Changes in Behavioral Sequences

The shelf provides a complex behavioral task because the cockroach can either climb over or tunnel under it. Naïve cockroaches (Harley et al., 2009) and individuals prior to the lesion climb in a higher percentage of trials run in the dark than in the light. However, in the whole set of lesioned insects, the incidence of climbing decreased, so that there was no longer a significant difference under the two light conditions (Figure 3-7a). In a previous study, we found that climbing occurred when both antennae touched the top of the shelf,

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and tunneling occurred when both antennae touched the underside (Harley et al., 2009).

Changes in responses to antennal contact or the contact pattern itself could explain the differences in climbing and tunneling in the dark and the light (Harley et al., 2009). To assess

whether or not similar changes occurred in individuals with brain lesions, we created four

new ethograms of this behavior for pre-lesion and post-lesion trials run under the two light

conditions. Despite the striking similarity between the pre-lesion shelf ethogram and that of naïve individuals (compare Figure 3-7b,d to Figure 5b,e in Harley et al., 2009, changes were noted in the ethogram after the brain lesion (Figure 3-7c,e). These changes included the addition of new transitions (Figure 3-7c,e shown in red), increases in rare transitions (shown in orange) and decreases in other transitions (shown in blue). Individuals exhibiting these

behaviors all had lesions to the CC or the ACT (Figure 3-7 f,g,h).

Changes in Turning Behavior

Similar to the shelf, where antennal contact directs the decision to climb or tunnel, antennal contact with a wall will induce turning. Furthermore, the antennal contact also influences turning direction as cockroaches will typically turn away from antennal contact with a wall. Here, we used a U-shaped arena to encourage turning behavior. While before the lesion most cockroaches turned in response to antennal contact with a wall (98% of pre- lesion trials), this failed to occur in 40% of the post-lesion trials. This failure resulted in the cockroach hitting its head on the wall and only then did the cockroach execute a turn. This delayed turning behavior increased significantly under both lighting conditions in individuals with lesions to the MB (p<0.001), PB (p<0.001), FB (p<0.001), EB (p<0.001), and LAL

(p<0.001) (Figure 3-8a,b). Only two individuals with lesions to the FB failed to show this

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behavioral change. Both of those cockroaches had lesions to the center of the FB (black dot

symbols in Figure 3-8c,d).

Delayed turning is not the only turning abnormality noticed after the lesion. Some individuals completely failed to turn or turned in the wrong direction. While in delayed turning, individuals would hit their head on the wall and subsequently turn, individuals which fail to turn would hit their head on the wall and then never make a turn greater than 30 degrees. In pre-lesion trials, most individuals turned at least 30 degrees over the course of 8

steps (Figure 3-9a). However, in post-lesion trials some individuals failed to turn more than

30 degrees while others turned more than 30 degrees in the wrong direction (Figure 3-9).

The increase in failure to turn was present in individuals with lesions to the EB (p<0.05),

and LAL (p<0.001) (Figure 3-11a). Interestingly, while this increase was statistically

significant in the lateral FB, it was not for individuals with lesions to the center of the FB

(Figure 3-11a inset).

There were also some individuals which turned but in the wrong direction. This

behavior was significant in individuals with lesions to the FB (p<0.05) and EB (p<0.05)

(Figure 3-11b). However, here a more detailed examination is revealing. Individuals with lesions in the lateral regions of the FB often turned in the wrong direction whereas those with lesions to the middle of the fan-shaped body did not show significant changes in

turning behavior (p<0.05) (Figures 3-9D and 3-11B inset).

Were these turns in a single direction or both? To examine this question, we

examined the turning direction of individuals with lesions to the lateral fanshaped body and

LAL relative to the side of their brain the lesion was on (Figure 3-10). Most of the abnormal

turns in individuals with lesions to the FB were in the direction contralateral to the lesion

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(Figure 3-10a). In individuals with lesions to the LAL abnormalities occurred in both directions, with a greater number occurring in the direction ipsilateral to the lesion (Figure 3-

10b). Because of these potential differences in the directionality of the deficit, we scored whether or not either failure to turn or turning in the wrong direction occurred when turning in the direction ipsilateral to the lesion, contralateral or in both directions. Half of the individuals with lesions to the EB and LAL showed bidirectional deficits (Figure 3-11e). The remaining EB lesions turned normally, while the remaining LAL lesions failed to turn contralateral to the lesion site. Lesions to the PB and FB resulted in deficits in turns in the direction contralateral to the side of the lesion but not when turning ipsilateral to the lesions.

Thus, for example a lesion on the right side of the structure would result in difficulty turning left but would have no difficulty turning to the right (Figure 3-11e).

We examined changes to the mean turn angle for individuals with lesions to the FB and LAL (Figure 3-10). This change in angle was not consistent for individuals with lesions outside of the major neuropils of the central complex. However, it was significant for individuals with lesions to the LAL (p<0.001) and approached significance for individuals with lesions to the FB. When the FB was separated into its medial and lateral components, the mean angle exhibited a significant change from pre lesion trials in individuals with lesions to the lateral FB, but not the medial FB (p<0.05, P>0.2, respectively). When turn angles in individuals with lesions to the lateral FB are further divided into ipsilateral and contralateral components, we found that turns in the direction ipsilateral to the lesion show no difference from pre lesion trials. Whereas, the turn angle of those contralateral to the lesion show a significant change from pre tests, while turns in the direction ipsilateral to the lesion

(p<0.0001, p<0.2, respectively). This same trend was not present in individuals with lesions to the LAL; those individuals showed a significant change in mean turn angle for both

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ipsilateral and contralateral directions (p<0.0001, p<0.0001). These mean turn directions

were not significantly different from one another.

Problems transitioning from a wall

When climbing tall obstacles, cockroaches must transition from the vertical surface to the horizontal surface. This behavior requires a degree of body flexion for the front feet

to reach the horizontal surface (Ritzmann et al., 2004) (Figure 3-12a). Cockroaches with bilateral circumoesophageal connective lesions no longer generate this flexion (Ritzmann et

al., 2005). If one prevents body flexion by attaching a brace to dorsal surface of the thorax,

the cockroaches have difficulty making the transition between vertical and horizontal

surfaces and this problem can lead to falling. Similar problems were reported for

circumoesophageal lesioned cockroaches climbing on steep steps (Ritzmann et al., 2004).

Before the lesion only one out of our 47 individuals fell off of this obstacle (Figure 3-12b).

After the lesion, this number increased to 5. In addition, 4 individuals displayed moderate

deficits resulting from delayed flexion (Figure 3-12b). Individuals which fell off of this

obstacle while attempting to transition had lesions to the FB, EB, or LAL (Figure 3-12c).

Those with moderate deficits had lesions within the same region with the exception of one

individual which had a lesion to the ACT.

Changes in local reflexes

In addition to the behaviors described above, at least one reflex has been previously

shown to be altered by removal of descending connections from the head ganglia. The

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femoral chordotonal organ (FCO) monitors femur-tibia (FTi) joint position and movement.

Stretching the FCO reflexively activates the slow extensor motor neuron that extends the

FTi joint (SETi) as well as the slow depressor of the trochanter (Ds) which extends the more

proximal coxa-trochanter (CTr) joint (Fourtner and Pearson, 1977; Mu and Ritzmann,

2008b). When the FCO is relaxed (as occurs during extension of the femur), activity in

SETi returns to baseline and Ds activity is actually depressed below baseline levels,

suggesting an active inhibition. In cockroaches which experienced bilateral ablation of the

cervical connectives, thereby removing all descending connections, the Ds reflex is reduced

and the inhibition associated with relaxation may actually reverse (Mu and Ritzmann, 2008b).

These changes in the reflex suggest that brain circuits have the ability to modulate the

thoracic reflexes.

Our lesions provide an opportunity to examine exactly where these modulatory brain

circuits may exist. We examined the FCO reflexes of a subset of our brain lesioned

cockroaches (n=19). Almost half of the individuals that we examined (6 of 19) showed signs of altered FCO reflexes. More importantly, the altered reflexes were confined to lesions in the FB and LAL neuropils. Both of the LAL lesions and 4/6 FB lesions showed these changes. Here we found that 2/2 of individuals with lesions to the LAL and 4/6 of individuals with lesions to the FB exhibited changes in this reflex. These changes were either a reversal (as seen in neck connective lesions), or the appearance of a previously uncharacterized motor units in the response. These latter were units that were never seen previous recordings of intact cockroaches (Mu pers. Comm.). Lesions to the MB, ACT, PB and EB did not result in any change to local reflexes.

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Discussion

Negotiating obstacles in a complex environment requires the ability to modify motor patterns. Even a small change in terrain height may require changes in posture (Watson et al.,

2002b) which are associated with changes in the pattern of motor activity in the legs (Watson et al., 2002a). When dealing with small barriers, an animal may simply place its front legs on top of the object through its normal swing movements. Increases in motor activity would then occur through reflexes limited to the thoracic ganglia (Watson et al., 2002a; Watson et al., 2002b). Even more substantial objects can be negotiated without active adjustments if the insect is running at high speed (Koditschek et al., 2004). However, during most of the cockroach’s life, it deals with barriers at slow speed when a larger barrier requires an active adjustment of posture and motor activity.

Directed changes in movement typically take advantage of sensory structures located on the head and require connection with the brain regions that process that information and use it to formulate appropriate descending commands. While much of the walking motor program in insects exists in the thoracic ganglia, when the descending influence is removed, insects are no longer capable of modifying their behavior to perform these complex tasks

(Graham, 1979b; Ridgel and Ritzmann, 2005; Ritzmann et al., 2005; Roeder, 1937). It has been suggested previously that the central complex (CC) is involved in motor planning

(Huber, 1960; Okada et al., 2003; Strausfeld, 1999b). Indeed, insects which have had large lesions of the CC show difficulty turning, climbing, , adjusting step length, creating calling song, and in flight control (Ilius et al., 1994; Ridgel et al., 2007; Strauss and Heisenberg,

1993; Weinrich et al., 2008). However, the involvement of these individual structures in complex obstacle negotiation behaviors was unknown. Here we examined deficits in

122 obstacle negotiation behaviors in individuals with lesions to the CC and surrounding areas.

We compared the involvement of these structures in several different behaviors, each of which had previously been shown to have deficits associated with gross lesion of the CC

(Pick and Strauss, 2005; Ridgel et al., 2007). In addition, we were able to investigate how different areas within a single structure, the FB, are involved in these behaviors.

We described our data in the Results section according to the behaviors that were tested. In this discussion section, we will re-examine these effects but now from the standpoint of each neuropil that was damaged.

Protocerebral Bridge

The protocerebral bridge receives visual information and has recently been shown to possess a topographic map of polarized light (Heinze and Homberg, 2007; Homberg, 2004).

However, this structure is still found in blind cave beetles (Ghaffar et al., 1984), suggesting that it is not solely involved in processing of visual information When this structure was lesioned, individuals exhibited deficits that could be associated with problems obtaining sensory information or problems interpreting it. These individuals exhibited delayed turning

(Figure 3-8), as well as head contact with the block (Figure 3-5). The latter only occurred in the light condition indicating that it is possible that these individuals may have been able to compensate in the dark, likely because their eyes are better adapted to seeing under those conditions. They also exhibited all three abnormalities when presented with a shelf (Figure 3-

7). The shelf is a task which is directed by the manner in which the antennae contact it

(Harley et al., 2009). This sort of deficit is consistent with a reduced ability to respond to sensory information. It is possible that integration of information from the right and left

123 antennae is faulty leading to these abnormalities. However, lesions to the PB also result in turning deficits in the direction contralateral to the location of the lesion itself (Figure 3-11), suggesting that there is some crossing over of mechanosensory information entering this brain region. A similar involvement of the PB in the integration of information from the two hemispheres of the brain was noted with visual stimuli (Bausenwein et al., 1994).. Such connections could perhaps even explain why some lesions to this structure result in behavioral abnormalities, while others do not.

Fan-shaped Body

Individuals with lesions to the FB exhibited deficits on all obstacles they faced. In the case of both the block and the turning arena, they showed a delayed response to the obstacle resulting in collisions. A similar delay in behavioral response to obstacles was previously noted in genetic lesions of the CC in Drosophila (Ilius et al., 1994). After collision with the block, FB lesioned cockroaches proceeded to climb; however, this was done in a less successful and less targeted manner than in the pre-lesion trials. Furthermore, both brute force and elevator climbing strategies both lack targeting of the limb toward the top of the block. In the case of the elevator strategy the limb is eventually targeted toward the top of the block, but only after several re-targeting events. This may suggest that these attempts are not guided in the same manner as controlled rearing attempts. In contrast, after their collision with the turning arena some individuals failed to turn or turned the wrong direction, while others exhibited relatively normal turning behavior.

The amount of the turning deficit in the FB lesions was related to their specific location. Individuals with lesions to the center of the FB exhibited no or slight turning

124 deficits while those with lesions to the lateral FB turned in the wrong direction or even failed to turn at all. Interestingly, these deficits were mostly limited to an inability to turn in the direction contralateral to the lesion. This observation means that individuals with lesions to the right side of the FB showed an inability to turn left, and vice versa, but typically had little problem turning toward the side on which the lesion existed.

It does appear that the lateral FB has a different role in these behaviors than the medial FB. During transitions on the wall barrier (Figure 3-9), the instance of abnormality was higher in individuals with laterally located FB lesions than those with central lesions. In contrast, there was no differences in deficits associated with block and shelf climbing between individuals with medially located versus laterally located FB lesions (Figure 3-4,3-

5,3-6, 3-7). This suggests that different regions within the same structure can be involved in different behaviors. It does not appear that this same gradient is present along the ellipsoid body or the protocerebral bridge; however, our data set contains fewer EB and PB lesions and more data is needed to state that definitively.

Ellipsoid Body

While individuals with lesions to the EB exhibited no deficits on the block (Figures

3-4, 3-5, 3-6), they exhibited profound deficits in turning (Figures 3-9,3- 10, 3-11). In contrast to individuals with lesions in the FB, these individuals showed turning deficits in both directions of movement. It is possible that these differences are present because of the differences in how these structures connect to the PB. Each FB column receives information from columns located on the right and left of the PB. In contrast, with the EB the left columns of the PB connect to odd numbered EB columns, and the right to the even

125 numbered columns(Heinze and Homberg, 2009; Muller et al., 1997; Strausfeld, 1999b;

Williams, 1975). Because of these connections it is possible that lesions to the EB result in destruction of large amounts of information from both the right and the left columns. With no additional connections between the specific area of the PB and another region of the EB, this information is lost completely. This hypothesis could also explain the abnormalities seen in these individuals when faced with a shelf (Figure 3-7). Whether or not this is the case it would seem that the EB and FB have different roles in these behaviors. .

Lateral Accessory Lobe

Individuals with lesions to the LAL exhibited striking abnormalities in obstacle negotiation behaviors relating to all obstacles we tested them on. This even includes changes in local reflexes (Figure 3-13). This region contains connections leading to and from the CC

(Heinze and Homberg, 2009). Furthermore, it contains direct connections to the ventral nerve cord (Homberg, 1994). As a linkage between the CC and thoracic ganglia, it is not surprising that individuals with LAL lesions have profound deficits in obstacle negotiation behaviors.

Control of Complex Behavior

We found that there is not a simple answer as to how the brain controls these complex behaviors, but rather, it seems that control varies for each type obstacle negotiation behavior. One example of this is that while lesions to the EB had little impact on climbing behavior, they were shown to have a profound effect on turning behavior. These lesions

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resulted in a statistically significant increase in delayed turning (Figure 3-8), failure to turn

(Figures 3-9, 3-11), and turning in the wrong direction (Figures 3-9, 3-11).

Furthermore, the control of these obstacle negotiation behaviors seems to have

some specificity for different elements of behavior. While individuals with lesions to the

ACT, FB, and LAL seemed to make more climbing attempts close to the block (Figure 3-4)

and changed to different climbing strategies (Figure 3-6), success of climbs was only affected

in individuals with lesions to the FB and LAL (Figure 3-4). In some cases this specificity

even extended itself to different conditions. For example, in the light, individuals with

lesions to the ACT, PB, EB, and LAL all had a significantly higher rate of collision with the

block than they had in pre-lesion tests. Whereas, in the dark, this difference only remained

statistically significant in individuals with lesions to the FB and LAL (Figure 3-5).

Interestingly, a very similar collision with an obstacle occurred in turning behavior. However, there were no areas that showed a significant increase in the light but not the dark (Figure 3-

8). However, it is possible that this effect was amplified by the failure of some individuals to turn (Figure 3-11), whereas failure to climb did not occur.

Reflexes

How might activity descending from the CC or LAL re-direct leg movements? One possibility is to alter reflex activity. It is known that reflexes in insects as well as mammals can change sign as their behavioral state is altered (Bässler and Büschges, 1998; Pearson and

Collins, 1993). Indeed, in stick insects inter-joint reflexes reverse their sign when the insect walks backward (Akay et al., 2007). It has been suggested that changes in these reflexes might start a cascade of changes leading to a new stable movement pattern such as turning

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(Mu and Ritzmann, 2008a). Removal of all descending activity through bilateral ablation of neck connectives causes profound changes in the reflex activation of Ds motor neurons upon stretch and relaxation of the femoral chordatonal organ (FCO). To determine whether the brain regions that we lesioned could contribute to these reflex changes, we recorded

FCO evoked reflexes in a subset of our subjects. Comparison with intact recordings made previously revealed that a substantial number of FB, EB and LAL lesions showed altered reflexes. These included reflex reversals similar to those seen in neck lesioned animals and reflexes that activated units that had not been previously recorded.

Because these observations could only be carried out after the lesion was made, it is not clear whether similar reflex reversals initiate the changes that lead to re-directed leg movements. Nevertheless, they are encouraging and should lead to more controlled experiments in which CC and LAL neurons are stimulated or blocked while recording FCO and other reflexes. For example, experiments in which muscarinic agonists are injected into the CC of grasshoppers induce stridulation (Heinrich et al., 2001), whereas activation of nitric oxide releasing neurons in the CC suppresses such behavior (Weinrich et al., 2008).

Potential for Non-specific Effects

Lesion experiments have inherent limitations. It is possible that the surgical process created unknown damage. However, a previous study which employed the same technique to open and reclose the head found that the procedure itself did not increase turning abnormalities over levels seen in pre-lesion tests (Ridgel et al., 2007). Furthermore, none of our 9 lesions that were outside the CC, mushroom bodies, and ACTs showed any significant changes in behavior. Another possibility is that the electrode used for our lesions caused damage upon its insertion. Individuals that had had the probe inserted without electrical

128 current, exhibited similar abnormalities to those with lesions to the same region. Most of these abnormalities were less severe than they were in individuals experienced the electrolytic lesion. However, as our lesions were identified histologically by where the tip of the probe was, we cannot rule out the possibility that the path of the probe caused additional abnormalities. Severe deficits such as inability to turn, difficulty climbing when close to the block, and falling off of the climbing wall are rare among our entire lesion population.

However, these abnormalities are consistent in individuals with lesions to certain brain regions. Together, this suggests that these deficits are likely a result of the location of the lesion, rather than a complication of the technique.

To truly understand the role of the CC in behavior we must use a variety of techniques. No experimental method is without consequence. Neurogenetic lesions have become a powerful tool for studying the neural basis of behavior (Strauss, 2002). Yet, these procedures can have unspecified effects also. The hope is that each technique has different difficulties. Then by combining studies which employ the use of genetics, chemical lesions, recordings, and temperature effects it will be possible to overcome the drawbacks of any single technique.

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Figure 3-1 Lesions are located in multiple brain regions. This is a depiction of the brain lesions referenced in the majority of these data (A). For clarity these lesions will be separated into two figures, one of the central complex (B), and one of lesions outside the central complex (C). Also the central complex figure (B) zooms in on the structures and adds separation between them (lower). While it is possible for a lesion to damage more than one structure, we designate the structure receiving the majority of the damage as the lesion location.

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Figure 3-2 A schematic of the obstacles used in this study: block (a), shelf (b), turning arena (c), and vertical to horizontal transition (d).

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Figure 3-3: Depiction of different turning behaviors. The change from the body axis at the starting position (antennal contact) is measured at each step (annuli). In pre-test trials only 1.7% of trials failed to reach a 30 degree turn in 8 steps, and only 0.9% of trials reached more than 30 degrees in the wrong direction. We used these criteria to determine whether or not an individual turned normally (a), turned the “wrong direction” (b), or “failed to turn” (c). 5 representative trials from the post-test are located on each graph.

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Figure 3-4: Change in success of climbs is related to lesion location. Climbing attempts can be separated into ‘success’ where the foot reaches the top of the block or a ‘miss’ where it does not. The horizontal distance from the front of the block to the cockroach’s head was measured for the initial climbing attempt for 54 individuals in each trial (n=147, n=146) in pre (A) and post tests (B) respectively. These attempts were then divided into groups according to the location of the lesion. The average number of attempts for each individual in each of the following categories was calculated: number of attempts occurring under 5mm from the block (C), number of attempts per trial (D), and success of attempts under 5mm (E). This average for each individuals post test was subtracted from the pre-test average to account for individual differences. That value for each individual within a group was then averaged to get the average change (depicted in graphs C-E). Statistics were performed with a chi squared test on raw data. The error bars in this and other figures represent the standard deviation for the group. * in this and other figures represents a significance level of p<0.05 while ** represents p<0.001.

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Figure 3-5: After the lesion more individuals hit their head on the block in the light(A) and dark (B). For each individual, the percent of trials in which the head contacted the block before climbing was calculated for both pre and post tests. We then subtracted the pre-lesion value from the post lesion value for each individual. This value was averaged among each lesion group to get the mean depicted by the bar graphs here. Statistics were performed using a chi-squared test on raw data. In figures C and D the standard deviation of the pretest data was used to color code the lesions.

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Figure 3-6: Climb strategy changes in some individuals after the lesion. The percent of climbing attempts (A) employing a given strategy was calculated for both pre lesion (n= 226 attempts) and post lesion (n=181 attempts) trials in 54 individuals. The percent of trials using brute force (B), elevator (C), and controlled rearing (D) strategies were calculated for each individual pre lesion and post lesion. Then the pre lesion value was subtracted from post lesion to calculate the change in the use of each of these strategies. This was averaged between individuals in each lesion category. Statistical significance was calculated using a χ2 on the raw data from each lesion group.

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Figure 3-7: Behavioral variability changes after the lesion in individuals presented with a shelf. Individuals can either climb over or tunnel under the shelf. The average percent of climbs was calculated for each individual and then averaged over all individuals. A χ2 test was used to determine significance. This figure represents 28 individuals (96 trials dark, 93 trials light) pre lesion and 25 individuals (84 trials light, 83 trials dark). For the ethograms (B-E) the numbers represent the proportion of trials that exit a given state and transition to the next state. Significance of changes in the transitions between the pre lesion and post lesion ethograms was calculated using a χ2 test. These significant differences were then labeled as a ‘decrease’ (blue), ‘increase’ (yellow), or ‘new behavior’ (red).

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Figure 3-8: Delayed turning increases after the brain lesion. In some individuals turning was delayed resulting in them hitting their head on the wall prior to turning. These individuals did eventually turn. The proportion of trials where this occurred for each individual was calculated for a given direction in the light and the dark. The directions were averaged such that an individual’s deficiencies in an individual direction would not bias the data then the pre lesion mean was subtracted from the post lesion mean for each individual. The data for individuals in a given group was averaged to create the mean data shown here for the light condition (A) and dark condition (B). Lesions are encoded depending on their deficit (light (c), dark (d)). The standard deviation of the raw pretest data was used to create this code. This deficit could occur in turns to the right (triangle toward right), left (triangle pointing left), or both directions (square). This increase could be 1 sd (orange) or 2 sd (red). Individuals with no change in behavior are shown in black (circles).

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Figure 3-9: Turning angles become more variable after lesions to certain brain regions. The change in turning angle (r) was measured relative to the position at antennal contact at each subsequent step (annuli) with the middle legs. The pre lesion traces (black) (A) are relatively consistent for the population. Whereas, post lesion traces (red) in individuals with lesions to the LAL (B), EB(C), and FB(D) show increased variability after the lesion. In the FB this increase in variability is solely noted in individuals with lesions to the lateral FB. We also show traces from trials in individuals with lesions to the PB (E), ACT (F), MB (G), and Other (H). The final position data were divided into 10 degree bins. Color coded dots were used to mark the frequency of each final position.

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Figure 3-10: Abnormalities in the FB and LAL relative to lesion location. The change in body angle relative to the position at antennal contact is plotted for each step after antennal contact for individuals with lesions to the lateral FB (A) and LAL (B). Here the turning direction is color coded relative to the location of the brain lesion. The final position data were divided into 10 degree bins. Color coded dots were used to mark the frequency of each final position. For instance, a lesion of the right side of the brain is ipsilateral for turns to the right. Note that in the lateral FB data, 4 contralateral turns moved in the opposite direction from the onset, while two others began in the wrong direction and then corrected. This behavior was not present in ipsilateral turns for these individuals. For individuals with lesions to the LAL, only one ipsilateral turn started in the wrong direction and then corrected its course.

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Figure 3-11: Abnormalities in turning behavior increase in individuals with lesions to the FB, EB, and LAL. Failure to turn was determined as an individual failing to make a turn of at least 30 degrees in 8 or fewer steps (A). Turning in the wrong direction (B) was determined as a turn of 30 degrees or more toward (instead of away from) the obstacle. If more than one turning behavior was present, such as if an individual turned the wrong direction then corrected course, only the first behavior was recorded. Lesions are encoded depending on their deficit (C and D). The standard deviation of the raw pretest data was used to create this code. This deficit could occur in turns to the right (triangle toward right), left (triangle pointing left), or both directions (square). This increase could be 1 sd (orange) or 2 sd (red). Individuals with no change in behavior are shown in black (circles). To relate the directionality of these deficits to the lesion location we examined how many of these individuals had either (or both of these deficits) and in which direction they occurred. The proportion of trials exhibiting these deficits was not factored into these data. Here we just investigate if either or both deficits occurred ipsilateral to the lesion, contralateral to the lesion, in both directions, or in neither direction (E).

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Figure 3-12: Some individuals fail to reach the top of a wall. Here we have a vertical wall which cockroaches will climb up and then transition to a horizontal surface. Most individuals were able to make this transition by flexing their bodies (A). In some individuals this flexion did not occur resulting in them falling off of the obstacle . In others, this flexion was delayed making the transition difficult. Individuals which fell off of this obstacle all had lesions to the FB, EB, or LAL with the exception of one individual (out of 47) that fell off in one of the pre lesion trials (n=282) (B,C). Individuals which exhibited delayed climbing had lesions to the ACT, FB, EB, or LAL.

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Figure 3-13: Changes in local reflexes associated with some lesion locations. Typical responses to stretching and relaxing the femoral chordotonal organ (FCO) for each condition are present here (A-C) Under normal conditions when the FCO is stretched the activity of Ds will increase and during relaxation activity in Ds will decrease (A, arrow). After the lesion some individuals showed a reversal of this reflex whereby activity in Ds would increase during relaxation of the FCO (B, arrow). Cumulative histograms of the response of Ds to stimulation are present for normal (A) and reversed (B) reflexes. In some individuals a unit which had previously not been identified was recorded (C). Locations of these lesions are marked according to the reflex behavior (black= no change, red = reversal, yellow = new element) (D). The occurrence of these events is limited to individuals with lesions to the FB and LAL (E).

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Figure 3-14 Summary of Findings. Here significant findings are noted for each region and behavioral test. Note that it is possible to exhibit one abnormality on an obstacle, but not all of the possible abnormalities for that obstacle. Individuals with lesions to the FB and LAL exhibited abnormalities on all obstacles. Individuals with lesions to the PB only exhibited abnormalities on the shelf and turn obstacles. Those with lesions to the EB exhibited significant abnormalities on the shelf, turn, and wall obstacles, but showed no abnormality in reflexes or block behavior. Individuals with lesions to areas outside the CC showed fewer abnormalities overall compared to individuals with lesions within the CC. This suggests that the CC is involved in complex behaviors and that different sub-regions of the CC have different levels of involvement in these behaviors.

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Chapter 4:

Discussion

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In this thesis, we have discussed the role of various regions of the brain in complex behaviors. In order to accomplish this, the behavioral complexity first had to be understood.

A simple reflex would not require information from the brain and would have little variability. This is in contrast to complex behaviors where variability increases with behavioral complexity. It is possible that as one considers behaviors where brain circuits play an important role, that variability increases. That is, the more brain involvement, the more complex the behavior.

Many of the behaviors I described here could, in theory, be controlled by a simple reflex event. Indeed, the decision to climb or tunnel based on antennal contact pattern can be recreated using a very simple algorithm (Lewinger et al., 2005). However, as we have shown here, this behavior also takes into account light sensed by the ocelli. This means that multiple sensory systems are involved in guiding this behavior, suggesting that visual and tactile information have to be integrated. It is likely that this information becomes integrated within the brain. Here, we were able to show that new behaviors arise, and other elements within the sequence change in frequency after brain lesions, further supporting the notion of brain involvement in these behaviors. Furthermore, these occurrences are mainly associated with lesions to the central complex, suggesting this is a region of the brain that is involved in guiding these behaviors.

The intrinsic complexity of the behaviors that we studied is related to integration of multiple sensory inputs and higher-order processing. This leads to a greater variability, meaning that any attempt to study the role of the brain in a given behavior must first strive to characterize the variability of that behavior. Without first doing so, changes in behavior may be dismissed as normal or be lost in their natural variability. For instance, in climbing

148 behavior multiple strategies can be used. All of them are present in behavioral tests before and after lesions. However, the change associated with the behaviors is in the frequency of their occurrence. Some behaviors occur more often after the lesion, others occur less.

Moreover, if one simply asks the question, “Can a cockroach climb after brain connections are removed?” The answer is yes. However, they do so in a much less controlled manner.

Thus, if one can ask a more precise question, such as “Do cockroaches climb in the same way after removal of brain connections as before?” the answer is no. However, in order to ask that more precise question, it is important to know how the cockroach climbs under normal conditions or under a range of conditions that it might well be expected to encounter in its normal habitat.

Changes in behavioral variability

Complex motor behaviors recently have been described as motor synergies, simpler motor patterns that are grouped in different orders to lead to fundamentally different limb movements (d'Avella et al., 2003). Similarly, ethograms divide complex behaviors into simpler elements which, when grouped in different ways, may lead to fundamentally different behavioral outcomes. Furthermore, these simple behavioral elements may themselves represent synergies in a behavioral manner (Pick and Strauss, 2005). Changes to either the sensors available to the cockroach or to its brain resulted in changes in the behavioral synergies present. However, this was highly dependent on the behavior and, in the case of lesioned cockroaches, on the area of the brain lesioned. Furthermore, even within a single brain neuropils, a lesion in one region might not affect a given behavior, but a lesion

149 in another region will. We saw this in the fan-shaped body where lesions to the center did not result in turning deficits, but lesions to the lateral edges did. This is not true of all behaviors, namely, lesions anywhere within the FB led to difficulties in climbing. These changes in behavior could represent changes in the interaction of motor synergies. However, a more detailed study of movement in individuals with these lesions is necessary to determine whether or not this is the case.

That is not to say that without a full understanding of the coordination of the limbs that this study is invalid. Our ability to compare changes in these behavioral sequences enabled us to assess the presence of behavioral deficits in a more specific way than previous studies. This was enhanced by the use of multiple obstacles. The combination of these enabled us to compare the roles of different regions of the central complex in different obstacle negotiation behaviors.

The Central Complex and the Cerebellum:

The central complex in insects has been implicated in control of motor coordination and some investigators have suggested that it could play a role similar to that of the vertebrate cerebellum (Pick and Strauss, 2005; Strausfeld, 1999; Strausfeld, 2009).

Interestingly, both the cerebellum and the central complex have been implicated in processing sensory information in addition to motor information (Gao et al., 1996; Heinze and Homberg, 2007; Ridgel et al., 2007; Ritzmann et al., 2008; Strauss, 2002). Here we found that many of the lesions to the central complex resulted in behavioral deficits similar to those seen in individuals with lesions to the antennae. However, some of the deficits we noted

(such as failure to turn and failure to transition during wall climbing), seem more severe than

150 they would if the insect were just missing its head sensors. Moreover, the effect on block climbing was distinctly different in brain lesion and partial antennal ablation. Surgically shortening the antenna resulted in the cockroach starting to climb when it was closer to the front of the block. The same thing happened in the brain lesioned animals. However, with antennal shortening, this effect actually resulted in greater success in climbing, while brain- lesioned animals were less successful even though they started at a closer position. The reason for this difference is clear. With shortened antennae, the cockroach simply commences climbing closer to the block where it has a better chance of placing its foot on the top of the object. Since there is no damage to the central nervous system, there is no underlying detriment. However, the brain damage that brings the cockroach closer to the block also compromises its motor control system. Thus, it cannot take advantage of the more promising starting location. Rather my lesions suggest potential motor problems—an inability to perform the behavior or perhaps the inability to initiate it.

Genetic manipulation revealed similar problems coordinating smaller behavioral subunits in Drosophila (Pick and Strauss, 2005). Interestingly, imaging studies in humans suggest that the cerebellum may play an important role in coordinating subunits of movement (Ramnani et al., 2001). These structures are similar in architecture are both involved in active sensing and coordination of behaviors.

Perhaps we have more in common with insects than we previously realized. Future studies may reveal that the central complex plays a role similar to the cerebellum and the columnar anatomy of each structure makes such comparisons tempting. However, they must be done with care. Until more data is available, such comparisons are merely suggestions.

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Caveats

This study is not without its faults. Perhaps the biggest drawback of this study is our use of lesions. It is difficult to assess if the lesion itself is resulting in the changes in behavior seen, or if they are a result of the animal’s reaction to the lesion. Genetic lesions have been used, but can also have unknown non-specific effects on the system.

One way to get around this obstacle is to use either a chemical or temperature lesion which is reversible. Individuals could be “lesioned” and then tested again after the block has been removed to examine how they behave. This would potentially allow for non-specific effects of the lesion to be removed such that only those caused by the lesion are in the forefront. Even with that method, we would only be able to propose the involvement of specific regions in specific behaviors. This technique would have to be enhanced perhaps by recording activity in a number of cells while an insect is performing a variety of behaviors.

However, these techniques are at their infancy and are difficult if not nearly impossible to perform in freely moving insects. That said, recent genetic advances have enabled the visualization of populations of cells within the brain of freely moving Drosophila (Seelig et al.,

2008). Clearly, the only way to examine control of such a complex brain region is to examine it with a wide range of techniques, while always relating the resulting data to natural behavior.

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