A COMPARISON OF THREE-DIMENSIONAL SWIMMING PATHS DURING VISUALLY-

EVOKED VERSUS AUDITORY-EVOKED ESCAPE SWIMS IN LARVAL

By

Benjamin Bishop

A Thesis Presented to

The Faculty of Humboldt State University

In Partial Fulfillment of the Requirements for the Degree

Master of Arts in Psychology: Academic Research

Committee Membership

Dr. Ethan Gahtan, Committee Chair

Dr. Christopher Aberson, Committee Member

Dr. Andrew Kinziger, Committee Member

Dr. Christopher Aberson, Graduate Coordinator

May 2016 VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS

Abstract

A COMPARISON OF THREE-DIMENSIONAL SWIMMING PATHS DURING VISUALLY- EVOKED VERSUS AUDITORY-EVOKED ESCAPE SWIMS IN LARVAL ZEBRAFISH

Benjamin Harper Bishop

Escape behaviors have been studied in zebrafish and aquatic organisms by seeking cellular-level descriptions of neural circuits, but few studies have examined vertical swimming during escapes. I analyzed three-dimensional swimming paths of larval zebrafish during visually-evoked and auditory-evoked escapes while the were in a cubical tank with equal vertical and lateral range. A vertical component was found in both visually-evoked and auditory-evoked escapes. The initial 10 seconds of stimulation involved an equal amount of increased vertical and horizontal movement for both escape behaviors, followed by a decrease in total distance traveled below that of spontaneous swimming. These escapes differentiated only after the initial 10 seconds of stimulation, with visually-evoked escapes involving a greater amount of vertical distance travelled and greater decrease in horizontal movement when compared to that of auditory-evoked escapes. To determine how these develop across ages at which zebrafish larvae are commonly used in behavioral assays, I tested light dimming- evoked and tap-evoked escapes in groups of larvae at 4 different ages: 6, 8, 10, and 12 days post fertilization. Both behaviors were found to not change as the age of the zebrafish increased. A comparison of light dimming-evoked diving in zebrafish to similar behaviors of other aquatic animals suggests it is a protean defense against specific predation threats. These results also imply that future studies of the neural mechanisms of visual behavior in zebrafish should consider vertical movement control elements.

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Table of Contents

Abstract ...... ii

Table of Contents ...... iii

List of Tables ...... v

Table of Figures ...... vi

A Comparison of Three-Dimensional Swimming Paths During Visually-Evoked Versus Auditory-Evoked Escape Swims in Larval Zebrafish ...... 1

Neural Circuits for Escapes in Zebrafish ...... 2

Literature Review...... 5

Increasing our Understanding of the Zebrafish Repertoire ...... 5

Development of Visual Escape Response ...... 8

Directionality of Light Dimming-Evoked Escape Responses ...... 9

Behaviors Associated with the nMLF ...... 11

Statement of the Problem ...... 16

Hypotheses ...... 16

Hypothesis 1...... 16

Rational for Hypothesis 1...... 16

Hypothesis 2a...... 16

Hypothesis 2b...... 16

Hypothesis 2b...... 17

Rational for Hypothesis 2...... 17

Hypothesis 3...... 17

Rational for Hypothesis 3...... 17

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VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS

Summary of Research Purpose ...... 17

Method ...... 18

Data Analysis ...... 20

Results ...... 22

Total Distance Traveled ...... 22

Total Vertical Distance Traveled ...... 25

Total Horizontal Distance Traveled ...... 29

Vertical Displacement ...... 32

Horizontal and Vertical Component of Dimming-evoked Dive Response ...... 36

Discussion ...... 37

Zebrafish Larvae Escape Swims Include a Negative Vertical Movement ...... 37

Biphasic Activity Response to Startle Stimuli ...... 38

Ethological Interpretations of Vertical Escape Swimming ...... 38

Age Effect on Total Distance Traveled ...... 39

Limitations ...... 39

Conclusion ...... 40

References ...... 41

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VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS

List of Tables

Table 1. effects on total swimming distance ...... 23

Table 2. Stimulus effects on vertical swimming distance...... 27

Table 3. Stimulus effects on horizontal swimming distance ...... 30

Table 4. Stimulus effects on vertical displacement ...... 34

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VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS

Table of Figures

Figure 1. Schematic of a 6 dpf zebrafish larvae highlighting the axonal and dendritic projections of the MeL and MeM of the nucleus of the medial longitudinal fasciculus (nMLF) ..... 13

Figure 2. Photograph of behavior recording apparatus ...... 19

Figure 3. Difference in total distance traveled between spontaneous and post-stimulus ...... 24

Figure 4. Difference in vertical distance traveled between spontaneous and post-stimulus ...... 28

Figure 5. Difference in horizontal distance traveled between spontaneous and post-stimulus .... 31

Figure 6. Difference in vertical displacement between spontaneous and post-stimulus ...... 35

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VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 1

A Comparison of Three-Dimensional Swimming Paths During Visually-Evoked Versus

Auditory-Evoked Escape Swims in Larval Zebrafish

The microstructure of neural circuits, including the physical patterning of synaptic connections among neurons in the circuit, is essential to how neural circuit’s processes information. But neural circuit microstructure is difficult to study in complex animals because of the large number of neurons and connections and behavioral complexity, which complicates efforts to correlate circuit properties with behavioral functions. Neuroscientists have therefore long studied simple reflex behaviors, especially escapes, in simple model organisms (Domenici,

Blagburn, & Bacon, 2011), because stereotyped movements suggest a dedicated and relatively simple underlying neural circuit. Many studies of neural circuits for escape have used invertebrate animal models such as snails (Croll, 2003), but zebrafish have become a popular model for studying neural circuits (Zottoli, 1978), and have the advantage, as a , of greater homology in neural mechanisms with humans (Burgess & Granato, 2007). In zebrafish, escape swims can be elicited reliably and repeatedly with auditory (Zeddies & Fay, 2005;

Spulber et al., 2014), tactile (Liu et al., 2012), or visual stimuli (Burgess & Granato, 2007;

Temizer, Donovan, Baier, & Semmelhack, 2015; Fernandes et al., 2012). The initial escape swim can vary depending on stimulus type and directionality (Spulber et al., 2014; Liu et al., 2012). In zebrafish, escape responses to different stimuli are distinguishable by kinematic parameters such as swim speed and turn angle. C-turns (Satou et al., 2009), S-turns (Liu et al., 2012), and O-turns

(Burgess & Granato, 2007) are all distinct patterns of escape swimming responses that have been described in zebrafish.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 2

Zebrafish are a standard model animal for developmental research of (Easter

& Nicola, 1996). Spontaneous swimming, beginning at 4 dpf, is characterized by bouts of activity separated by inactive intervals, and activity increases as zebrafish develop (Spulber et al., 2014). Auditory-evoked escape responses have been reliably observed in zebrafish 5 dpf

(Zeddies & Fay, 2005; Spulber et al., 2014). While visually-evoked escape responses have been

observed in zebrafish as young as 68 hours post fertilization (hpf) unreliably, with reliable

responses occurring 79 hpf (Easter & Nicola, 1996). A visual stimulus commonly used to

produce startle responses in zebrafish is light dimming, the abrupt decrease in ambient

illumination.

Dark photokinesis is the movement triggered by light dimming (Fernandes et al., 2012).

In zebrafish, dark photokinesis is characterized by an initial period of hyperactivity, known as

the visual motor response (VMR) (Fernandes et al., 2012; Burgess & Granato, 2007). It has been

observed by Fernandes et al. (2012), that zebrafish perform a dive response when a light

that the fish have acclimated to is suddenly extinguished. When observed from above, it has been

found that zebrafish perform large slow lateral turns, referred to as O-turns (Burgess & Granato,

2007). Both of these observations were done with a two dimension view of the fish obtained

from a single camera. This current research attempts to quantify the behavior in a three-

dimensional perspective by measuring both lateral and vertical movement within the same assay.

Neural Circuits for Escapes in Zebrafish

Extensive research has been published on the neurons and circuits that are involved with

the , most focusing on initial movements that occur within 1 second following an

effective startle stimulus (Eaton & Emberley, 1991; Kimmel, Patterson, & Kimmel, 1974; Liu et

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 3 al., 2012). The Mauthner , a neuron that has been studied for over 100 years, has been linked to escape responses from multiple sensory modalities. The Mauthner neuron consists of two large, easily identifiable, bilateral neurons located in the 4th rhombomere of the in zebrafish (Koyama et al., 2010). It has been linked to the stereotypical C-bend behavior of the startle response that is characterized by the C shape the body forms during the behavior (Eaton &

Emberley, 1991; Burgess & Granato, 2007; Monesson-Olson, Troconis, & Trapani, 2014). The

Mauthner neuron has been found to mediate a C-bend escape response to auditory, vibrational, tactile, and some visual stimulation (Satou et al., 2009; Liu et al., 2012). While the Mauthner neuron was found to be active during a visual looming stimuli (Temizer et al., 2015), it has been found not to be active during a response to light dimming (Burgess & Granato, 2007). This distinction suggests a separate neural circuit is used for this variety of visually evoked escape response.

The nucleus of the medial longitudinal fasciculus (nMLF) is a cluster of neurons in the midbrain that, like the Mauthner neurons, have long that descend into the , and nMLF neurons are also associated with motor control and escape responding (Semmelhack et al.,

2014; Thiele, Donovan, & Baier, 2014). The nMLF has been shown to be active during multiple behaviors, including the C-bend escape response resulting from tactile stimulation (Gahtan,

Sankrithi, Campos, & O’Malley, 2002), as well as visual prey tracking (Semmelhack et al., 2014;

Gahtan, Tanger & Baier, 2005) and spontaneous behaviors involving slow swims, tail flips, and struggles (Thiele, Donovan, & Baier, 2014). Neurons in the nMLF have been grouped into mesencephalic medial (MeM), mesencephalic lateral (MeL), and the Mesencephalic Small

(MeS) (Kimmel, Powell, & Metcalfe, 1982; Thiele, Donovan, & Baier, 2014). Each category has

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 4 been researched, and differences in their anatomy and function have been found. of the

MeM neurons cross the midline into contralateral midbrain destinations, while dendrites of MeL neurons project only within the ipsilateral midbrain (Gahtan & O’Malley, 2003; Gahtan, Tanger,

& Baierm, 2005). The axons of MeM and MeL neurons follow the same pattern as their dendrites, with MeM axons crossing the midline and MeL axons remaining ipsilateral. These axonal and dendritic projections suggest that MeL neurons contribute to behaviors unilaterally, while MeM neurons affect bilateral spinal motor circuitry. The MeL is found to have dendrites ascending into the optic tectum, which is the main visual brain area in fish. Consistent with that finding, research has shown MeL neurons are active during visually-evoked behaviors while

MeM neurons were not (Sankrithi & O’Malley, 2010).

The Mauthner neurons and nMLF are two extensively-studied central components of a larger escape circuit that also includes peripheral elements such as sensory receptors and muscles. Specific escape-related functions have been described for other structures including specific spinal (Bhatt, Mclean, Hale, Fetcho,

2007; Downes & Granato, 2006) and neurons in the brain (Takahashi, Narushima, Oda, 2002;

Lacoste et al., 2015). All of these studies use sophisticated methods to visualize or manipulate specific neuronal elements within the escape circuit, and all of them also measure escape-related swimming movements in some way. This thesis focuses on vertical movements during escapes in zebrafish, a feature of escape movement that is most often not measured in studies of neural circuit mechanisms for escape. My data show prominent vertical swimming in some escape responses and imply that future neural circuit models of zebrafish escape control must include vertical swimming control elements.

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Literature Review

Neuroethology, a scientific field established by in the 1950’s, focuses on the neural circuit mechanisms controlling innate animal behaviors such as sensory reflexes, predator-prey interactions, and reproductive behavior. Tinbergen’s ‘hierarchy hypothesis’ of the brain suggested that neural circuits for different behaviors are anatomically and functionally distinct and are hierarchically organized (Tinbergen, 1951). This model led to influential concepts in behavioral neuroscience including the concept

(Ierusalimsky & Balaban, 2007; Bullock, 2000), in which activation of a single neuron triggers a larger circuit that results in a specific behavior, as well as other theoretical principles of neural circuit organization (Marder & Abbot, 1995). What links these studies in is the emphasis on accurate and thorough characterization of innate behavior. Innate behaviors evolved in natural environments, so experiments that seek to reveal the neural circuit for an innate behavior should measure the behavior in a way that corresponds to its natural expression. To put it otherwise, if the description or expression of a natural behavior is incomplete, any neural circuit model for the behavior must also be incomplete (Tinbergen, 1951). In regards to the focus of the present research, previous studies of neural circuits controlling escape reflexes in zebrafish have mainly measured escapes in environments that restrict vertical movement. If vertical movement is a natural part of the escape response, neural circuit elements that control vertical movement may be missed or misinterpreted.

Increasing our Understanding of the Zebrafish Escape Response Repertoire

In the zebrafish’s natural environment, having a larger repertoire of escape behaviors to utilize during different types of threats increases survivability. Liu, Bailey, and Hale’s (2012)

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 6

examined the flexibility of the startle response through tactile stimulation of different locations

on the body of zebrafish. The fish were placed in petri dishes and their movements were

recorded from a single camera mounted above that dish. They observed the commonly

documented C-start, which is characterized by the simultaneous activation of motor neurons on

one side of the body, resulting in a bend of the body resembling the letter C, and the less

commonly documented S-start, which is characterized by the activation of rostral motor neurons

on one side of the fish, and caudal motor neurons on the opposite side, resulting in a bend of the

body resembling the letter S. Tactile stimulation was done by touching either the head or tail of

the fish. Their results showed consistent C-starts when the head of the fish was stimulated, while

varying C- and S-starts were observed when the tail was stimulated. The C-starts result in a fast

180° turn followed by quick propulsion away from the threat, opposite the original trajectory.

The S-start, being stimulated from behind, results in a quick withdrawal of the tail away from the threat and propulsion forwards, in the original trajectory. The variability in escape responses,

elicited in this way, was interpreted as support for idea that zebrafish larva use a variety of

responses in natural environments to escape a variety of threats.

Beyond the flexibility of escape responses to a single type of startle, distinctly varying

behaviors are exhibited when stimuli that act on different sensory modalities are presented.

Spulber et al. (2014) observed the responses of zebrafish to multiple types of stimuli, including

visual and auditory. Larvae were placed in individuals wells (10mm diameter) of a plastic tissue

culture plate and swimming movements in the lateral plane were recorded from video camera

above the plate. Three types of behaviors were observed, spontaneous activity, visual motor

responses evoked by light dimming, and auditory/vestibular evoked responses produced by a

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 7

tapper that hit the recording chamber’s chassis. During light dimming trials (sudden and

complete darkness for 10 minutes), zebrafish showed a vigorous increase in swimming-bout frequency with a decrease in amplitude (angle of tail during bout) for an average of three minutes of darkness, followed by a decrease bellow baseline of 50 bouts per minute for the remainder of the darkness trial. During auditory-evoked escapes (10 taps with 1 tap per minute), zebrafish performed a single bout per tap with increased amplitude of 2.5 times that of a spontaneous swimming bout. One limitation of this study was that the visual and auditory stimuli had different durations (10 minutes of continuous light dimming versus 10 auditory taps at a rate of one per minute) so the responses may not be directly comparable. Nonetheless, this research suggests that both light dimming and auditory tap trials produce behaviors distinct from each other and from spontaneous swimming.

Different types of visual stimuli have been found to produce distinct behaviors in zebrafish. Stimuli that resemble looming shadows, such as a growing dark circle on an illuminated computer screen, produces a C-bend escape response of a 180° turn and propulsion forward (Temizer et al., 2015). The looming stimulus, representing an approaching object from the front of the fish, was found to stimulate a C-bend, followed by a fast swim to propel away from the looming object. These results show the versatility of the C-bend to be even greater than previous thought, as visual stimuli were not previously believed to produce a C-bend. Although the looming shadow stimulus is similar to the light dimming stimulus, the behaviors evoked are distinct. They also included a gradual light dimming trial by showing a large light grey circle slowly increasing in darkness till completely black, but this did not reliably produce a C-bend.

By monitoring the activity of retinal ganglion cells (RGC) terminals in the brain, the

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 8

researchers found that both looming and gradual dimming stimuli trigger activation in certain

brain areas that receive retinal input (RGC arborization fields 6 (AF6) and 8 (AF8)), while other

brain areas (AF7) were active only by looming shadows. One obvious way in which these stimuli

differ is that there is visual motion in the looming shadow (since the shadow grows) but not in

the gradual dimming stimulus. Visual motion is known to be processed in different brain areas

from other visual features such as color in many animals (Zeki, 2008) so this result is not

surprising. However, these studies in zebrafish are noteworthy because of the potential for

precise, cellular-level mapping of sensorimotor brain circuits in zebrafish. Arborization fields 6,

7 and 8 are three of nine RGC arborization fields that have been described in zebrafish. Their

different sensitivities to different visual stimuli may explain the distinct swimming responses that

are evoked by different visual stimuli. The study by Temizer et al. (2015), and many other

studies on zebrafish sensorimotor systems, exemplifies the neurotheological framework by

beginning with behavioral analyses that distinguish and classify ethologically-based behaviors and then using neurobiological methods to illuminate the circuits and circuit properties that support them. These studies have already demonstrated several distinct escape responses in zebrafish and have begun to reveal the underlying neural pathways and mechanisms.

Development of Visual Escape Response

Easter and Nicola (1996) conducted an experiment to discover the earliest onset of visual

escape responses. They observed responses to light dimming from above by tracking swimming

movements in the lateral plane from a single camera. Startles were first observed in fish as young

as 68 hpf unreliably, however once the fish reach 79 hpf visual startles occurred more reliably.

By also running trials with zebrafish genetically modified to not produce eyes, they were able to

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 9

conclude that the visual startle from an abrupt light dimming requires a retina. This was

important because other light-responsive cells, such as cells in the hypothalamus that can

respond to light transmitted directly into the brain through the head, were also candidates for

mediating responses to whole-field illumination changes, but this study showed that the

dimming-evoked escape response requires a neural pathway that originates with the retina. While they were able to document at what age the VMR begins, little is known of the change in this behavior as the age of the zebrafish larva increases.

Directionality of Light Dimming-Evoked Escape Responses

A distinct light dimming-evoked behavior in zebrafish, termed an O-bend, has been described in zebrafish larvae. Burgess and Granato (2007) attempted to find evidence that motor activities caused by visual stimuli are utilized to seek well-lit regions, where the possibility of feeding is increased. The experiment utilized a camera from above, and filmed lateral swimming movements of multiple fish in individual wells. This setup allowed for high throughput (many fish tested at once) and the high temporal and spatial resolution of the video recordings allowed quantitative analyses of body turns and other kinematic measurements. The light dimming assay involved 5mm white LEDs that illuminated from below, and turned off for 500ms. Findings indicated that this stimulus resulted in large slow turns, termed O-turns by the authors, for the entirety of the dimming, and would cease when the light turned back on. This behavior was distinct from the motor patterns that were observed during other visual stimuli, suggesting a distinct maneuver. The design limited the directions the fish could swim to the lateral plain. This restriction possibly prohibited a vertical component of the O-turn that may or may not be

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 10 involved. This discovery warrants further investigation into the neural circuits used in the performance of this distinct behavior.

When observing zebrafish from the side of a tank that allows vertical movement, a dive response has been observed in zebrafish after light dimming stimuli. This behavior has been observed in larval zebrafish 7 dpf (Fernandes et al., 2012). The researchers compared this dive response of control zebrafish to those inoculated (eyes removed) and with their pineal ablated.

Eyes and pineal are photosensitive tissue known to process visual information, and Fernandes et al. (2012) ran an experiment to discover if zebrafish are able to respond to light dimming stimuli if both tissues were disabled. When comparing control zebrafish to enucleated fish with ablated pineal photoreceptors, no change to the dive was discovered. This suggests that the presence of photosensitive tissue exists beyond the eyes and pineal that mediate this response. Their apparatus used to measure vertical movement consisted of a tank filled with 70 ml of E3 medium at a depth of 30 millimeters. Five larva were placed in the tank and allowed to acclimate for 3 minutes. The design consisted of 10 cycles of 60 seconds with the lights on and 60 seconds with the lights off. Having the light source above or below the tank resulted in a similar dive response from the larva, which the authors argue suggests this response is not a form of phototaxis. With the focus being only on the effects of removing the eyes and pineal, no further focus was put into the dive response. This research shows a vertical component is present in a light dimming- evoked behavior. Therefore, studies that only analyze lateral movement could not detect neural mechanisms for these vertical behaviors.

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Behaviors Associated with the nMLF

Figure 1 shows a schematic of the MeM and MeL neurons within the nMLF. Lateral neurons of the nMLF have been associated with visually stimulated behavior. Gahtan, Tanger, and Baier (2005), through ablation studies, discovered that ablating MeL neurons had a similar effect on visually-evoked swimming as ablating the entire tectum, which is the primary destination for RGC axons in the brain. This result suggests that tectum and MeL neurons are part of the same visual-motor pathway. More specifically, since the tectum is the first stage of visual processing in the brain, and since MeL dendrites project into the tectum, this finding suggests that MeL neurons receive input from the tectum and transform it into appropriate output signals in to spinal motor control circuits. One visual-motor assay used in this study was depended on the ability of larvae to use vision to capture prey (swimming paramecia) and analyses focused on whether different ablations disrupted the visual recognition or motor control components of the task. By comparing bilateral MeL ablated, bilateral tectum-ablated, and control fish, they found that the ability to control orientation toward prey was reduced in both

MeL-ablated and tectum ablated fish. Anatomical data suggests that both MeLr and MeLc possess projections into ipsilateral tectum regions. To test if both areas are in the same neural pathway, Gahtan, Tanger, and Baier (2005), conducted ipsilateral and contralateral ablations of the tectum and MeL. It was found that contralateral ablations (tectum on one side of the brain and MeL on the other side) greatly reduced prey capture success, while unilateral ablations showed no statistically significant difference than control groups. Also, bilateral ablations of the MeL have less of an effect on pray capture than contralateral ablation of MeL

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 12 and tectum. The researchers explain this as evidence that the tectum sends visual information to other premotor neurons in the brain, not just MeL, but that MeL plays an important role in the behavior.

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Figure 1. Schematic of a 6 dpf zebrafish larvae highlighting the axonal and dendritic projections of the MeL and MeM neurons of the nucleus of the medial longitudinal fasciculus (nMLF). The contralateral projection from the eye to the optical tectum (Opt Tect) is shown in light gray at the top of the image. There are approximately 300 brain neurons (150 per side) that descend into the spinal cord in 6dpf larvae, most contained within the hindbrain, but for clarity only the 2 descending neurons that will be targeted for ablation in this study are shown. The MeM neuron is shown on the right (black cell body) with dendrites that project contralaterally (to both sides) within the midbrain and axonal outputs that project controlaterally within the spinal cord. The MeL neuron is shown.

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The nMLF has been associated with more than visual orientating control in zebrafish.

Thiele, Donovan, and Baier (2014) implicated the nMLF as having a major role in the steering

behaviors as well. Utilizing two-photon calcium imaging to record neural activity, they observed

activation of MeL and MeS neurons within the nMLF during spontaneous swims, tail flips, and

complex motor sequences termed struggles that are frequently observed in head-immobilized

zebrafish. Activation of both left and right nMLF was observed during each behavior. The

researchers also studied these nMLF neurons using optogenetic stimulation. A light-sensitive ion channel was expressed in nMLF neurons allowing the neurons to be activated (caused to fire action potentials) when a light of the correct wavelength turned on over the brain (the larvae are transparent so the light reaches the neurons). Optogenetic stimulation of nMLF neurons on one side of the brain caused smooth ipsilateral tail deflections in the zebrafish larva with the amplitude of tail movements dependent on the intensity of light stimulation. To test behaviors resulting in optogenetic activation of medial regions, they repositioned the light to the midline, equal distant from the two lateral activation sites. No change in kinematics was observed during these trials. Activation of the entire nMLF region resulted in long uncoordinated bouts of swimming. These movements usually lasted for the entire light activation, resulting in swims that continue longer than spontaneous movement. These results suggest that simultaneous bilateral activation of nMLF neurons is not part of natural motor circuits.

Sankrithi and O’Malley (2010) found nMLF activation during C-bend escape responses caused by tactile stimulation, a response that is known to also be associated with Mauthner cell activation. This finding led them to investigate whether the Mauthner cell responds during light

dimming-evoked swims as well. They found no calcium response from the Mauthner cells, or

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 15 their homologs MiD2cm and MiD3cm. This result, shows that co-activation between the

Mauthner cells and nMLF exists for some stimulated behavior but not all.

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Statement of the Problem

While much has been learned about the ethology and neural mechanisms of zebrafish escape responses, most research has analyzed only lateral movements and has tested behavior in settings that prohibit vertical movement. This limits our understanding of the complete behavior, and the neural circuit elements that control these behaviors may be missed or misinterpreted.

This current experiment’s apparatus utilized two cameras situated 90 degrees from each other on separate sides of a 10 cm cubed tank. This allows for the observation of behavior in the x, y, and z plane, allowing for a more accurate representation of the behavior than has been previously reported. In addition, to learn more about the development of visual-motor responses, larvae were tested at ages 6, 8, 10, and 12 days post fertilization, ages at which larvae are often used in experimental analyses.

Hypotheses

Hypothesis 1. Both tap and light dim sensory stimuli will trigger an escape response. This will be represented by an increase in total distance traveled post-stimulus versus pre-stimulus.

Rational for Hypothesis 1. This hypothesis is based on previous demonstrations of distinct sensory-evoked swimming patterns in zebrafish (Spulber et al., 2014)

Hypothesis 2a. Auditory tap-evoked swimming will have a higher frequency of swim bouts but smaller amplitude tail oscillations compared to dim-evoked swimming, but total distance traveled 60 seconds post stimulus will be equivalent for tap and dim evoked swims.

Hypothesis 2b. Vertical distance traveled will be greater following light dimming-evoked swims compared to tap-evoked swims.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 17

Hypothesis 2b. Light dimming-evoked swims will be characterized by negative vertical displacement and horizontal movement.

Rational for Hypothesis 2. This hypothesis is based on previous descriptions of auditory and visually-evoked swimming responses in zebrafish larvae (Spulber et al., 2014), including the observation of distinct O-turns during light dimming-evoked swims (Burgess and Granato,

2007). The O-turn is observed when the response is viewed from above, and a dive is observed when viewing from the side. This allows for the inference that the fish will perform a downward dive with long slow turns throughout when allotted equal space in all directions.

Hypothesis 3. As age increases, the difference between spontaneous swimming activity and sensory-evoked activity will decrease.

Rational for Hypothesis 3. This hypothesis is based on previous observations that spontaneous swimming activity increases as zebrafish larvae develop (Spulber et al., 2014).

Summary of Research Purpose

With the equal availability of movement in the x, y, and z plane a more accurate representation of light dimming and auditory tap responses will be observed. Zebrafish have become an important model species in behavioral neuroscience research, highlighting the importance of accurately measuring behaviors and accounting for factors such as stimulation type, physical environment, and age that may modify responses.

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Method

Zebrafish embryos were obtained from mating crosses of wildtype adults maintained as

brood stock. Adults were maintained, and larvae raised, under standard laboratory conditions

(Westerfield, 2000). Larvae were raised in 60mm petri dishes (maximum 40 larvae per dish)

containing approximately 20ml of egg water at a depth of ~5mm. Larvae were fed powdered

zebrafish food twice daily beginning on day 5 post fertilization, and were tested individually

during the light phase of the light-dark cycle between ages 6 and 12 dpf.

Larvae were transferred to the behavioral recording tank using a transfer pipette. Larvae were tested individually (N=12). The goal was to test 4 larvae at each age, 6, 8, 10, and 12 dpf, although at ages 10 and 12 dpf, only 2 larvae were tested. The tank was a 10cm acrylic cube with white translucent plastic (to diffuse stimulus lights and enhance the fish’s contrast against the background) on 2 adjacent walls and on the top and bottom, as shown in Figure 2. Two other adjacent walls of the tank were left clear to allow for video imaging from cameras (Pixelink PL-

B 741, each with a Computer 12mm, F1.4 lens fitted with a visible light blocking, infrared-pass filter) positioned 30cm away, facing each wall. The entire apparatus was contained within a sound- and light-attenuating cabinet and illuminated with diffused infrared light to allow for video imaging in the dark.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 19

Figure 2. Photograph of behavior recording apparatus. Translucent plastic top containing tapping mechanism excluded from photograph to allow image of tank.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 20

Escapes were elicited either visually, by sudden dimming of ambient light, or by a non-

directional auditory-vibrational ‘tap’ produced by a mechanical motor on the top of the tank.

Taps were given once per second for the 60-second trial. The stimulus light was an array of 20,

white light LEDs arranged in a 4x5 grid (2.5x3 cm), pointed toward the recording tank from a

distance of 30cm directly above the tank.

A custom computer program written in DaqFactory controlled stimulus presentation and image acquisition, and a LabJack U3 was used for computer-hardware interface. Four trial

blocks were run, each consisting of a top dim and tap trial with 30 minutes between trials (8

trials per fish). Cameras recorded swimming movements at 1Hz frame rate starting 60 seconds

before the stimulus (dimming or taps) and for 60 seconds after. The visible light remained on

during taps.

Data Analysis

Zebrafish movement was quantified using ImageJ (NIH). The average background of the

4 trial block was subtracted from each frame to separate the zebrafish movement from the

background. The XY coordinates corresponding to the larvae’s position were manually extracted

for each video frame from each camera using the point tool in ImageJ. Coordinate pairs from the

2 cameras were then combined in Matlab to generate 3-dimensional plots of the swimming path, and in Microsoft Excel to calculate total distance, vertical distance, horizontal distance, and vertical displacement.

All statistical analyses were performed using SPSS (version 22.0.0.0). A two by two

Repeated Measures ANOVA was used to analyze total distance, vertical distance, horizontal distance, and vertical displacement. Effects of two independent variables were examined:

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 21

Stimulus type (2 levels, within subjects: auditory tap or light dimming) and Time (2 levels, within subjects: total pre-stimulus swimming, total post-stimulus swimming. A paired samples t- test was used to analyze the horizontal and vertical components of the dimming-evoked dive response. A mixed model ANOVA was used to analyze age’s effect on total distance traveled after each stimulus. Behaviors were analyzed in three separate time bins, the total 60 seconds, the first 10 seconds, and the last 10 seconds of stimulation. All stimulated behavior was analyzed with an equal amount of spontaneous swimming time. To achieve a power of 0.8 or above, a total sample size of 10 fish was needed.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 22

Results

Total Distance Traveled

There was no main effect of stimulation time on total swimming distance, meaning that the distance traveled during 60 seconds of stimulation was equivalent to that during the 60 seconds of pre-stimulus spontaneous swimming, F(1, 11) = 3.137, p = .104, partial η2 = .222, as shown in Table 1. There was also no effect of stimulus type (dim or tap) on total swimming distance traveled, F(1, 11) = 0.579, p = .463, partial η2 = .050, and no interaction between stimulus time and stimulus type, F(1, 11) = 0.045, p = .836, partial η2 = .004.

To examine possible change in swimming activity across the 60 second period of stimulation I performed an additional analysis with the variable stimulus time further divided into 3 levels: pre-stimulus distance, distance during the first 10 seconds of stimulation, and distance during the last 10 seconds of stimulation. The total distance traveled during the first 10 seconds of stimulation was significantly greater than spontaneous swimming, F(1, 11) = 32.59, p

< .001, partial η2 = .748, but there was no effect of stimulus type, F(1, 11) = 0.677, p =.428, partial η2 = .058, and no time by stimulus type interaction, F(1, 11) = 0.236, p =.637, partial η2 =

.021. The total distance traveled during the last 10 seconds of stimulation was significantly lower than spontaneous swimming, F(1, 11) = 12.540, p = .005, partial η2 = .533, with no difference between stimulus type, F(1, 11) = 2.60, p = .135, partial η2 = .191, and no interaction, F(1, 11) =

1.09, p =.318, partial η2 = .090. In summary, total swimming distance showed an initial increase and a later decrease below baseline after stimulation for both auditory and visual startle stimuli.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 23

Table 1

Stimulus effects on total swimming distance. Means and standard deviations are shown for the difference in total distance travelled (in mm) during spontaneous swimming and periods of stimulation for each stimulus and time bin.

Pre-Dim Post-Dim Dim Effect Pre-Tap Post-Tap Tap Effect

60 Seconds 331.3 (76.1) 301.7 (60.3) -29.7 (93.9) 341.2 (86.8) 317.7 (82.5) -23.5 (40.6)

First 10 Seconds 55.2 (12.7) 71.1 (17.6) 15.8 (19.8)* 56.9 (14.5) 76.2 (22.1) 19.3 (12.2)**

Last 10 Seconds 55.2 (12.7) 42.4 (10.6) -12.8 (15.7)* 56.9 (14.5) 48.8 (14.1) -8.1 (9.2)* Note. *p < .05, **p < .01. Univariate unadjusted statistics were used for all analyses.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 24

Figure 3. Difference in total distance traveled between spontaneous and post-stimulus. Means swimming distance is shown in 10 second time bins. Error bars represent standard error. An initial increase of movement during the first 10 seconds of stimulation is followed by a decrease in movement below that of spontaneous swimming.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 25

Total Vertical Distance Traveled

When analyzing only cumulative vertical distance traveled, there was no main effect of

stimulus time (60 seconds pre-stimulus versus 60 seconds post-stimulus), F(1,11) = 2.00, p

=.185, partial η2 = .154, but there was a main effect of stimulus type, F(1,11) = 14.99, p =.003,

partial η2 = .577, with interaction between stimulus time by stimulus type, F(1,11) = 15.54, p

=.002, partial η2 = .585, as shown in Table 2, showing that tap and dim stimuli affected vertical swimming differently. Vertical distance during 60 seconds of dim stimulation was greater than that during spontaneous swimming, F(1,11) = 16.98, p =.002, partial η2 = .607, while the vertical

distance during tap stimulation was not significantly different from that during spontaneous

swimming, F(1,11) = 3.01, p =.111, partial η2 = .215.

The main effect of total vertical distance traveled during the first 10 seconds of

stimulation was greater than that of spontaneous swimming, F(1,11) = 55.89, p < .001, partial

η2 = .836. The responses to both stimuli included vertical movement, F(1,11) = 3.01, p =.111,

partial η2 = .215, with an interaction between the two variables, F(1,11) = 9.33, p =.011, partial

η2 = .459. More vertical distance was traveled during the first 10 seconds of light dim, F(1,11) =

46.10, p <.001, partial η2 = .807, than tap, F(1,11) = 32.55, p <.001, partial η2 = .757. The main

effect of total vertical distance traveled during the last 10 seconds of stimulation was greater than

that of spontaneous swimming, F(1,11) = 8.07, p =.016, partial η2 = .423. There was a significant

difference in total vertical distance traveled between the two stimulus types, F(1,11) = 17.07, p

=.016, partial η2 = .608, and an interaction between the two variables was found F(1,11) = 24.49,

p < .001, partial η2 = .690. The dimming stimulus maintained vertical movement in the last ten

seconds F(1,11) = 40.30, p < .001, partial η2 = .786, while tap stimulus did not, F(1,11) = 1.124,

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 26 p =.312, partial η2 = .093. In summary, cumulative vertical distance showed an initial increase after stimulation for both auditory and visual startle stimuli. This increase continued throughout the 60 seconds of visual startle stimuli but returned to baseline after the initial increase for auditory startle stimuli.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 27

Table 2

Stimulus effects on vertical swimming distance. Means and standard deviations are shown for the difference in vertical distance travelled (in mm) during spontaneous swimming and periods of stimulation for each stimulus and time bin.

Pre-Dim Post-Dim Dim Effect Pre-Tap Post-Tap Tap Effect

60 Seconds 78.2 (37.6) 117.8 (30.1) 39.6 (33.3)* 88.6 (48.8) 69.4 (22.5) -19.2 (38.4)

First 10 Seconds 8.8 (5.5) 25.4 (9.1) 16.6 (8.4)** 11.3 (7.3) 20.8 (7.2) 9.5 (5.8)**

Last 10 Seconds 8.8 (5.5) 18.5 (7.2) 9.7 (15.7)** 11.3 (7.3) 9.2 (4.7) -2.1 (7.0) Note. *p < .05, **p < .01. Univariate unadjusted statistics were used for all analyses

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 28

Figure 4. Difference in vertical distance traveled between spontaneous and post-stimulus. Means distance is shown in 10 second time bins. Error bars represent standard error.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 29

Total Horizontal Distance Traveled

When analyzing only cumulative horizontal distance traveled, a main effect was found

for stimulus time, F(1,11) = 6.24, p =.030, partial η2 = .362, as shown in Table 3, indicating that horizontal swimming decreased during the stimulation period. There was no main effect of stimulus type, F(1,11) = 1.40, p =.262, partial η2 = .113, and no interaction between the two

variables, F(1,11) = 1.16, p =.305, partial η2 = .095. While horizontal swimming decreased

overall during the entire 60 second stimulation period, it was significantly greater than

spontaneous swimming during the first 10 seconds of stimulation, F(1,11) = 22.91, p =.001,

partial η2 = .676. There was no main effect of stimulus type F(1,11) = 0.758, p =.402, partial η2 =

.064, and interaction effects, F(1,11) = 0.580, p =.462, partial η2 = .050, when examining the

first 10 seconds of stimulation. Horizontal distance during the last 10 seconds of stimulation was

significantly lower than baseline, F(1,11) = 20.32, p =.001, partial η2 = .649, with a main effect

of stimulus type, F(1,11) = 6.27, p =.029, partial η2 = .363, and an interaction between time and

stimulus type, F(1,11) = 6.06, p =.032, partial η2 = .355, reflecting the fact that the decrease in

horizontal swimming during the last 10 seconds of tap stimulation F(1,11) = 11.74, p =.006,

partial η2 = .516, was less than the decrease in horizontal swimming during the last 10 seconds

dimming, F(1,11) = 15.67, p =.002, partial η2 = .588. In summary, cumulative horizontal distance

showed an initial increase and a later decrease below baseline after stimulation for both auditory

and visual startle stimuli, with a greater decrease for visual startle stimuli.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 30

Table 3

Stimulus effects on horizontal swimming distance. Means and standard deviations are shown for the difference in horizontal distance travelled (in mm) during spontaneous swimming and periods of stimulation for each stimulus and time bin

Pre-Dim Post-Dim Dim Effect Pre-Tap Post-Tap Tap Effect

60 Seconds 319.4 (70.8) 267.1 (57.4) -52.4 (95.3)* 324.8 (77.7) 302.8 (78.9) -22.0 (31.9)*

First 10 Seconds 53.2 (11.8) 64.3 (16.3) 11.1 (19.8)* 54.1 (13.0) 70.9 (21.1) 16.7 (12.0)*

Last 10 Seconds 53.2 (11.8) 35.4 (8.8) -17.8 (15.6)* 54.1 (13.0) 46.9 (13.3) -7.2 (7.3)* Note. *p < .05, **p < .01. Univariate unadjusted statistics were used for all analyses.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 31

Figure 5. Difference in horizontal distance traveled between spontaneous and post-stimulus. Means distance is shown in 10 second time bins. Error bars represent standard error. Horizontal distance decreased overall during the 60 seconds of stimulation, with an initial increase followed by a continued decrease bellow spontaneous swimming levels. Horrizontal distance traveled decreased more during the last 10 seconds of dim stimulation compared to taps.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 32

Vertical Displacement

The analysis vertical displacement took into consideration the direction of vertical

movement by summing negative distance scores for downward movements and positive distance

scores for upward movements, whereas the previous analysis of cumulative vertical distance

summed the absolute values of all vertical movements. There was a significant main effect of

stimulus time on vertical displacement, F(1,11) = 31.54, p <.001, partial η2 = .741, as shown in

Table 4, reflecting greater negative vertical displacement during stimulation. There was a main

effect of stimulus type, with the light dimming stimulus producing more negative vertical

displacement than the auditory stimulus, F(1,11) = 10.09, p =.009, partial η2 = .479. There was

also a stimulus time by stimulus type interaction, F(1,11) = 20.53, p =.001, partial η2 = .651, as light dimming resulted in more negative displacement than tap, F(1,11) = 32.73, p <.001, partial

η2 = .748, even though the tap stimulus also resulted in significant negative displacement

compared to spontaneous swimming, F(1,11) = 5.30, p =.042, partial η2 = .325.

Negative vertical displacement was found to be significantly greater during the first 10

seconds of the stimulation period than spontaneous swimming, F(1,11) = 49.22, p < .001, partial

η2 = .817. The amount of negative displacement during the first 10 seconds was not significantly

different between the two stimuli, F(1,11) = 3.01, p = .109, partial η2 = .217, but there was an

interaction between stimulus type and stimulus time, F(1,11) = 5.24, p = .043, partial η2 = .323.

The simple effects show the dim stimulus triggered a larger increase of negative vertical displacement, F(1,11) = 33.20, p < .001, partial η2 = .751, than the tap stimulus, F(1,11) =

19.007, p = .001, partial η2 = .633. The vertical displacement during the last 10 seconds of the

stimulus was similar to that of spontaneous swimming, F(1,11) = 0.552, p = .473, partial η2 =

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 33

.048 (Table 4). This is true for both stimuli types, F(1,11) = 0.201, p = .663, partial η2 = .018, with no interaction between the two variables, F(1,11) = 0.163 p = .694, partial η2 = .015.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 34

Table 4

Stimulus effects on vertical displacement. Means and standard deviations are shown for the difference in vertical displacement (in mm) during spontaneous swimming and periods of stimulation for each stimulus and time bin

Pre-Dim Post-Dim Dim Effect Pre-Tap Post-Tap Tap Effect

60 Seconds 1.8 (12.2) -70.4 (41.5) -72.1 (43.7)** -7.1 (9.8) -21.8 (19.1) -14.7 (22.2)*

First 10 Seconds .29 (2.0) -21.1 (12.2) -21.4 (12.9)** -1.2 (1.6) -12.5 (9.2) -11.3 (9.0)*

Last 10 Seconds .29 ± 2.0 1.6 (14.5) 1.3 (14.5) -1.2 (1.6) 1.5 (5.5) 2.7 (6.2) Note. *p < .05, **p < .01. Univariate unadjusted statistics were used for all analyses.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 35

Figure 6. Difference in vertical displacement between spontaneous and post-stimulus. Means distance is shown in 10 second time bins. Error bars represent standard deviation. Both stimuli produced greater negative vertical displacement during the 60 seconds of stimulation, with dimming producing significantly greater displacement. This greater vertical displacement continues during the dimming stimulus up until the last 10 seconds of stimulation, while the tap response returns to baseline more quickly.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 36

Horizontal and Vertical Component of Dimming-evoked Dive Response

Stimulus effect was calculated by subtracting distance traveled for each direction by the

average distance traveled during spontaneous swimming. During the 60 seconds of light

dimming, there was significantly greater vertical distance traveled (M=39.59, SD=33.28) than

horizontal distance (M = -52.36, SD = 95.25), t(11) = 3.70, p = .003, d = 1.07 The dive consisted

of equal vertical (M = 16.56, SD = 8.45) and horizontal (M = 11.07, SD = 19.80) movement

during the first 10 seconds of stimulation, t(11) = 1.086, p = .301, d = 0.313. During the last 10

seconds of stimulation vertical movement was still greater than during spontaneous swimming

(M = 9.70, SD = 5.29), while horizontal movement fell below that during spontaneous swimming

(M = -17.85, SD = 15.62), t(11) = 6.23, p < .001, d = 1.80.

Effect of Age on Total Distance Traveled

There was no significant effect of age on total distance traveled during the 60 seconds of

stimulation, F(3,8) = 2.56, p = .128, partial η2 = .490, and no significant interaction between age

and stimulus type, F(3,8) = 1.54 p = 279, partial η2 = .365. No effect of age was found on total

distance traveled during the first 10 seconds, F(3,8) = 1.07, p = .416, partial η2 = .286, with no interaction between age and stimulus type, F(3,8) = 2.79, p = .109, partial η2 = .511. No effect of

age was found on total distance traveled during the first 10 seconds, F(3,8) = 2.56, p = .128,

partial η2 = .490, with no interaction between age and stimulus type, F(3,8) = 1.15, p = .387,

partial η2 = .301.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 37

Discussion

The main goal of this study was to characterize three dimensional swimming paths of

zebrafish larvae as they responded to escape-eliciting stimuli. Many previous studies have analyzed sensory-evoked escapes in zebrafish, primarily to reveal neural mechanisms underlying

those behaviors, but most have tested larvae in tanks that prohibit vertical swimming. The

current results show that swimming responses to both light dimming and auditory tap stimuli

include a significant negative vertical component, i.e., dives.

Zebrafish Larvae Escape Swims Include a Negative Vertical Movement

Both tap and dim stimuli evoked dives within the first 10 seconds of stimulation, as

shown in Figure 4. This is important because most studies of zebrafish escape responses analyze

early components of the behavior during which these dives would be occurring. However, while

initial tap and dim responses were similar, they became markedly different at later post-stimulus

time points. Most strikingly, dives were sustained throughout the dim response but were transient

during taps, and total vertical displacement across the post-stimulus period was much greater

(more negative) during dim responses, as shown in Figure 6. Overall swimming distance, however, was equivalent between the two stimulus conditions, as shown in Figure 3. This is consistent with the previous finding that auditory/vibrational stimuli evoke swims with high frequency and low amplitude tail beats while light stimuli evoke swims with lower frequency and higher amplitude tail beats, predicting no overall difference in speed (Spulber et al., 2014).

My results extend this previous report in showing that swimming direction is also affected by stimulus type (dimming produced more negative displacement). This finding also highlights the

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 38 importance of 3-dimensional motion tracking by showing that the distribution of movement can change in a way not detectible when analyzing total movement.

Biphasic Activity Response to Startle Stimuli

An unexpected finding was that swimming speed increased initially and then decreased below the spontaneous activity baseline for both stimulus types. To my knowledge, this biphasic response to startle stimuli has not been described previously in zebrafish larvae. The early increase in swimming activity agrees with previous studies of escape behavior in zebrafish, many of which focus on the initial, high-velocity turns and swims that occur within the first 100ms of a startle stimulus (Spulber et al., 2014). The later decrease in swimming activity observed may reflect a form of reflexive freezing, a common anti-predator strategy that can be combined in sequence with fleeing in some cases (Eilam, 2005). Further research should be conducted to determine potential ethological rational for this behaviors in zebrafish.

Ethological Interpretations of Vertical Escape Swimming

The continuous dive performed by the zebrafish, while being exposed to a dimming stimulus, shows significant horizontal movement, as shown in Table 3 and Figure 5. During the first 10 seconds of the response, an equivalent amount of horizontal and vertical distance was traveled. However, as the response continued horizontal movement lessened and vertical movement continued. A possible explanation for this initial horizontal movement may be an evasion tactic against aerial predation. Zebrafish mainly reside in slow moving shallow streams and still pools, where the water is typically clear (Parichy, 2015). Zebrafish tend to remain towards the surface of the water where their prey (larval and adult insects) is most visible due to light penetration (Colwill & Creton, 2011). Known aerial predators of zebrafish include

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 39

kingfisher and heron (Parichy, 2015). At the onset of sudden light dimming, which may indicate

an aerial predator overhead, a quick dive with horizontal components is likely to be a successful

technique for evasion (Domenici et al., 2011). Previous research shows an o-turn behavior during

this stimulus when the fish is observed from above (Burgess & Granato, 2007). The vertical dive

with horizontal movement found in this current experiment suggests that this o-turn may be part

of a corkscrew-like dive response.

Age Effect on Total Distance Traveled

The intended sample size in each age category was not obtained. During data collection,

the apparatus was needed for another experiment within the lab. Due to time constraints for both

projects, an initial analysis was done on the 12 data files already recorded, and a decision was

made to stop data collection. The small effect of time on distance traveled lead to the conclusion

that significance was not going to be found with the additional 4 fish. Neither spontaneous

movement nor stimulated movement was effected by the age of the larvae. Previous research

suggests that spontaneous movement increases with age (Spulber et al., 2014), however this data

suggests that the increased movement does not take place between ages 6 and 12 dpf.

Limitations

Several methodological and theoretical limitations of this study should be considered.

Whole-field diming is not a natural stimulus: The dimming stimulus caused a complete elimination of light around the testing apparatus. This was done to produce the strongest possible response to characterize the stimulus effect. However, the instantaneous and complete dimming

of the entire whole-visual-field that larvae experienced in this study would not occur in natural

environments so it is possible the responses observed are not representative of responses to light

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 40

dimming in nature. It would be interesting to examine this ethological question in future studies

that used a more ecologically relevant stimulus, such as a looming shadow (Temizer et al.,

2015). But the current results are still relevant to zebrafish research studies using taps and whole-

field brightness stimuli and suggest that vertical swimming should be measured. The 60 sec

duration of sensory stimulation is also unlikely to parallel natural stimuli but this duration was

chosen to promote reliable responding and to give enough time for potential differences in tap

and dim responses to be observed We did not control the starting location of the larvae so our

apparatus did not guarantee the larvae had equal range of motion in vertical and horizontal axes.

Our apparatus still limited the vertical range of motion and larvae frequently reached the bottom

of the tank after dim stimuli

Conclusion

These results signify the differences in escape responses within the zebrafish repertoire,

and the involvement of a vertical component in escapes. To properly investigate a neural

circuit’s involvement with the response, an accurate assay representing the natural behavior is

critical (Tinbergen, 1951). The lack of investigation into the vertical component of these commonly studied escape responses may bias the neuroscience research being done on these behaviors. When investigating neuronal involvement in escape behaviors, such as that of the

nMLF and Mauthner neurons (presented in detail in the Introduction), zebrafish are commonly

observed within vertically restricting containers such as petri dishes. This suggests an accurate

representation of the behavior was not previously observed, and further research into the

involvement of previously categorized neural circuits in the vertical component is needed.

VISUAL AND AUDITORY-EVOKED ESCAPE SWIMS 41

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