The influence of exercise on anxiety-like behavior in zebrafish (Danio rerio)

Accepted Manuscript Title: The influence of exercise on anxiety-like behavior in zebrafish (Danio rerio) Authors: C. DePasquale, J. Leri PII: DOI: Reference:

S0376-6357(17)30543-0 https://doi.org/10.1016/j.beproc.2018.04.006 BEPROC 3649

To appear in:

Behavioural Processes

Received date: Revised date: Accepted date:

10-11-2017 12-2-2018 9-4-2018

Please cite this article as: DePasquale C, Leri J, The influence of exercise on anxiety-like behavior in zebrafish (Danio rerio), Behavioural Processes (2010), https://doi.org/10.1016/j.beproc.2018.04.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The influence of exercise on anxiety-like behavior in zebrafish (Danio rerio)

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C. DePasqualea*, J. Leria

Department of Biology, Pennsylvania State University – Altoona, Altoona, PA, USA

* Corresponding author: C. DePasquale (+1 814-949-5287, [email protected], 207

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Hawthorn Building, Penn State-Altoona, Altoona, PA 16601)

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HIGHLIGHTS

Zebrafish from exercise and control treatment groups were compared for anxiety

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Two different tests of anxiety were used; the novel tank test and the light-dark test

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Exercised fish exhibited reduced anxiety-like behaviors

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Exercised fish spent more time in the top and were quicker to enter the top of the

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novel tank test

Exercised fish spent more time in the light compartment of the light-dark test

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compared to control fish

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Abstract

In non-human mammals, exercise has been shown to decrease anxiety-like behavior. Conversely, a number of studies have reported no effect or even an increase in anxiety-

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like behavior after exercise, however, inconsistent training regimes and behavioral paradigms across studies may be confounding the results. Zebrafish (Danio rerio) are a well-established animal model in neurobehavioral research, and have the potential to shed

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new insight into the effects of exercise on anxiety-like behavior where previous research has been limited, due to the ability to precisely control intensity and duration of exercise,

and the validation of tests for measuring different aspects of anxiety-like behaviors. In the current study, fish were split between two treatment groups; Exercised and Control. Fish in the exercised condition were aerobically challenged (max water velocity: 0.5 m/s)

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using a swim tunnel one hour a day, five days a week, for six weeks. Control fish spent

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an equal amount of time in the swim tunnel but were not aerobically challenged (max

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water velocity: 0.05 m/s). After six weeks, all fish were tested individually in two

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standard complimentary anxiety tests for zebrafish: the novel tank test and the light-dark test. Exercised fish exhibited reduced anxiety-like behaviors in the novel tank test; they

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spent more time in the top and were quicker to enter the top of a novel tank compared to

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Control fish. In addition, Exercised fish spent more time in the light compartment of the

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light-dark test compared to Control fish. Our results demonstrate the beneficial effect of

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exercise on anxiety-like behavior in zebrafish.

Keywords: anxiety; physical exercise; light-dark test; novel tank diving test; zebrafish

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(Danio rerio)

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1. INTRODUCTION

In mammalian models of human physiology research, exercise has been shown to

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decrease anxiety-like behavior by mediating changes in the hypothalamic-pituitary-

adrenal (HPA) axis (Fulk et al., 2004; Patki et al., 2014; Lalanza et al., 2015). However,

a number of studies have also reported minimal effects or even an increase in anxiety-like behavior (Burghardt et al., 2004; Fuss et al., 2010; Lalanza et al., 2015). Different

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training regimes (voluntary wheel running versus forced treadmill running, duration and

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intensity of exercise training) and behavioral paradigms (different tests of anxiety, such

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as the open field test, elevated plus maze or light-dark test) may play a part in the

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inconsistent results across studies. For example, many rodent studies that have looked at the effects of exercise on anxiety-like behavior have focused on the effects of voluntary

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wheel-running. Although the voluntary exercise paradigm has many benefits, including a

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reduction in human-subject interactions, there are a number of drawbacks, especially concerning the consistency of wheel-running across individuals (Otsuka et al., 2016).

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Fish represent a novel alternative for studying the effects of exercise on the brain

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and behavior because exercise intensity and frequency can be tightly controlled. It has been suggested that the neural mechanisms underlying the effects of exercise on

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cognition and stress resistance are conserved among vertebrates, including fish (Nicastro and Greenwood, 2016). In particular, the neuronal pathways of the serotonergic system (which control the HPA axis) seem to show some degree of evolutionary conservation, with many of the behavioral functions of serotonin being highly conserved, including anxiety behavior (Herculano and Maximino, 2014). Moreover, previous opinion has been

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that fish possess simple brains that could only undergo basic neuronal processes of primitive behavior (Dugatkin and Wilson, 1993). However, a recent surge of interest in fish cognition has provided growing evidence that the fish brain is more sophisticated

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than this, and the utility of this complexity in fish behavior is only now emerging (Bshary et al., 2002; Braithwaite, 2005; Stewart et al., 2012). Recently, exercise has been shown to have beneficial effects on learning in zebrafish, Danio rerio (Luchiari and Chacon, 2013), and has been shown to increase boldness, exploration and aggression in

mosquitofish, Gambusia holbrooki (Sinclair et al., 2014), however, the effects of exercise

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on anxiety-like behavior in fish have yet to be explored.

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Zebrafish are becoming a widely used animal model in neurobehavioral research

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due to their low cost, quick reproductive cycle, ease of genetic manipulation and the

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availability of well-developed behavioral paradigms. More recently, zebrafish have become an established model for looking at the effects of exercise on brain and behavior

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(Luchiari and Chacon, 2013; Gilbert et al., 2014). Traditionally, the swimming

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performance of fish has been measured using standard techniques for assessing aerobic

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swimming capacity (critical swimming velocity, Ucrit), and usually involves one to several stepped increases in flow speed that are intended to cause fish to fatigue (Brett,

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1964). Ucrit as a measure of aerobic capacity has been used extensively in studies on zebrafish (e.g. Plaut, 2000; Uliano et al., 2009; Palstra et al., 2010; Massé et al., 2013).

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Thus, values of Ucrit can be used to consistently and accurately determine different swimming regimes for exercising zebrafish. A number of paradigms have been developed as effective tests of anxiety-like behaviors in zebrafish, and are designed to elicit behaviors that parallel anxiety-like

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behavior in rodents (Stewart et al., 2012). However, it has been suggested that each test assesses different aspects of anxiety (Blaser and Rosemberg, 2012; Stewart et al., 2012). For example, the novel tank test which looks at the amount of time a fish spends at the

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bottom of an unfamiliar tank, has been suggested to parallel thigmotaxic behavior (wallhugging) in the open field test in rodents (Rosemberg et al., 2011; Blaser and Rosemberg, 2012; Stewart et al., 2012). However, the light-dark test which evaluates the amount of time the fish spends in either a white or black compartment of a test tank, has been

suggested to parallel scototaxic (light avoidance) behavior in rodents (Maximino et al.,

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2010; Stewart et al., 2012). Both of these tests have been used independently as measures

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of anxiety in zebrafish, but the validation of using one of these behavioral tests of anxiety

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itself (Blaser and Rosemberg, 2012).

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over the other is difficult since no single behavioral measure reliably reflects anxiety by

Therefore, the overarching goal of the current study was to determine the effects

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of exercise on anxiety by using a combination of tests that effectively evaluate different

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aspects of anxiety-like behaviors in zebrafish. We hypothesized that exercised fish would

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show reduced anxiety-like behavior exhibited by increased time in the light compartment of the light-dark test and decreased time at the bottom of the tank during the novel tank

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test.

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2. METHODS

2.1 Ethical Note

of The Pennsylvania State University; IACUC no. 44578.

2.2 Treatments

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This research complied with all requirements of the animal care and use protocols

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One year old offspring of wild-type zebrafish (n=52) were randomly distributed

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across 10 home tanks (35 x 19 x 28 cm) with equal numbers of each sex in each tank.

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Each home tank had a biofilter, heater and gravel substrate. Each tank of fish was

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randomly assigned to one of two treatment groups (5 tanks per treatment); Exercised or Control. The fish were maintained on a 12 L : 12 D cycle with a water temperature of 25

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± 1°C. Fish were transferred from their home tanks to a multi-channel swim tunnel (Fig.

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1) for one hour per day, five days a week for 6 weeks. In order for all replicate tanks to be trained every day, two groups of 5 tanks were established for the procedure; the groups

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alternated daily between an ‘Early’ (8:00- 9:00 am) and ‘Late’ (9:00-10:00 am) training

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session. Both groups contained Exercised and Control tanks whose channel assignment (1 through 5) was randomized each week. Fish were placed into assigned swim chambers

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after appropriate water flows had been established and measured. Following swim tunnel exposure fish were returned to home tanks and fed (commercial flake food and live brine shrimp). Exercised fish were aerobically challenged by swimming against a water velocity ranging from 0.05 to 0.5 m/s. A maximum water velocity of 0.5 m/s was determined

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based on previously reported Ucrit values for adult zebrafish (Palstra et al., 2010). Control fish were allowed to move freely in little to no water flow (water velocity range 0-0.05 m/s). Flow measurements (Global Water Instruments Flow Probe, Model: FP111) were

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taken at the front (Exercised: Mean = 0.37 m/s, S.E. = 0.01 m/s; Control: Mean = 0.02 m/s, S.E. = 0 m/s), middle (Exercised: Mean = 0.20 m/s, S.E. = 0.02 m/s; Control: Mean

= 0 m/s, S.E. = 0 m/s), and back of each swim channel (Exercised: Mean = 0.08 m/s, S.E. = 0 m/s; Control: Mean = 0 m/s, S.E. = 0 m/s) every day before fish were placed in the

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channels.

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2.3 Anxiety Assay 1: The Light-Dark Test

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On day 1 of week 7, all fish from each treatment were screened for anxiety

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behaviors in the light-dark test (Fig. 2a). The experimental tank was divided into three

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equal sized compartments (25 x 25 x 30 cm) using opaque acrylic and water depth was maintained at 20 cm. The walls and floor of the center compartment were covered in gray

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tape and represented a neutral space where fish could choose to enter either the light or dark compartments. The walls and floor of the dark compartment were covered with

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black tape. The walls and floor of the light compartment were covered in white tape with a 360-lumen light placed squarely above. Each compartment had a rectangular door (5 x

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7 cm) that allowed free access between compartments. To start a trial, individual fish were placed into the center compartment and given 10 minutes to move freely between the compartments. Data collection began as soon as a fish was placed in the water. A camera suspended from the ceiling recorded behavior. After each trial a third of the water

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was replaced and mixed. Videos were analyzed using Behavioral Observation Research Interactive Software, BORIS (Friard and Gamba, 2016). Variables measured included total time spent in each of the three compartments (dark, neutral, light), number of entries

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into the light and dark compartments, latency to enter the light compartment and total number of compartment changes.

2.4 Anxiety Assay 2: The Novel Tank Test

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On day two of week 7, all fish from each treatment were screened for anxiety

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behaviors in the novel tank test (Fig. 2b). The test tank (24 x 20 x 17 cm, water depth 14

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cm) was covered on three sides with black plastic. A camera facing the uncovered side of

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the tank recorded behavior. Each fish could explore the tank for 5 minutes. After each

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trial, a third of the water was exchanged with new sump water and mixed before a new

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fish was tested.

Videos were analyzed using Behavioral Observation Research Interactive

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Software, BORIS (Friard and Gamba, 2016). The test tank was visually divided into bottom, middle, and top zones using a 3 x 3 cm grid superimposed on the computer

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monitor. When first released, all fish swam to the bottom of the tank. Data collection began once a fish reached the bottom (approximately 5 seconds). Following DePasquale

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et al. (2016), variables measured included latency to enter top (s), ratio of entries into top/bottom, time spent in top (s), average top entry duration (s), freezing duration (s) and movement rate (number of grid lines crossed/min).

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2.5 Statistical Analysis

Data were analyzed using general linear models. Data were tested for equality of

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variance and transformed when necessary. When data could not be transformed, the nonparametric Kruskal-Wallis test was used. Light-dark variables were compared across treatments with exercise regime (exercised or control) as a fixed factor and tank as a

random factor. Diving variables were compared across treatments with exercise regime (exercised or control) as a fixed factor and tank as a random factor. Time spent in the

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bottom of the tank was compared across treatments, with minute of observation as a

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fixed-effect variable and individual fish as a random-effect variable. One fish from the

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Exercised treatment group died during training, and was thus excluded from the analyses.

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are quoted as mean ± s.e.m.

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Analyses were performed in SPSS (v. 21) and significance was tested at α = 0.05. Values

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3. RESULTS

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3.1 Anxiety Assay 1: The Light-Dark Test

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Exercised fish spent significantly more time in the light compartment (F1,41 =

19.66, P < 0.01; Fig. 3a) and significantly less time in the dark compartment (F1,41 = 14.32, P = 0.01; Fig. 3b) compared to Control fish. There was no difference between treatment groups in the amount of time spent in the central (neutral) compartment of the test tank (F1,41 = 0.01, P = 0.91; Fig. 3c). There was also no difference between

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treatments in the number of entries into the light compartment (F1,41 = 3.55, P = 0.07), number of entries into the dark compartment (F1,41 = 1.30, P = 0.31), latency to enter light compartment (F1,41 = 1.64, P = 0.25) or total number of compartment changes (F1,41

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= 0.04, P = 0.86).

3.2 Anxiety Assay 2: The Novel Tank Test

Across the full 5 minute trial, there was a trend toward Exercised fish spending

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less time in the bottom of the tank compared to Control fish (F1,48 = 3.59, P = 0.06; Fig.

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4). There was a significant minute x treatment effect; fish reared in the Control treatment

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spent more time in the bottom of the test tank in the first 3 minutes of the trial, showing

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increased signs of anxiety compared with fish that experienced exercise (F4,48 = 2.73, P =

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0.03; Fig. 4). There was no significant difference in time spent at the bottom of the tank

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across the 5 minute trial, regardless of treatment (F4,48 = 1.72, P = 0.15; Fig. 4). There was no overall difference in the rate of movement between treatments (F1,41 = 0.02, P =

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0.97; Fig. 5a), but fish that experienced exercise moved into the top quicker (F1,41 = 7.51, P = 0.04; Fig. 5b) and stayed at the top for longer (F1,41 = 15.95, P < 0.01; Fig. 5c).

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There was a significant tank effect on time to move to top; Exercised tank 2 moved slightly faster into the top on average than all other tanks (F1,41 = 6.59, P = 0.05). There

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was no difference between treatments in the number of entries into the top/bottom (F1,41 = 0.34, P = 0.59), average top entry duration (F1,41 = 1.47, P = 0.29), or freeze duration (χ2 = 0.17, P = 0.68).

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4. DISCUSSION

As predicted, zebrafish that were aerobically challenged exhibited reduced

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anxiety-like behavior in two different tests of anxiety compared to Control fish that were not subjected to aerobic exercise. Exercised fish moved into the top quicker and spent

more time in the top of the novel tank test overall compared to Control fish. In addition, Exercised fish spent increasingly less time in the bottom of the novel tank in the first three minutes of the trial compared to Control fish. In terms of the light-dark test,

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Exercised fish spent more time in the light compartment and less time in the dark

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compartment (with no difference in time spent in the neutral compartment) compared to

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Control fish. Taken together, the results of both behavioral tests suggest that aerobic

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exercise decreases anxiety-like behavior across multiple measures in zebrafish. The effects of physical exercise on anxiety-like behavior in fish has not been

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studied before, however, mosquitofish that were exercised (water velocity, 0.06 m/s for

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28 days continuously) showed an increase in boldness, exploration and aggression

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compared with control fish (Sinclair et al., 2014). Thus, even at low water velocities (similar to the Control conditions in the current study), changes in fish behavior were

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observed. If we consider that stress can drive changes in anxiety behavior (Greenwood and Fleshner, 2008), it is also interesting to note that Atlantic salmon (Salmo salar)

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exposed to exercise using a moving light stimulus set at 1.5 BL/s exhibited a decrease in resting plasma cortisol levels after 28 days (Herbert et al., 2011). Moreover, rainbow trout (Oncorhynchus mykiss) displayed a decrease in cortisol in response to an acute bout of forced exercise (5 minutes of chasing over 28 days) compared with control conspecifics (Hernandez et al., 2002), however, no measure of swim speed was reported. Page 11

Several studies have reported a beneficial effect of exercise on anxiety-like behavior in mammalian models of human research (Duman et al., 2008; Trejo et al., 2008; Dubreucq et al., 2015), however, some have reported minimal or no effect

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(Burghardt et al., 2004; Pietropaolo et al., 2006) and a number have even reported an increase in anxiety-like behavior (Burghardt et al., 2004; Leasure and Jones, 2008; Fuss

et al., 2010). It is difficult to compare results across studies and draw conclusions due to

the different exercise regimes and paradigms used to assess anxiety behavior. In terms of voluntary wheel-running, frequency, duration and intensity of exercise bouts can vary

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across individuals presenting difficulties when interpreting results (Otsuka et al., 2016).

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Where forced treadmill running has been used to control training intensity in rodents,

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inconsistent training paradigms across studies have made it difficult to make comparisons

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and draw conclusions (e.g. 5 days/week at 12 m/min for 40 mins over 3 weeks, Trejo et al., 2008; 5 days/week at 20 m/min at 5% grade for 45 mins over 8 weeks, Burghart et

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al., 2004). In terms of behavioral measures, it has been suggested that a combination of

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tests should be used to measure anxiety in rodents, since no one test alone reflects a full

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analysis of anxiety-related behavior (Green, 1991). However, many studies have focused on only one test of anxiety (novelty-suppressed feeding test: Trejo et al., 2008, elevated

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plus-maze: Costa et al., 2012) or different studies have used different combinations of tests (elevated plus maze and open field test: Burghardt et al., 2004; Pietropaolo et al.,

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2006; Duman et al., 2008, light-dark test and open field test: Fuss et al., 2010; Salim et al., 2010). Since so many studies have utilized different exercise regimes and behavioral paradigms, it is not surprising that the literature shows such divergent results.

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Similarly, it has been suggested that a combination of behavioral tests should be used to look at anxiety-like behavior in zebrafish (Blaser and Rosemberg, 2012). Thus, in the current study, two different behavioral tests were used to assess anxiety; the novel

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tank test (to measure thigmotaxic aspects of behavior) and the light-dark test (to measure scototaxic aspects of behavior). Exercised fish spent more time in the light compartment of the light-dark test and spent more time in the top of the novel tank test compared to

Control fish, showing that exercise can affect both thigmotaxic and scototaxic aspects of behavior in zebrafish. Interestingly, time in the bottom of the tank during the novel tank

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test was not significantly different at the start compared to the end of the 5 minute trial

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for either Exercised or Control fish. This result was contrary to our expectations,

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however, the increase in time spent at the bottom of the tank during minute 4 may be due

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to disturbances by the experimenter when preparing the next fish for trial. Moreover, fish from both treatment groups spent more time in the light side than the dark side during the

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light-dark test, indicating that Control fish did not exhibit substantial light-avoidance as

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we predicted. Facciol et al. (2017) recently suggested that inconsistent results in the light-

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dark test probably stems from the modification of three factors; illumination level, background shade and ‘openness’ (e.g. dark side covered with a lid versus uncovered).

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Facciol et al. (2017) found that zebrafish exhibited a preference for black over white backgrounds, but not illuminated over dark. Therefore, our results further highlight the

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importance of using multiple measures and standard techniques to assess anxiety behavior. Zebrafish are emerging as a candidate animal model for exercise physiology and behavior research because they provide many advantages over rodent animal models. For

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example, the use of swim tunnels to tightly control flow velocities and the use of Ucrit as a standard measure of aerobic exercise allows protocols to be standardized across studies. Moreover, zebrafish have a natural tendency to schoal, thus making it easier to use group

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exercise protocols. In rodent studies that have compared the effects of forced treadmill running (where exercise is tightly controlled) and voluntary wheel running on anxiety,

results have been confounding. Burghardt et al. (2004) found an increase in anxiety-like

behaviors in the open field test in mice that experienced voluntary exercise but not forced exercise (treadmill running). However, Leasure and Jones (2008) reported an increase in

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anxiety-like behavior in the open field test in rats that experienced forced exercise

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(motorized wheel) but not in voluntary wheel runners when compared to sedentary

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controls. Thus the effects of exercise on anxiety behavior are not always clear cut, and

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emphasizes the importance of using standard exercise regimes that can be compared across studies.

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The results of the current study show that exercise can reduce anxiety-like

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behavior in fish, and provides an exercise regime which utilizes standard Ucrit values that

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can be applied across different studies. The results also highlight the importance of using multiple tests to look at different components of anxiety behavior in order to fully

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understand the impact of exercise on anxiety-like behavior in zebrafish.

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Declarations of interest: none

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ACKNOWLEDGEMENTS

We would like to thank Peggy Hubley of the Jack Gittlen Cancer Research

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Foundation in the College of Medicine at Hershey for supplying the zebrafish embryos and Jennifer Sturgill for fish husbandry and maintenance. We would also like to thank Dr. James Chen (Kansas State University) and Vada Palochko for initial design and

construction of the swim tunnel. This work was supported by The Pennsylvania State

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University.

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REFERENCES

M

Blaser, R. E. & Rosemberg, D. B. 2012. Measures of Anxiety in Zebrafish (Danio rerio): Dissociation of Black/White Preference and Novel Tank Test. PLoS ONE. DOI:

TE

D

https://doi.org/10.1371/journal.pone.0036931

EP

Braithwaite, V. A. 2005. Cognitive ability in fish. Fish Physiology, 24, 1–37.

CC

Brett, J. R. 1964. The respiratory metabolism and swimming performance of young

A

sockeye salmon. Journal of Fisheries Research Board of Canada, 21, 1183-1226.

Bshary, R., Wickler, W. & Fricke, H. 2002. Fish cognition: A primate’s eye view. Animal Cognition, 5, 1-13.

Page 15

Burghardt, P. R., Fulk, L. J., Hand, G. A. & Wilson, M. A. 2004. The effects of chronic treadmill and wheel running on behavior in rats. Brain Research, 1019, 84–96.

SC RI PT

Costa, M. S., Ardais, A. P., Fioreze, G. T., Mioranzza, S., Botton, P. H. S., Portela, L. V., Souza, D. O. & Porciúncula, L. O. 2012. Treadmill running frequency on anxiety and hippocampal adenosine receptors density in adult and middle-aged rats. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 36, 198-204.

U

Dubreucq, S., Marsicano, G. & Chaouloff, F. 2015. Duration- and environment-

N

dependent effects of repeated voluntary exercise on anxiety and cued fear in mice.

M

A

Behavioural Brain Research, 282, 1-5.

Dugatkin, L. A. & Wilson, D. S. 1993. Points of view: Fish behavior, partner choice

TE

D

experiments and cognitive ethology. Reviews in Fish Biology and Fisheries, 3, 368-372.

EP

Duman, C. H., Schlesinger, L., Russell, D. S. & Duman, R. S. 2008. Voluntary exercise produces antidepressant and anxiolytic behavioral effects in mice. Brain

CC

Research, 1199, 148-158.

A

Facciol, A., Tran, S. & Gerlai, R. 2017. Re-examining the factors affecting choice in the light–dark preference test in zebrafish. Behavioural Brain Research, 327, 21-28.

Page 16

Friard, O. & Gamba, M. 2016. BORIS: a free, versatile open-source event-logging software for video/audio coding and live observations. Methods in Ecology and

SC RI PT

Evolution, 7: 1325–1330. doi:10.1111/2041-210X.12584.

Fulk, L. J., Stock, H. S., Lynn, A., Marshall, J., Wilson, M. A. & Hand, G. A. 2004.

Chronic physical exercise reduces anxiety-like behavior in rats. International Journal of Sports Medicine, 25(1), 78-82.

U

Fuss, J., Abdallah, N. M.-B. B., Vogt, M. A., Touma, C., Pacifici, P. G., Palme, R.,

N

Witzemann, V., Hellweg, R. & Gass, P. 2010. Voluntary Exercise Induces Anxiety-

A

Like Behavior in Adult C57BL/6J Mice Correlating With Hippocampal Neurogenesis.

M

HIPPOCAMPUS, 20, 364-376.

D

Gilbert, M. J. H., Zerulla, T. C. & Tierney, K. B. 2014. Zebrafish (Danio rerio) as a

TE

model for the study of aging and exercise: physical ability and trainability decrease with

EP

age. Experimental Gerontology, 50, 106-113.

CC

Green, S. 1991. Benzodiazepines, putative anxiolytics and animal models of anxiety.

A

Trends in Neurosciences, 14 (3), 101-104.

Greenwood, B. N. & Fleshner, M. 2008. Exercise, Learned Helplessness, and the Stress-Resistant Brain. NeuroMolecular Medicine, 10, 81–98.

Page 17

Herbert, N. A., Kadri, S. & Huntingford, F. A. 2011. A moving light stimulus elicits a sustained swimming response in farmed Atlantic salmon, Salmo salar L. Fish Physiology

SC RI PT

and Biochemistry, 37, 317-325.

Herculano, A. M. & Maximino, C. 2014. Serotonergic modulation of zebrafish

behavior: Towards a paradox. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 55, 50–66.

U

Lalanza, J. F., Sanchez-Roige, S., Cigarroa, I., Gagliano, H., Fuentes, S., Armario,

N

A., Capdevila L. & Escorihuela, R. M. 2015. Long-term moderate treadmill exercise

A

promotes stress-coping strategies in male and female rats. Nature Scientific Reports, 5:

M

DOi: 10.1038/srep16166

D

Leasure, J. L. & Jones, M. 2008. Forced and voluntary exercise differentially affect

EP

TE

brain and behavior. Neuroscience, 156, 456-465.

Luchiari, A. C. & Chacon, D. M. M. M. 2013. Physical exercise improves learning in

CC

zebrafish, Danio rerio. Behavioral Processes, 100, 44-47.

A

Massé, A. J., Thomas, J. K. & Janz, D. M. 2013. Reduced swim performance and aerobic capacity in adult zebrafish exposed to waterborne selenite. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 157 (3), 266-71.

Page 18

Maximino C., Marques de Brito, T., Dias, C. A. G. M., Gouveia, A., Morato, S. 2010. Scototaxis as anxiety-like behavior in fish. Nature Protocols, 5, 209–216.

SC RI PT

Nicastro, T. M. & Greenwood, B. N. 2016. Central monoaminergic systems are a site of convergence of signals conveying the experience of exercise to brain circuits involved in cognition and emotional behavior. Current Zoology, 62 (3), 293-306.

Otsuka, T., Nishii, A., Amemiya, S., Kubota, N., Nishijima, T. & Kita, I. 2016.

U

Effects of acute treadmill running at different intensities on activities of serotonin and

N

corticotropin-releasing factor neurons, and anxiety- and depressive-like behaviors in rats.

M

A

Behavioural Brain Research, 298, 44–51.

Palstra, A. P., Tudorache, C., Rovira, M., Brittijn, S. A., Burgerhout, E., van den

D

Thillart, G. E. E. J. M., Spaink, H. P. & Planas, J. V. 2010. Establishing Zebrafish as a

TE

Novel Exercise Model: Swimming Economy, Swimming-Enhanced Growth and Muscle

EP

Growth Marker Gene Expression. PLoS ONE, 5. DOI: 10.1371/journal.pone.0014483

CC

Patki, G., Li, L., Allam, F., Solanki, N., Dao, A. T., Alkadhi, K. & Salim, S. 2014. Moderate treadmill exercise rescues anxiety and depression-like behavior as well as

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memory impairment in a rat model of posttraumatic stress disorder. Physiology & Behavior, 130, 47-53.

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Pietropaolo, S., Feldon, J., Alleva, E., Cirulli, F. & Yee, B. K. 2006. The Role of Voluntary Exercise in Enriched Rearing: A Behavioral Analysis. Behavioral

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Neuroscience, 120 (4), 787-803.

Plaut, I. 2000. Effects of fin size on swimming performance, swimming behaviour and routine activity of zebrafish, Danio rerio. Journal of Experimental Biology, 203, 813820.

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Rosemberg, D. B., Rico, E. P., Mussulini, B. H. M., Piato, Â. L., Calcagnotto, M. E.,

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Bonan, C. D., Dias, R. D., Blaser, R. E., Souza, D. O. & de Oliveira, D. L. 2011.

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Differences in Spatio-Temporal Behavior of Zebrafish in the Open Tank Paradigm after a

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Short-Period Confinement into Dark and Bright Environments. PLoS ONE, 6 (5). DOI:

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http://doi.org/10.1371/journal.pone.0019397.

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Salim, S., Sarraj, N., Taneja, M., Saha, K., Tejada-Simon, M. V. & Chugh, G. 2010.

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Moderate treadmill exercise prevents oxidative stress-induced anxiety-like behavior in

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rats. Behavioural Brain Research, 208, 545–552.

Sinclair, E. L. E., Noronha de Souza, C. R., Ward, A. J., W. & Seebacher, F. 2014.

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Exercise changes behaviour. Functional Ecology, 28, 652–659.

Stewart, A., Gaikwad, S., Kyzar, E., Green, J., Roth, A. & Kalueff, A. V. 2012. Modeling anxiety using adult zebrafish: A conceptual review. Neuropharmacology, 62

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(1), 135–143.

Trejo, J., Llorens-Martin, M., & Torres-Aleman, I. 2008. The effects of exercise on

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spatial learning and anxiety-like behavior are mediated by an IGF-I dependent mechanism related to hippocampal neurogenesis. Molecular and Cellular Neuroscience, 37, 402-411.

Uliano, E., Cuccaro, F., Galbano, S. & Agnisola, C. 2009. Factors affecting critical

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swimming speed (Ucrit) in zebrafish: A preliminary study. Comparative Biochemistry and

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Physiology A: Molecular and Integrative Physiology, 154 (1), S25-S26.

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Figure Captions

Figure 1: Diagram of the multi-channel swim tunnel. The swim tunnel was

constructed of opaque acrylic with 5 channels. Each channel had a drain at one end and

received water from an inflow pipe at the other end (Lifegard Aquatics Customflo Water System). Both the inflow and drainage areas were blocked from fish access by a

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honeycomb plastic mesh that allowed for more laminar water flow, and thus making the

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area in which fish were held 35 cm in length. Water in each channel was maintained at a

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constant depth of 8 cm. Water was transported from a sump tank located below the swim

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channels through inflow pipes to each channel by variable-flow pumps (Rio+ Aqua

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Pump/Powerheads 1100-1400). The sump contained a biofilter and a heater. Water

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temperature was maintained at 25 ± 1°C.

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Figure 2: Diagrams of the behavioral test apparatus for (a) The light-dark test and (b) The

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novel tank test. Dotted blue line represents water level.

Figure 3: Differences in behaviors between fish from control or exercised treatments in

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the light-dark test. (a) Time in the light compartment, (b) Time in the dark compartment and (c) Time in the central (neutral) compartment. Mean ± s.e.m. * Denotes significant difference.

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Figure 4: Time spent at the bottom during the novel tank test during each minute of the trial. Solid black line, control; Dotted black line, exercised. Mean ± s.e.m. * Denotes

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significant difference.

Figure 5: Differences in behaviors between fish from control or exercised treatments in the novel tank test. (a) Movement rate, (b) Latency to top and (c) Time in top. Mean ±

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s.e.m. * Denotes significant difference.

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