Behavioural Brain Research 210 (2010) 1–7

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Behavioural Brain Research

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Research report

Parametric analyses of anxiety in zebrafish scototaxis

Caio Maximinoa,b,c,∗ , Thiago Marques de Britod,e , Rafael Colmanetti f , Alvaro Antonio Assis Pontesg ,Henrique Meira de Castro f, Renata Inah Tavares de Lacerdaf, Silvio Moratod, Amauri Gouveia Jra,h

a Laboratório de Neurociências e Comportamento, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém/PA, Brazilb Programa de Pós-Graduacão em Neurociências e Biologia Celular, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém/PA, Brazilc Laboratório de Neuroendocrinologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém/PA, Brazild Laboratório de Comportamento Exploratório, Departamento de Psicologia & Educacão, Universidade de São Paulo, Ribeirão Preto/SP, Brazile Programa de Pós-Graduacão em Psicobiologia, Departamento de Psicologia & Educacão, Universidade de São Paulo, Ribeirão Preto/SP, Brazilf Departamento de Psicologia, Universidade Estadual Paulista “Julio de Mesquita Filho”, Bauru/SP, Brazilg Faculdade de Artes, Arquitetura e Comunicacão Social, Universidade Estadual Paulista “Julio de Mesquita Filho”, Bauru/SP, Brazilh Núcleo de Teoria e Pesquisa do Comportamento, Instituto de Psicologia, Universidade Federal do Pará, Belém/PA, Brazil

a r t i c l e i n f o

Article history:Received 22 December 2009Received in revised form 18 January 2010Accepted 22 January 2010Available online 29 January 2010

Keywords:ZebrafishFearAnxietyBehaviorAvoidance

a b s t r a c t

Scototaxis, the preference for dark environments in detriment of bright ones, is an index of anxiety inzebrafish. In this work, we analyzed avoidance of the white compartment by analysis of the spatiotem-poral pattern of exploratory behavior (time spent in the white compartment of the apparatus and shuttlefrequency between compartments) and swimming ethogram (thigmotaxis, freezing and burst swimmingin the white compartment) in four experiments. In Experiment 1, we demonstrate that spatiotemporalmeasures of white avoidance and locomotion do not habituate during a single 15-min session. In Experi-ments 2 and 3, we demonstrate that locomotor activity habituates to repeated exposures to the apparatus,regardless of whether inter-trial interval is 15-min or 24-h; however, no habituation of white avoidancewas observed in either experiment. In Experiment 4, we confined animals for three 15-min sessions in thewhite compartment prior to recording spatiotemporal and ethogram measures in a standard preferencetest. After these forced exposures, white avoidance and locomotor activity showed no differences in rela-

ScototaxisMotivationDrive

tion to non-confined animals, but burst swimming, thigmotaxis and freezing in the white compartmentwere all decreased. These results suggest that neither avoidance of the white compartment nor approach

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. Introduction

Scototaxis, a preference for dark environments in detriment ofrightly lit ones, is a behavioral trait that is present in many speciesf teleost fishes [1–6], and it has been suggested that it repre-ents a species-specific defense pattern that exploits crypsis withhe substratum as a strategy for avoiding predators [4–6]. As such,cototaxis can represent “anxiety” or “fear”-like behavior in thesepecies, including the zebrafish [5,7–10].

All procedures described so far to the establishment of dark pref-

rence use free exploration of a black and white tank, measuringime spent in both compartments as a proxy for preference, andsing diverse measures of total locomotion as a control for activ-

ty effects [3–7,9–13]. As it happens with unconditioned models in

∗ Corresponding author at: Laboratório de Neurociências e Comportamento, Insti-uto de Ciências Biológicas, Universidade Federal do Pará, r. Augusto Correa 01,6075-110 Belém/PA, Brazil. Tel.: +55 91 8180 2446; fax: +55 91 3201 7568.

E-mail address: caio.maximino@gmail.com (C. Maximino).

166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2010.01.031

unt for the behavior of zebrafish in the scototaxis test.© 2010 Elsevier B.V. All rights reserved.

rodents [14], the definition of what are the controlling stimuli inthe scototaxis task is still needed [10]. Without extensive analyses,it is not clear what are the reinforcers which are involved in sco-totaxis – whether a particular behavior observed in this model isdriven by positive (approach the dark compartment) or negative(avoid the light compartment) drives.

An approach to determining the underlying motivations whichcontrol behavior in a given task is “behavioral validation” [15,16], astrategy in which analysis of the organization of the ethogram of theanimal in the test situation reveals ethologically relevant dimen-sions of defensive behavior. This approach was taken by Blaser et al.[10], using the dark–light tank in zebrafish. The authors establishedpreference for a dark environment in this species, and proceededto confine subjects to each compartment and record their behav-ior while confined. After confinement, animals were then exposed

to the preference test once again. Animals were separated in“high-avoidant” and “low-avoidant” using a median split of theircombined pre- and post-test scores for time spent in the white com-partment. Confinement to the white compartment elicited freezingin high avoidance animals, but not in low-avoidance subjects;

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rratic movement presented a weak relationship with avoidance,nd thigmotaxis and locomotor behavior were poor predictors ofigh- versus low-avoidance. Based on these results, Blaser andolleagues suggested that freezing is a viable measure of whitenvironment-induced anxiety, while erratic movement, thigmo-axis and locomotor behavior are not.

In this work, we attempt a similar approach with the scototaxisest in zebrafish, evaluating intra- and intersession habituation ofhite avoidance and locomotor behavior, as well as the effects

f forced exposures to the white compartment on spatiotemporaleasures of the test. We observe no signs of intra- or interses-

ion habituation of white avoidance, with intersession habituationf locomotor activity. We also analyzed habituation and effects oforced exposures to the white compartment on burst swimming,higmotaxis and freezing. In the intrasession experiment, thigmo-axis shows a U-shaped function, while freezing shows an inverted-shaped function, but burst swimming does not change across

he session. In the intersession habituation experiment with 15-in inter-trial intervals, no habituation was observed in ethogrameasures. In the intersession habituation experiment with 24-h

nter-trial intervals, burst swimming habituated, but thigmotaxisnd freezing did not. All ethogram measures decreased after ani-als were forcefully exposed to the white compartment three

onsecutive times.

. Methods

.1. Subjects

Subjects were 50 adult wild-type zebrafish of mixed genders, purchased fromlocal aquarium supply store. Subjects were housed in collective 30 L tanks, in

roups of 20–25 individuals, for 3 months prior to experiments, at Laboratórioe Neurociências e Comportamento at Universidade Federal do Pará. Tanks pos-essed a recirculation filtration system using mechanical, biological, and chemicalltration. The temperature of the tanks was held at 25 ± 2 ◦C, and the room wasaintained on a 12/12 h light/dark cycle (photoperiod starting at 0700). Subjectsere fed once daily on a diet of pellet ration (Oscar Gold©). Housing conditions and

xperimental protocols were approved by Universidade Federal do Pará’s Researchthics Committee, and complied to ASAB/ABS [17] and SBCAL/Brazil standards [18].

.2. Apparatus

The apparatus used in all experiments was the same as that described else-here [5,6]. Briefly, a tank (15 cm × 10 cm × 45 cm) constructed with both black

nd white matte acrylic was used; half of the tank comprised a white compartment15 cm × 10 cm × 22.5 cm), and the other half comprised a black compartment. Theolored material chosen was not reflective, to avoid shoaling tendencies. The tankontained sliding central doors, colored with the same color of the aquarium side,efining a central compartment with 15 cm × 10 cm × 10 cm. During experiments,he water column was kept to 10 cm, and the tank was rotated in 180◦ after eachrial, so as to eliminate orientation effects. The tanks were illuminated by environ-

ental light (60 W light bulb, located at 1.80 m above the tank), which kept thellumination uniform and constant between trials.

.3. Experiment 1

In Experiment 1, 10 subjects were individually placed in the central compart-ent of the preference tank for a 5-min acclimation interval. After that, both doors

etween the black and the white compartments were removed, allowing the fisho make and initiate choice and then to freely explore the two compartments.he subjects’ behavior was video-recorded with a Sony DCR-DVD610 camera forhe entire 15-min of the preference test, and digital MPEG2 files transferred tocomputer for viewing; records for each measure (see Section 2.7 below) wereade for each 3-min. Even though the confinement in the central compartment

or acclimation allowed for contact with both sides of the tank, data from thosenimals that did not cross the midline in the 900 s session were discarded, touarantee that animals sampled both alternatives (black and white compartments)19].

.4. Experiment 2

In Experiment 2, 10 subjects were tested in the standard preference test, asescribed in Section 2.3. After the first trial ended, animals were transferred backo the acclimation tank, where they stayed for a 15-min interval. After that inter-al, subjects were individually placed in the central compartment for the 5-min

in Research 210 (2010) 1–7

acclimation interval, and subsequently tested again. When the second trial ended,animals were then transferred back to the acclimation tank, and the whole proce-dure was iterated. Subjects were exposed to a total of 3 trials, comprising 60 min persubject.

2.5. Experiment 3

In Experiment 3, the same procedure used in Experiment 2 was applied, withthe exception that inter-trial intervals had duration of 24-h, and 5 trials were done.

2.6. Experiment 4

In Experiment 4, 10 subjects were individually transferred to the white compart-ment of the aquarium for the first “flooding” trial, and were not allowed to leave thewhite compartment. A “flooding” trial lasted 15-min, and trials were repeated twomore times (with a 3-min interval between them) before animals were tested fordark preference. After “flooding” trials were completed for a single subject, theywere transferred to the central compartment of the apparatus, and tested in thestandard protocol, as described in Section 2.3.

2.7. Spatiotemporal and behavioral measures

All measures were recorded using a video camera (Sony DCR-DVD610) posi-tioned above the tank, and transcribed using X-Plo-Rat 1.1.0 [20]. Spatiotemporalmeasures included Proportion black (%): the proportion of the trial spent in the blackcompartment; Proportion white (%): the proportion of the trial spent in the whitecompartment; Shuttle (n): the total number of times that the center-point of theanimal crossed through the center of the tank that divided the black and whitecompartments.

Behavioral measures (swimming ethogram) were recorded solely for behavioralunits taking place in the white compartment. The swimming ethogram was partiallybased on Blaser et al. (in press), and included; Duration burst swimming (%): theproportion of the time spent in the white compartment used in unsteady, transientswimming with a duration of <2 s [21]; Duration thigmotaxis (%): the proportion ofthe time spent in the white compartment used in sustained swimming at a distanceof up to 2 cm of the nearest wall; Duration freezing (%): the proportion of the timespent in the white compartment that the animal is immobile (moving less than0.75 cm/s).

2.8. Statistical analysis

Since normality was not assumed, preference for the black compartment wasestablished using Mann–Whitney’s U-test for proportion white against the nullhypothesis that it is equal to 50%. Habituation was assessed using linear regres-sions followed by F-tests for the slopes against the null hypothesis that the slopeequals zero. In Experiments 2 and 3, the consistency and stability of multiple testingacross individuals was assessed by intra-class correlation coefficients. Spatiotem-poral measures from Experiment 4 were analyzed using Mann–Whitney’s U-test,while ethogram measures were analyzed using one-way Kruskal–Wallis analysesof variance.

3. Results

3.1. Experiment 1

Fig. 1 shows the results for the intrasession habituation experi-ment. No habituation effect was observed for either spatiotemporalmeasures (proportion white: r2 = 0.38, slope = 0.12 ± 0.092,F[1,3] = 1.8, NS; shuttle: r2 = 0.58, slope = 0.13 ± 0.061, F[1,3] = 4.1,NS) or ethogram measures (burst swimming: r2 = 0.00,slope = 0.00 ± 0.10, F[1,3] = 0.00, NS; thigmotaxis: r2 = 0.00073,slope = −0.033 ± 0.71, F[1,3] = 0.0022, NS; freezing: r2 = 0.029,slope = –0.13 ± 0.45, F[1,3] = 0.088, NS). In terms of the cumulativespatiotemporal measures of preference, animals spent signifi-cantly more time in the black compartment than in the whitecompartment (U[df = 9] = 100, p = 0.0001; data not shown).

3.2. Experiment 2

Fig. 2 presents results for the intersession habituation exper-

iment with 15-min inter-trial intervals. Across trials, shuttlefrequency habituated (r2 = 1.0, slope = −19 ± 0.58, F[1,1] = 1100,p = 0.0193), but not burst swimming, thigmotaxis or freezing(burst swimming: r2 = 0.94, slope = −3.5 ± 0.87, F[1,1] = 16, NS; thig-motaxis: r2 = 0.75, slope = 0.50 ± 0.29, F[1,1] = 3.0, NS; freezing:

C. Maximino et al. / Behavioural Brain Research 210 (2010) 1–7 3

Fig. 1. (Top panel) Zebrafish (Danio rerio) do not present intrasession habituationof white avoidance (time spent in white compartment, black circles, scale at lefty-axis) nor shuttle frequency (number of midline crossings, gray squares, scale atright y-axis) in the scototaxis test. (Bottom panel) Swimming ethogram measures(burst swimming, black circles; thigmotaxis, light gray squares; freezing, dark graytriangles) do not show intrasession habituation, with thigmotaxis showing a U-shaped function and freezing showing an inverted U-shaped function. In both panels,error bars represent mean ± s.e.m.

Fig. 2. (Top panel) Zebrafish (Danio rerio) shows habituation of shuttle frequency(number of midline crossings, gray squares, scale at right y-axis), but not of whiteavoidance (time spent in white compartment, black circles, scale at left y-axis)across trials in a repeated exposures experiment using 15-min inter-trial intervals.Ethogram measures (burst swimming, thigmotaxis and freezing) do not habituate(Bottom panel). In both panels, error bars represent mean ± s.e.m.

Table 1Matrices of intra-class correlations between fixed effects of repeated exposure onspatiotemporal and ethogram measures across three consecutive trials (15-mininter-trial interval).

Trial 1 Trial 2 Trial 3

White avoidanceTrial 1 1.0 0.707 0.748Trial 2 0.707 1.0 0.833Trial 3 0.748 0.833 1.0

Shuttle frequencyTrial 1 1.0 0.897 0.790Trial 2 0.897 1.0 0.812Trial 3 0.790 0.812 1.0

Burst swimmingTrial 1 1.0 0.416 0.668Trial 2 0.416 1.0 0.498Trial 3 0.668 0.498 1.0

ThigmotaxisTrial 1 1.0 0.707 0.746Trial 2 0.707 1.0 0.830Trial 3 0.746 0.830 1.0

Freezing

Trial 1 1.0 0.658 0.534Trial 2 0.658 1.0 0.405Trial 3 0.534 0.405 1.0

r2 = 0.25, slope = −0.50 ± 0.87, F[1,1] = 0.33, NS) nor proportion white(r2 = 0.00, slope = 0.00 ± 0.12, F[1,1] = 0.00, NS). Animals also spentmore time in the black than in the white compartment across all tri-als (trial 1: U[df = 9] = 102, p < 0.05; trial 2: U[df = 9] = 97, p < 0.05; trial3: U[df = 9] = 99, p < 0.05; data not shown). Regarding intra-subjectstability, all measures showed consistent (r2 > 0.4) intra-class cor-relation coefficients across trials (Table 1).

3.3. Experiment 3

Fig. 3 presents results for the intersession habituationexperiment with 24-h inter-trial intervals. No significant habit-uation effect was observed for proportion white (r2 = 0.20,slope = 0.20 ± 0.23, F[1,3] = 0.73, NS), but it was observed in shut-tle frequency (r2 = 0.96, slope = −5.6 ± 0.63, F[1,3] = 80.0, p = 0.003),and burst swimming (r2 = 0.94, slope = −2.7 ± 0.38, F[1,3] = 51.0,p = 0.0057). No habituation effect was observed in both freez-ing (r2 = 0.05, slope = −0.10 ± 0.25, F[1,3] = 3.0, NS) and thigmotaxis(r2 = 0.50, slope = 0.40 ± 0.23, F[1,3] = 3.0, NS). Animals also spentmore time in the black than in the white compartment across all tri-als (trial 1: U[df = 9] = 99, p < 0.05; trial 2: U[df = 9] = 95, p < 0.05; trial 3:U[df = 9] = 80, p < 0.05; trial 4: U[df = 9] = 86, p < 0.05; trial 5: U[df = 9] = 70,p < 0.05; data not shown). Regarding intra-subject stability, allmeasures showed consistent (r2 > 0.4) intra-class correlation coef-ficients across trials (Table 2).

3.4. Experiment 4

Fig. 4 presents results for the forced exposure experiment. Noeffect of the forced exposure was observed on spatiotemporalmeasures (proportion white: U[df = 18] = 40, NS; shuttle frequency:U[df = 18] = 31, NS). A significant decrease was observed in allbehaviors (F[2,54] = 4.00, p = 0.0233); Bonferroni post-hoc tests

demonstrated very significant differences between groups inburst swimming (t[df = 18] = 7, p < 0.001), thigmotaxis (t[df = 18] = 7,p < 0.001), and freezing (t[df = 18] =3, p < 0.01). Animals spent moretime in the black than in the white compartment in both controland forced exposure groups (control: U[df = 9] = 100, p < 0.05; forcedexposure: U[df = 9] = 89, p < 0.05; data not shown).

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Fig. 3. (Top panel) Zebrafish (Danio rerio) habituate shuttle frequency (number ofmidline crossings, gray squares, scale at right y-axis), but not avoidance of the whitecompartment (time spent in the white compartment, black circles, scale at righty-axis) when exposed to the scototaxis apparatus for 5 consecutive days, with a24-h interval between trials. (Bottom panel) Thigmotaxis (light gray squares) andfreezing (dark gray triangles), but not burst swimming, also do not habituate in the24-h inter-trial interval repeated exposures experiment. In both panels, error barsrepresent mean ± s.e.m.

Table 2Matrices of intra-class correlations between fixed effects of repeated exposure onspatiotemporal and ethogram measures across three consecutive trials (15-mininter-trial interval).

Day 1 Day 2 Day 3 Day 4 Day 5

White avoidanceDay 1 1.0 0.774 0.874 0.824 0.722Day 2 0.744 1.0 0.790 0.812 0.732Day 3 0.874 0.790 1.0 0.936 0.668Day 4 0.824 0.812 0.936 1.0 0.582Day 5 0.722 0.732 0.668 0.582 1.0

Shuttle frequencyDay 1 1.0 0.732 0.671 0.790 0.732Day 2 0.732 1.0 0.738 0.812 0.806Day 3 0.671 0.738 1.0 0.919 0.789Day 4 0.790 0.812 0.919 1.0 0.789Day 5 0.732 0.806 0.789 0.789 1.0

Burst swimmingDay 1 1.0 0.663 0.671 0.789 0.663Day 2 0.663 1.0 0.706 0.785 0.748Day 3 0.671 0.796 1.0 0.919 0.805Day 4 0.789 0.785 0.919 1.0 0.712Day 5 0.663 0.748 0.805 0.712 1.0

ThigmotaxisDay 1 1.0 0.673 0.689 0.781 0.615Day 2 0.673 1.0 0.672 0.710 0.847Day 3 0.689 0.672 1.0 0.835 0.634Day 4 0.781 0.710 0.835 1.0 0.555Day 5 0.615 0.847 0.634 0.555 1.0

FreezingDay 1 1.0 0.666 0.692 0.774 0.607Day 2 0.666 1.0 0.670 0.713 0.815Day 3 0.692 0.670 1.0 0.835 0.566Day 4 0.774 0.713 0.825 1.0 0.504Day 5 0.607 0.815 0.566 0.504 1.0

Fig. 4. White avoidance (Top panel) and shuttle frequency (Middle panel) do notchange after three 30-min forced exposures to the white compartment of thescototaxis tank. All behavioral measures (burst swimming, thigmotaxis and freez-

ing) decrease after forced exposures (Bottom panel). In all panels, bars representmean ± s.e.m.

4. Discussion

The present article described the effects of intra- and interses-sion habituation and forced exposures to the white compartmenton spatiotemporal and behavioral measures of anxiety in the sco-totaxis test in zebrafish. Overall, neither intra- nor intersessionhabituation was observed in the proportion of the trial durationthat the animals spent in the white compartment, but there was asignificant decrease in locomotor activity across sessions, regard-

less of the extent of the inter-trial interval. Within a given session,the three behavioral measures described did not habituate, butthere was a decrease in the proportion of time that animals spentin burst swimming. Forced exposures (post-confinement trials) didnot alter the time spent in the white compartment or general loco-

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otor activity (shuttle frequency), but it decreased the proportionf time that animals spent burst swimming, sticking to the walls,r freezing while in the white compartment.

The spatiotemporal distribution of exploratory behavior dur-ng the session, as assessed by Experiment 1, does not suggest thatabituation for any measure takes place (Fig. 1, top panel). This con-rasts, for example, with the spatiotemporal distribution of bottomersus top of the tank in the novel tank diving test in zebrafish7–9,12,22–24], in which zebrafish gradually increase their explo-ation of the top of the tank as the session passes. Using thisest, Wong et al. [12] found that zebrafish steadily increases timepent in the top of the apparatus and decreases the frequency anduration of freezing bouts; by about the 10th minute of a 30-minovel tank diving session, the number of and duration of transi-ions to the top, as well as number and duration of freezing bouts,eaches a steady level, without further increases or decreases [12].ollowing classic exploratory behavior theory [25–29], it seemshat intrasession habituation in the novel tank diving test is con-rolled by decreasing novelty; however, increasing basal anxietyy exposing animals to anxiogenic treatments (alarm substance,ub-convulsant dose of pentylenetetrazole) diminishes or abol-shes habituation, while decreasing basal anxiety by exposingnimals to anxiolytic treatments (acute ethanol, acute morphine,nd chronic fluoxetine) enhances habituation in the novel tankiving test. In the present work, we could not reliably record theertical distribution of zebrafish in the apparatus due to techni-al difficulties (since the apparatus is not transparent, recordingsust be made from the top, rendering quantitative analyses of

ertical distribution impossible), and thus we could not assessf that is also the case in the scototaxis test. Nonetheless, therincipal measure which is thought to represent anxiety in thisest – namely, white avoidance – did not show signs of intrases-ion habituation, which could indicate that exploration of thehite compartment is driven primarily by fear/anxiety than byovelty.

Burst swimming, thigmotaxis and freezing also do not showntrasession habituation (Fig. 1, bottom panel), but the last two

easures show an interesting two-phase profile. Thigmotaxisecreases in the 3 first time blocks of Experiment 1, increasingfterwards (U-shaped pattern); freezing, on the other hand, shownd inverted U-shaped pattern, increasing in the 3 first time blocksnd decreasing afterwards. This latter temporal pattern of freezings suggestive of an initial hyperarousal caused by fear, followedy a reduction in arousal, as predicted by optimal arousal the-ry [30]. Thigmotaxis, on the other hand, shows the U-shapedunction that suggests that it is controlled mainly by decreasingovelty [25,27]. Although evidence on stimulus control of thigmo-axis in zebrafish is sparse, these results suggest that thigmotaxisn the white compartment may be related to “risk assessment”ehavior.

The results from both intersession habituation experiments androm forced exposures suggest that, while the white compartmentoes present a negative reinforcer, as discussed by Blaser et al.10], the scototaxis presented by zebrafish in this test cannot bettributed solely to an avoidance drive. Time spent in the whiteompartment does not habituate, even when trials are separatedrom each other by a 24-h interval. The results from the ethogramnalysis are also consistent with this hypothesis, since no habit-ation was observed in these (putative) measures of anxiety-likeehavior. The exception is burst swimming, a fast and transientwimming mode that fish assume when fleeing predators or strik-

ng prey [21]. Work regarding burst swimming elicited by auditory,isual or nociceptive stimuli, this mode of swimming is sometimesnterpreted as indicating fright and not arousal [31]. This mode ofwimming habituated in the first 24 h and last 48 h of Experiment. A similar measure (erratic swimming) also shows intrases-

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sion habituation (blocked by pentylenetetrazole and caffeine) inthe novel tank diving test [12]. It is unlikely that this decreasein burst swimming in scototaxis is related to habituation offear/anxiety, since white avoidance and freezing do not habituate.The hypothesis that burst swimming is related to arousal cannot bediscarded.

Interestingly, both spatiotemporal and ethogram measuresshow a high degree of within-subjects consistency across trials(Tables 1 and 2); thus, animals which show high avoidance ofthe white compartment, for example, have increased avoidance inall sessions, and animals which show increased freezing while inthe white compartment will show high freezing (relative to theother animals) in all sessions. Visual inspection of individual datapoints (data not shown) suggest, however, that habituation (or lackthereof) of spatiotemporal and ethogram measures does not varybetween subjects.

The significance of the three behavioral measures of theethogram is more clearly interpreted in light of the results fromthe forced exposure experiments. The three behavioral measuresused in this study – burst transient swimming, thigmotaxis, andfreezing – were chosen because they were explored in two previ-ous experiments [6,10]. All these behaviors occur more frequentlyin the white compartment than in the black compartment of thescototaxis tank [6,10]. Also, during a single 30-min forced expo-sure to the white compartment and a single 30-min exposureto the black compartment, high-avoidant zebrafish (i.e., animalswhich consistently preferred the dark compartment) spend moretime freezing in the white environment than in the black envi-ronment [10]; however, the black compartment elicited morethigmotaxis than the white compartment in those animals. Blaseret al. suggested that this represents avoidance of not only the whitecompartment but also of the center of the dark compartment [10].Using a different methodology, we demonstrated that burst swim-ming, thigmotaxis and freezing responses are all decreased afterthree 15-min forced exposures to the white compartment, fol-lowed by the preference test. These behaviors were also analyzedin other contexts, with varying results. In an open-field, thigmo-taxis occurs frequently, while freezing is not consistently observed[11,24,32–34]. When presented with a live sympatric predator,zebrafish did not show differences in avoidance (as measured bydistance from the predator), freezing, or thrashing [32]; a preda-tor model, on the other hand, decreases freezing in comparisonto a no-stimulus control [11] (another study, however, reporteddecreased burst swimming and no effect on freezing [35]). Freezingwas also not altered significantly by exposure to an alarm substance[24].

In experimental psychopathology and biological psychiatry,anxiety and fear are understood as two different constructs, basedon different circuitry, neurochemistry, and genetic underpinnings[14–16,36–41]. From the point of view of stimulus control, anxi-ety is related to the exposure to potentially threatening or “risky”contexts (such as novel and mildly aversive environments), whilefear is related to distal or proximal threat (such as highly aversivesituations, predators or partial predator stimuli) [14–16,41–46].Thus, the fact that freezing is not significantly altered by preda-tors or partial predator stimuli, while erratic/burst swimming is,indicates that both measures are derived from different processes– anxiety in the first case, and fear or hyperarousal in the last[47]. Thigmotaxis, on the other hand, is still poorly understood and(apart from its pharmacological sensitivity to benzodiazepines andethanol [10,11,34,48–51]), what are its controlling stimuli is still an

open question.

Since scototaxis is a trait that is present in other teleosts (includ-ing characids, poecilids, anabantids, gymnotids, and other cyprinids[2,4–6]), and since there are some intriguing species differencesin scototaxis (e.g., female Nile tilapias show increased locomotion

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n an all-black tank, while other species show increased shut-le frequency in an all-white tank [5]), it would be interesting tonvestigate whether the results reported here can be replicatedsing other species of teleost fish. Currently, we are reproducinghe experiments described here with the goldfish Carassius aura-us, a related cyprinid which was used in avoidance conditioningxperiments [52–56]. It would also be interesting to test drugshich present anxiolytic and panicolytic profile in human clini-

al contexts and see if they alter some of the measures which wereefined in this work; results using chronic [7] and acute [57] fluox-tine treatments, buspirone, chlordiazepoxide and diazepam [58],thanol [35,59], the glutamate antagonist MK-801 [60,61], caffeine7], and the nicotinic receptor antagonists MLA and DH�E [22,23]ere already described using thigmotaxis in the open-field and

ottom-dwelling in the novel tank diving test – results which areromising, but as of yet inconclusive. The parametric dissection ofcototaxis put forth by this article will likely contribute to furthernderstanding on the pharmacological and neural bases of anxiety

n this taxon.

cknowledgments

C.M. and T.M.B. are recipients of post-graduation scholarshiprants by CAPES. R.I.T.L. is recipient of undergraduate scholarshiprant by CNPq. Part of this research was supported by grants fromNPq to A.G.

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