Behavioural Processes 149 (2018) 35–42

Contents lists available at ScienceDirect

Behavioural Processes
journal homepage: www.elsevier.com/locate/behavproc

Extending the analysis of zebrafish behavioral endophenotypes for modeling T

psychiatric disorders: Fear conditioning to conspecific alarm response
⁎⁎
Caio Maximinoa,b, , Daniele L. Meinerzc, Barbara D. Fontanac,d, Nathana J. Mezzomoc,e,
Flavia V. Stefanelloc, Alessandro de S. Prestesd, Cibele B. Batistac, Maribel A. Rubind,
⁎
Nilda V. Barbosad, João Batista T. Rochad, Monica G. Limaf, Denis B. Rosembergb,c,d,
a
Laboratório de Neurociências e Comportamento “Frederico Guilherme Graeff”, Instituito de Estudos em Saúde e Biológicas, Universidade Federal do Sul e Sudeste do Pará,
Avenida dos Ipês, S/N, Marabá, PA, Brazil
b
The International Zebrafish Neuroscience Research Consortium (ZNRC), 309 Palmer Court, Slidell, LA 70458, USA
c
Laboratório de Neuropsicobiologia Experimental, Departamento de Bioquímica e Biologia Molecular, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa
Maria, Avenida Roraima, 1000, 97105–900, Santa Maria, RS, Brazil
d
Programa de Pós-graduação em Bioquímica Toxicológica, Universidade Federal de Santa Maria, Avenida Roraima, 1000, 97105–900, Santa Maria, RS, Brazil
e
Programa de Pós-graduação em Farmacologia, Universidade Federal de Santa Maria, Avenida Roraima, 1000, 97105–900, Santa Maria, RS, Brazil
f
Laboratório de Neurociências e Comportamento “Frederico Guilherme Graeff”, Departamento de Morfologia e Ciências Fisiológicas, Universidade do Estado do Pará,
Avenida Hileia, S/N, Agrópolis do INCRA, Marabá, PA, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Anxiety, trauma- and stressor-related disorders are severe psychiatric conditions that affect human population
Fear conditioning worldwide. Given their genetic tractability, evolutionarily conserved neurotransmitter systems, and extensive
Alarm substance behavioral repertoire, zebrafish have become an emergent model organism in translational neuroscience. Here,
Conditioned place aversion we investigate whether a single exposure to conspecific alarm substance (CAS) produces fear conditioning in
Psychiatric disorders
zebrafish using a conditioned place aversion (CPA) paradigm, as well as the persistence of aversive responses at
Zebrafish
different time intervals. While CAS elicited freezing and erratic movements at conditioning phase, zebrafish
showed a robust avoidance for the CAS-paired compartment and increased risk assessment up to 7 days post-
conditioning. Additionally, we observed the existence of two behavioral phenotypes (high- and low-avoider fish)
that present different fear-like responses at conditioning phase and evasion of the conditioning side at post-
conditioning trials. Collectively, we show a prolonged conditioned place aversion in zebrafish after a single CAS
conditioning session, reinforcing the use of fear conditioning protocols as valuable strategies for modeling
psychiatric disorders-related phenotypes in zebrafish.

1. Introduction et al., 2017). Fear conditioning protocols elicit traumatic memories

Anxiety, trauma- and stressor-related disorders are severe psychia-
tric conditions that affect human population worldwide (Shalev, 2009).
Since the evolution theory has been closely associated with the beha-
vioral neuroscience, animal models of affective disorders attempt to
reproduce features of human psychiatric disorders in laboratory ani-
mals, correlating the physiological and behavioral changes associated
to specific emotional states (Campos et al., 2013). One approach for
modeling anxiety, trauma- and stressor-related disorders in animals is
avoidance conditioning, in which subjects learn to minimize and/or
prevent contact with aversive events (Krypotos et al., 2015; LeDoux
after exposure to an intense stressor, modeling a psychological stress in
the absence of noxious stimuli. These models have been associated with
a vulnerability to phobic fears and other anxiety-related disorders, such
as panic disorder (PD), agoraphobia, acute stress disorder (ASD), and
posttraumatic stress disorder (PTSD) (Battle, 2013).
Avoidance learning depends on a Pavlovian component which in-
volves the association of a neutral conditioned stimulus (CS), (e.g., tone
and visual cues) with a threat signal (unconditioned stimulus, US). Re-
exposure to the CS will elicit the expectancy of an aversive outcome and
thereby activates conditioned defensive responses (e.g., physiological
arousal, avoidance tendencies) in the absence of US (Bolles et al., 1970;

⁎
Corresponding author at: Laboratório de Neuropsicobiologia Experimental. Departamento de Bioquímica e Biologia Molecular, Centro de Ciências Naturais e Exatas, Universidade
Federal de Santa Maria. Avenida Roraima, 1000, 97105–900, Santa Maria, RS, Brazil.
⁎⁎
Corresponding author at: Laboratório de Neurociências e Comportamento “Frederico Guilherme Graeff”, Instituito de Estudos em Saúde e Biológicas, Universidade Federal do Sul e
Sudeste do Pará. Avenida dos Ipês, S/N, Marabá, PA, Brazil.
E-mail addresses: cmaximino@unifesspa.edu.br (C. Maximino), dbrosemberg@gmail.com (D.B. Rosemberg).

https://doi.org/10.1016/j.beproc.2018.01.020
Received 10 October 2017; Received in revised form 29 January 2018; Accepted 30 January 2018
Available online 02 February 2018
0376-6357/ © 2018 Elsevier B.V. All rights reserved.
C. Maximino et al.
Dinsmoor, 1977, 2001; Fernando et al., 2014; Krypotos et al., 2015;
LeDoux et al., 2017). Escaping behavior persists depending on instru-
mental learning with multiple sources of reinforcement (Krypotos et al.,
2015) and impaired safety signal learning has been proposed as an
endophenotype for several brain disorders (Jovanovic et al., 2012;
Jovanovic et al., 2010; Jovanovic et al., 2009).
Avoidance conditioning has been observed in different vertebrate
species (Vargas et al., 2012) to assess various behavioral en-
dophenotypes (de Mooij-van Malsen et al., 2011). In goldfish, for ex-
ample, active avoidance depends on the dorsomedial telencephalon
(Portavella et al., 2004a; Portavella et al., 2004b), a basolateral
amygdala homolog (Maximino et al., 2013). Although inhibitory
avoidance protocols have been proposed using electric shocks as US in
zebrafish (Blank et al., 2009; Manuel et al., 2014), a different approach
consists in using aversive chemical cues as fear-inducing stimuli. Con-
specific alarm substance (CAS) is a pheromone-like mixture released in
the water as consequence of damage to the epithelial club cells (Doving
and Lastein, 2009; von Frisch, 1938). In zebrafish, CAS elicits fear-like
behaviors, such as erratic movements and freezing (Mathuru et al.,
2012; Quadros et al., 2016; Speedie and Gerlai, 2008). Moreover, CAS
serves as US in Pavlovian fear conditioning, because it increases fear
responses to CS after paired with a neutral visual stimulus (flashing red
light) (Hall and Suboski, 1995a; Hall and Suboski, 1995b). Although
the re-exposure to the context substantially increases erratic swimming
24 h after conditioning (Ruhl et al., 2017), the persistence of escaping
responses after distinct time intervals remains unexplored. Since CAS
can also sensitize anxiety-like behavior after a single exposure, this
pheromone is a potential stimulus for avoidance conditioning research.
A brief CAS exposure increases anxiety-like behavior and arousal 24 h
after the stimulus presentation, suggesting a time-dependent sensitiza-
tion for both domains (Lima et al., 2016). Thereby, using CAS in
avoidance conditioning protocols could expand the breadth of domains
to include trauma re-experience, improving the face validity of zebra-
fish models of psychiatric disorders. Here, we investigate the aversive
conditioning to a single CAS exposure in zebrafish using a conditioned
place aversion (CPA) paradigm and verify the stability of conditioned
responses after different time intervals.
2. Material and methods
2.1. Animals and housing
Male and female (∼50:50 ratio) wild type adult zebrafish (Danio
rerio) (4–6 months-old, blue short-fin [BSF] phenotype) were obtained
from a commercial distributor (Hobby Aquários, RS, Brazil). Since
short-fin phenotype fish are genetically heterogeneous, they are ex-
pected to better represent the natural populations, decreasing the ef-
fects of arbitrary genetic drifts (Speedie and Gerlai, 2008; Lima et al.,
2016; Parra et al., 2009). In the literature, a large amount of data using
heterogeneous zebrafish stocks from commercial sources has shown a
considerable replication among groups when tested in different beha-
vioral protocols (Egan et al., 2009; Song et al., 2018; Piato et al., 2011;
Quadros et al., 2016). Fish were previously housed for 14 days in 50-L
thermostatic aquaria to acclimate to laboratory conditions. Tanks were
filled with non-chlorinated water (temperature set at 27 ± 1 °C, pH
7.2) and kept under constant mechanical and chemical filtration. Fish
were raised in a 14/10 light/dark photoperiod cycle (lights on at 7:00
am and off at 9:00 pm) and lightning was provided by ceiling-mounted
fluorescent tubes. Animals were fed thrice daily with commercial flake
fish food (Alcon BASIC®, Alcon, Brazil) and kept in accordance with the
standard protocols of zebrafish maintenance (Lawrence, 2007). All
experimental protocols were approved by the Ethics Commission on
Animal Use of the Federal University of Santa Maria (process number
106/2014).
Two weeks before the experiments, fish were randomly transferred
from their housing tanks to an aquarium measuring
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Behavioural Processes 149 (2018) 35–42
50 cm × 35 cm × 6 cm (length × width × height), which had equal
divisions for each fish (6 cm × 6 cm × 6 cm – length × width ×
height) and small perforations (0.5 cm diameter) to allow free water
circulation inside the tank. These perforated Plexiglas partitions al-
lowed a precise identification of each animal during the experimenta-
tion and permitted fish to visualize conspecifics minimizing isolation
stress. Water conditions were similar to those of housing tanks, feeding
was not interrupted during this period, and animals were kept in the
task room to minimize the effects of context variation during the trials.
To minimize potential handling stress throughout the experiments, fish
were handled once daily.
2.2. Conditioned place aversion
Conditioned place aversion (CPA) is a protocol derived from con-
ditioned place preference, in which a drug is administered in a com-
partment of the apparatus but not in the other. Then, rewarding
properties of the drug may be assessed by increased occupancy of the
associated compartment in a posterior drug-free situation (Collier et al.,
2014; Prus et al., 2009). In CPA, a potentially aversive stimulus is
paired with one compartment, while its absence is associated with the
other compartment of the apparatus. Thus, explicit relationships be-
tween the presence and absence of an aversive US and context are es-
tablished, in which clear safe and danger signals are present during the
trials.
The dimensions of CPA apparatus were 30 cm × 13 cm × 12 cm
(length × width × height). Tank was divided into two equally sized
compartments (12 cm × 12 cm × 12 cm – length × width × height) by
a 5-cm gray central partition and two sliding guillotine-type doors
(12 cm × 12 cm). To ensure the contrast between experimental subjects
and tank allowing a precise automated behavioral analysis, chambers
were divided by opaque plastic self-adhesive films in white or yellow
colors, covering walls, floor and the corresponding sides of the sliding
guillotine-type doors. Two yellow- and white-covered tanks with
identical dimensions (12 cm × 12 cm × 12 cm – length × width ×
height) were coupled to the CPA apparatus and used for the con-
ditioning phase. Tanks were illuminated by a 15-W fluorescent lamp
located at 1.80 m above each apparatus, which kept the illumination
uniform and constant between trials. The CPA tanks and the experi-
mental protocol are shown in Fig. 1.
2.2.1. Preconditioning trial
The conditioned place preference literature recommends that
‘biased’ tanks can be used for conditioning because they are highly
selective, imposing a more “steep” barrier to avoidance conditioning. In
biased designs for CPA, the preference of each individual animal for a
particular compartment prior to conditioning is evaluated, and the
most-preferred compartment is further associated with the noxious
stimulus (Prus et al., 2009). We followed this method by introducing a
preconditioning trial, in which fish were placed individually into the
central partition of the apparatus. After an acclimation period to the
new environment of 30 s, the guillotine-type doors were removed and
the animal was free to explore the apparatus for 10 min. The basal
preference was assessed by measuring the time spent in each com-
partment.
2.2.2. Conspecific alarm substance extraction and conditioning trial
CAS was extracted as described previously (Canzian et al., 2017;
Lima et al., 2016), where a single donor fish was rapidly euthanized by
decapitation. Afterwards, 10–15 superficial shallow cuts were made on
each side of fish body and care was taken to avoid blood drowning. The
donor fish was further washed with 10 mL of distilled water to obtain
the skin extract preparation, which was reserved on ice for posterior
use.
For the conditioning trial, experimental fish were individually
placed into their most-preferred compartment, and 3.5 mL/L CAS was
C. Maximino et al.
Fig. 1. Schematic representation of the experimental protocol. Zebrafish were kept during 14 days before the experiments in perforated Plexiglas tanks and the basal preference was
assessed at the preconditioning trial. After 24 h, animals were exposed to conspecific alarm substance (CAS) for 5 min and changes on preference were tested at different postconditioning
sessions (DPC). The dimensions of the CPA tanks are shown.
further added to the water (Canzian et al., 2017; Egan et al., 2009; Lima
et al., 2016; Quadros et al., 2016). The animal was kept for 5 min in this
compartment and immediately further removed from the tank. Then,
the tank was washed to remove CAS residuals, and the same animal was
later transferred to the non-preferred compartment, where distilled
water was added. The animal was kept in this “safe” compartment for
5 min. Animals from control group were exposed to distilled water in
both compartments instead of CAS. To allow a better data reproduci-
bility, the order of test was randomized for each fish, and the exposure
to the first compartment occurred between 11:00–12:00, while the
exposure to the other chamber occurred between 14:00–15:00. Both
erratic movements and freezing events were measured at conditioning
phase. These endpoints are characteristic of fear consistently elicited
during CAS exposure (Canzian et al., 2017; Quadros et al., 2016;
Speedie and Gerlai, 2008) and serve the purpose of ‘quality control’.
Additionally, latent classes were analyzed to identify heterogeneous
trajectories using the pattern of behavior change over time (see below).
2.2.3. Postconditioning trials
At postconditioning sessions, occupancy was assessed to evaluate
the efficacy of the avoidance conditioning protocol. Animals changing
their preference to the “safe” compartment show signs of avoidance
learning. Erratic swimming and freezing were recorded simultaneously
in each compartment as indexes of CS-US conditioning. Preference was
scored at different postconditioning (DPC) periods: 24 h (DPC1), 48 h
(DPC2), 72 h (DPC3), and 7 days (DPC7) after conditioning. All tests
occurred in the same manner: first, each fish was placed into the central
partition for 30 s and then the guillotine-type doors were opened.
Animals had free access to all compartments for 10 min and the time
spent in each chamber was quantified. Aversion was estimated as the
difference (in seconds) of time spent in the CAS-paired compartment
during the post and preconditioning phases. We also analyzed the
number of risk assessment episodes, which were defined as fast (> 1 s)
entries in the conditioning compartment followed by re-entry in the
safe compartment, or as partial entries in the conditioning side (Kalueff
et al., 2013; Mezzomo et al., 2016). After each trial, animals were kept
into the perforated Plexiglas tank and manipulated once daily until the
end of the experimental period. Fish were further anaesthetized in
water at 4 °C and euthanized by decapitation.
2.3. Behavioral analysis
All experimental sessions were recorded during the same time frame
37
Behavioural Processes 149 (2018) 35–42
each day (between 10:00 and 16:00) using a webcam located above the
CPA apparatus. Two trained observers (inter-rater reliability > 0.85)
blind to the experimental condition analyzed the videos using auto-
mated video-tracking software system (ANY-maze™, Stoelting CO, USA)
at a rate of 30 frames/s. In order to provide a background noise, a fan
was kept on in the experimental room.
2.4. Statistics
Data normality and homoscedasticity were previously analyzed by
Kolmogorov–Smirnov and Bartlett’s tests, respectively, as basic pre-
requisites for running parametric analyses. The basal preference of
zebrafish was assessed using one-way analysis of variance (ANOVA)
followed by Tukey’s test. To ascertain that initial preference was ran-
domly distributed between the groups, a Fisher exact test was applied to
the proportions of animals preferring each compartment. Effect sizes
were reported as Cohen’s d and behavioral endpoints were expressed as
means ± standard error of the mean (S.E.M.). At conditioning phase,
CAS effects were evaluated by unpaired Student’s t test and effect sizes
were reported as Hedges’ g. Conditioning effects were analyzed by re-
peated measures ANOVA (RM-ANOVAs), with group (CTRL vs. CAS) as
fixed effect and trial period (Preconditioning, DPC1, DPC2, DPC3, and
DPC7) as the within-subjects factor. Differences between groups were
further assessed by Tukey’s test. P-values for trial effects were adjusted
for sphericity.
Latent Growth Modeling (LGM) methods were used to test for po-
pulation heterogeneities in avoidance conditioning over trials. LGM has
been applied to identify heterogeneous trajectories in longitudinal data
by identifying latent sub-populations within finite distributions
(Muthén, 2004; Proust-Lima et al., 2016). LGM provides a statistical
framework for determining the number of classes/sub-populations that
best fit the data (Muthén, 2004). LGM methods have been applied to
identify distinct patterns of symptoms following trauma exposure
(Bonanno et al., 2012; Galatzer-Levy et al., 2013), to avoidance
learning and threat extinction learning in rats (Galatzer-Levy et al.,
2013; Galatzer-Levy et al., 2014).
A heterogeneous linear mixed model (HLME) was employed using
the lcmm package from R (Lima et al., 2015) (https://cran.r-project.
org/web/packages/lcmm/index.html) to identify heterogeneous tra-
jectories by testing for discrete mixture distributions of avoidance
conditioning and retrieval. HLME is an extension of LGM methods that
uses fixed effects, and was employed to reduce the number of para-
meters being modeled – a necessity given that small sample sizes
C. Maximino et al.
decrease convergence of parameter fits. A piecewise model, which al-
lows modeling separate trajectories for the same subjects, was used
(Flora et al., 2008). Behavior at preconditioning was used as one piece,
and the retrieval piece covered the 4 time points of the post-
conditioning trials. Those two trajectories were connected by a single
intercept placed at DPC1. Competing models were compared using
linear slopes only for the preference trials. The best fit was assessed
based on Bayesian Information Criteria (BIC) and log-likelihood, with
parsimony and interpretability consistent with previous data on rodents
(Galatzer-Levy et al., 2014). The best model was selected based on
lower values for the criterion indices.
After the establishment of the latent classes, individual animal’s
probable class assignment was used in further analysis. Behavioral
endpoints measured at conditioning session between classes were
compared by unpaired Student’s t test with Welch’s correction and ef-
fect sizes were reported as Hedges’ g. Data analyzed across different
time periods were compared using RM-ANOVA with class assignment as
a fixed factor and trial as the within-subjects factor. The level of sig-
nificance was set at p ≤ 0.05 and effect sizes were reported as gen-
eralized eta squared (gεs).
3. Results
At preconditioning phase, one-way ANOVA showed that zebrafish
had a preference for the white compartment of the CPA apparatus
(F(2,93) = 9.164, p = .0002, d = 0.7221) (Fig. 2A). Animals were fur-
ther randomly distributed in such a way that CTRL and CAS groups had
similar baseline place preference (Fisher’s exact test, p = .65,
d = 0.1609) (Fig. 2B). At conditioning phase (Fig. 2C), unpaired Stu-
dent’s t test revealed that CAS increased freezing bouts
(t(df=30) = 2.425, p = .0215, g = −0.84), freezing duration
(t(df=30) = 2.693, p = .0115, g = −0.93), number of erratic move-
ments (t(df=30) = 3.778, p = .0007, g = −1.30) and duration of erratic
movements (t(df=30) = 4.333, p = .0002, g = −1.49) (Fig. 2D).
Behavioral data suggest significant changes regarding the color
preference in CAS-exposed animals, indicating aversion to the con-
ditioning side at different time intervals. RM-ANOVA using group and
trial period as factors yielded a significant group effect
(F(1,90) = 25.501, p < .00001, gεs = 0.222 for the group term). CAS-
exposed group had a stronger avoidance response to the white com-
partment when compared to CTRL at postconditioning trials (DPC1 to
DPC7) (Fig. 3A). Although distance traveled and crossings did not differ
between groups, a trial period effect was observed in distance traveled
(F(3,90) = 8.29, p = .001, gεs = 0.1327 for the trial term) and crossings
(F(3,90) = 5.9351, p = .0047, gεs = 0.07 for the trial term), showing
reduced locomotion across the trials (Fig. 3B). Fig. 4 depicts the
avoidance and fear-like behaviors in the conditioning compartment at
pre and postconditioning phases. In CAS-exposed animals, risk assess-
ment and freezing increased at DPC1–DPC7 (F(1,90) = 26.99,
p < .0001, gεs = 0.403 for the group term) and DPC1–DPC2
(F(3,90) = 5.7, p = .005, gεs = 0.11 for the interaction term), respec-
tively. Regarding the number of erratic movements, RM-ANOVA
yielded no statistical differences after correction for sphericity
(F(3,90) = 2.2, p = .119, gεs = 0.029 for the interaction term).
Using the HLME, we found that a 2-class model best fits the data
(Akaike’s information criteria: 836.21 for 2-class model, vs. > 841 for
1-class, 3-class, and 4-class models). The largest class (here noted as
“high avoiders”) was composed of 81.25% of animals and was char-
acterized by a steady decline in time in conditioning side 24 and 48 h
after CAS exposure, reaching an asymptote up to 7 DPC. The second
class (described herein as “low avoiders”) was composed of 18.75% of
animals and was characterized by an initial decrease in the time spent
in conditioning side 24 h after CAS exposure, followed by a return to
control levels. Fig. 5 shows the main behaviors observed for both
classes. Unpaired Student’s t test revealed that high avoiders had in-
creased freezing bouts (t(df=6.254) = 2.469, p = 0.0469, g = 1.00) and
38
Behavioural Processes 149 (2018) 35–42
decreased erratic movements (t(df=9.649) = 3.623, p = .0049,
g = −1.29) at conditioning session when compared to low avoiders. No
significant differences were observed in the duration of these behaviors
(t(df=10.42) = 0.7998, p = .4417, NS for freezing and
t(df=2.611) = 0.7142, p = .5336, NS for erratic movements) (Fig. 5A).
Finally, RM-ANOVA yielded significant differences between high and
low avoiders regarding the time spent in the conditioning side
(F(3,42) = 6.6, p = .0009, gεs = 0.26 for the interaction term). Both
classes did not differ in the number of risk assessment, freezing bouts,
erratic movements, distance traveled, and crossings across trials. In all
cases, except for erratic swimming, a trial effect was observed (Fig. 5B).
Table 1 depicts all experimental data obtained for high and low avoider
fish.
4. Discussion
Here, we report the use of CPA paradigm to assess the behavioral
effects of CAS in adult zebrafish. We demonstrate a persistence of CS
aversion (up to 7 DPC) following a single CAS exposure and increased
risk assessment and freezing episodes across postconditioning trials.
Interestingly, while freezing and erratic swimming increased at con-
ditioning phase, erratic swimming did not change after conditioning
session. Additionally, we observed the existence of two behavioral
phenotypes (high- and low-avoider fish) that present different fear-like
responses at conditioning phase and avoidance of the conditioning side
at postconditioning sessions. Considering the persistence of evasion
after a single exposure to a chemical cue that elicits fear (Canzian et al.,
2017; Nathan et al., 2015; Ogawa et al., 2014) and exerts a time-de-
pendent sensitization of anxiety-like behaviors (Lima et al., 2016), our
novel findings expand the analysis of aversive behaviors in zebrafish.
Because zebrafish display a high genetic and physiological con-
servation (Levin et al., 2015; Stewart et al., 2015), as well as robust
aversive behavioral responses (Kalueff et al., 2013), this species is a
suitable model organism in biological psychiatry and translational
neuroscience research (Caramillo et al., 2015; Lima et al., 2016;
Maximino et al., 2015; Stewart et al., 2014). Here, CAS exposure was
associated with the most preferred side of the CPA apparatus, eliciting
avoidance tendencies as well as fearful responses (freezing, risk as-
sessment) which were not observed in the ‘safe’ compartment. Since
avoidance conditioning carries at least two factors (Pavlovian con-
ditioning and instrumental conditioning of the avoidance response)
(Krypotos et al., 2015), the persistence of freezing are assumed to be
(classically) a conditioned contextual response. Despite the long-term
avoidance and fear responses are useful to assess aversive behaviors,
they help assess learning and memory in zebrafish. While it is not clear
the time intervals tested following CAS exposure previously (Hall and
Suboski, 1995a; Hall and Suboski, 1995b), classically conditioned CAS
responses are maintained after training (Lima et al., 2016). Although
changes in locomotor activity and exploration are not directly involved
in the preference response, they markedly reduced at postconditioning
trials, suggesting habituation to the test apparatus in control and CAS-
exposed fish.
We have also demonstrated heterogeneity in evasion responses, a
phenomenon observed in active avoidance in rodents (Galatzer-Levy
et al., 2014) and in inhibitory avoidance in zebrafish (Manuel et al.,
2014). Here, two distinct populations provide better model fit than a
single population estimate. Specifically, two phenotypes were un-
covered by latent growth modeling: the first (“high avoiders”, 81.25%
of animals) showed signals of avoidance 24 h after training (DPC1) and
achieved asymptotic levels of avoidance 48 h after training (DPC2). The
second phenotype (“low avoiders”; 18.75% of animals) showed evasion
at DPC1, but later return to baseline levels. Thus, we demonstrate that
both high and low avoiders display aversive responses following a
single CAS exposure, suggesting one-trial learning. Nonetheless, dif-
ferently than low avoiders, high avoiders showed pronounced escaping
behavior (DPC1 to DPC7), which reveals a clear persistence of evasion
C. Maximino et al. Behavioural Processes 149 (2018) 35–42

Fig. 2. Basal preference of zebrafish (preconditioning session) and CAS-induced fear-like behaviors (conditioning session). (A) Time spent in yellow, central, and white compartments of
the CPA apparatus. Data were expressed as means ± S.E.M and analyzed by one-way ANOVA followed by Tukey’s HSD test (* p ≤ 0.05, *** p ≤ 0.005, n = 32). (B) Initial preference of
zebrafish randomly distributed into control (CTRL) and conspecific alarm substance (CAS) groups. Data were expressed as means ± S.E.M and the proportion of animals preferring each
compartment was assessed using a Fisher exact test, considering p ≤ 0.05 as statistical significance. (C) Schematic cartoons of the conditioning phase. In a random order, CTRL group was
exposed to non-chlorinated water, while CAS group was conditioned to CAS in the most preferred compartment of the CPA apparatus and paired with non-chlorinated in the other
compartment. (D) Fear-related behaviors of zebrafish during conditioning. Data were expressed as means ± S.E.M and analyzed by unpaired Student’s t test (* p ≤ 0.05, *** p ≤ 0.005)
(n = 16 per group).

for at least 7 days postconditioning. While no other differences between
phenotypes were observed after conditioning, high avoiders froze more
and displayed less erratic swimming than low avoiders during CAS
exposure. Conversely, the occurrence of freezing during conditioning is
incompatible with a successful evasion in active avoidance conditioning
of rodents (Choi et al., 2010). It is unclear whether freezing in zebrafish
has the same function as freezing in rodents, because it is not con-
sistently observed during CAS exposure (Canzian et al., 2017; Quadros
et al., 2016; Ruhl et al., 2017; Speedie and Gerlai, 2008). Interestingly,
CAS had a higher effect on erratic swimming (g = −5.21) than freezing
(g = −3.34), suggesting that erratic swimming is a better index of
alarm reaction – and, therefore, increased fear levels at conditioning
phase could impair avoidance learning. Studying animals with high and
low avoidance is an interesting strategy to understand the mechanisms
underlying avoidance conditioning (Choi et al., 2010). Acquisition and
retrieval processes following CAS conditioning recruit the dorsomedial
telencephalon, while only retrieval recruits the dorsolateral tele-
ncephalon, a hippocampus homolog (Ruhl et al., 2017). Given the
homology between the dorsomedial telencephalon and the mammalian
basolateral amygdala, different activities in CNS regions may possibly
reflect the heterogeneity observed herein. However, more studies are
needed to elucidate how fear-like behaviors influence the persistence of

39
C. Maximino et al. Behavioural Processes 149 (2018) 35–42

Fig. 3. CAS induces conditioned place avoidance without changing locomotion. (A) Changes on preference scores at 1, 2, 3, and 7 DPC, calculated as the difference (in seconds) of time
spent in the CAS-paired compartment at post and preconditioning phases. Data were expressed as means ± S.E.M and analyzed using RM-ANOVA followed by Tukey’s HSD test (* p ≤
0.05, ** p ≤ 0.01). (B) Locomotor activity of zebrafish in the CPA apparatus. Data were expressed as means ± S.E.M and analyzed using RM-ANOVA (n = 16 per group).

Fig. 4. Avoidance and fear-like behaviors at preconditioning and postconditioning phases. The number of risk assessment episodes, freezing bouts, and erratic movements in CAS-paired
compartment were quantified at preconditioning and at different postconditioning intervals. Data were expressed as means ± S.E.M and analyzed using RM-ANOVA followed by Tukey’s
HSD test (n = 16 per group).

Fig. 5. Effects of CAS in zebrafish displaying high avoidance (HA) and low avoidance (LA) to the conditioning compartment. (A) Fear-related behaviors of HA and LA zebrafish at
conditioning phase. Data were expressed as means ± S.E.M and analyzed by unpaired Student’s t test with Welch’s correction (* p ≤ 0.05, ** p ≤ 0.01). (B) Avoidance, fear-like
behaviors, and locomotion of HÁ and LA at preconditioning and postconditioning phases. Data were expressed as means ± S.E.M and analyzed using RM-ANOVA followed by Tukey’s
HSD test.

avoidance responses in zebrafish.
5. Conclusion
Collectively, we report for the first time the persistence of a con-
ditioned place aversion across different time intervals in zebrafish after
a single CAS conditioning session. CAS elicits fear-related behaviors and
avoidance responses for at least 7 DPC, suggesting a persistence of
aversion. Our novel findings related to different escaping responses
observed across postconditioning trials (as described for high and low
avoiders) raise the possibility to investigate the molecular bases and the
neurochemical mechanisms underlying these phenotypes. Additionally,
we reinforce the use of CAS associated with CPA paradigm as a valuable
strategy for assessing anxiety, trauma- and stressor-related behavioral
endophenotypes in order to improve the face validity of zebrafish
models of psychiatric disorders.

40
C. Maximino et al. Behavioural Processes 149 (2018) 35–42

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