PBB-72117; No of Pages 9

Pharmacology, Biochemistry and Behavior xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Pharmacology, Biochemistry and Behavior

journal homepage: www.elsevier.com/locate/pharmbiochembeh

Anxiogenic-like effects of chronic nicotine exposure in zebrash

Adam Michael Stewart a,b, Leah Grossman a,c, Adam D. Collier d, David J. Echevarria b,d, Allan V. Kalueff a,b,e,
a

ZENEREI Institute, 309 Palmer Court, Slidell, LA 70458, USA

The International Zebrash Neuroscience Research Consortium (ZNRC), 309 Palmer Court, Slidell, LA 70458, USA
c
St. George's University School of Medicine, Grenada, West Indies
d
Department of Psychology, University of Southern Mississippi, 118 College Drive, Hattiesburg, MS 39406, USA
e
Research Institute of Marine Drugs and Nutrition, College of Food Science and Technology, Guangdong Ocean University, Zhanjiang, Guangdong, China
b

a r t i c l e

i n f o

Available online xxxx

Keywords:
Aquatic model
Nicotine
Chronic exposure
Anxiety
Neurotoxicology
Drug discovery

a b s t r a c t
Nicotine is one of the most widely used and abused legal drugs. Although its pharmacological prole has been
extensively investigated in humans and rodents, nicotine CNS action remains poorly understood. The importance
of nding evolutionarily conserved signaling pathways, and the need to apply high-throughput in vivo screens
for CNS drug discovery, necessitate novel efcient experimental models for nicotine research. Zebrash (Danio
rerio) are rapidly emerging as an excellent organism for studying drug abuse, neuropharmacology and toxicology
and have recently been applied to testing nicotine. Anxiolytic, rewarding and memory-modulating effects of
acute nicotine treatment in zebrash are consistently reported in the literature. However, while nicotine abuse
is more relevant to long-term exposure models, little is known about chronic effects of nicotine on zebrash behavior. In the present study, chronic 4-day exposure to 12 mg/L nicotine mildly increased adult zebrash
shoaling but did not alter baseline cortisol levels. We also found that chronic exposure to nicotine evokes
robust anxiogenic behavioral responses in zebrash tested in the novel tank test paradigm. Generally paralleling
clinical and rodent data on anxiogenic effects of chronic nicotine, our study supports the developing utility of
zebrash for nicotine research.
2015 Elsevier Inc. All rights reserved.

1. Introduction
Like alcohol and caffeine, nicotine (Fig. 1) is one of the most widely
used legal drugs (Crocq, 2003). Acting as a classical agonist of nicotinic
acetylcholine receptors, it modulates the release of several neurotransmitters, including dopamine, norepinephrine and serotonin (Dong et al.,
2010; Eddins et al., 2009, 2010), also see (Clarke and Reuben, 1996;
Ribeiro et al., 1993; Wonnacott, 1997) for details. Used as a mild
psychostimulant by healthy individuals, nicotine is also a major
substance of abuse (NIDA, 2014; WHO, 2013), causing strong dependence and harm to the users (Fig. 1AB; Gable, 2006; Morgan et al.,
2010; Nutt et al., 2010). Not surprisingly, nicotine is ranked among
the top 5 most dangerous abused drugs, after alcohol, heroin, cocaine
and methamphetamine (Carhart-Harris and Nutt, 2013; Nutt et al.,
2010).
Nicotine use is currently the main preventable cause of disease,
disability and death worldwide (CDC, 2014; NIDA, 2014). While
nicotine abuse occurs due to tobacco intake, tobacco contains a variety
of other toxic chemicals that account for much of the harm, in addition
to that caused by nicotine (Nutt et al., 2014; Rose et al., 2010). Nicotine
Corresponding author at: ZENEREI Institute, 309 Palmer Court, Slidell, LA 70458, USA.
Tel./fax: +1 240 328 2275.
E-mail address: avkalueff@gmail.com (A.V. Kalueff).

abuse is widespread, currently affecting ~ 30% of the global adult

population and in some countries exceeding 50% (Bank, 2014; WHO,
2013); Fig. 1C. Nicotine abuse is particularly increased among drug
addicts, operating as a gateway drug to increase brain susceptibility
to other drugs of abuse, such as cocaine (Levine et al., 2011). Nicotine
use is also highly comorbid with other psychiatric disorders, with
bidirectional inuences observed between the disorder and nicotine
dependence (Griesler et al., 2011). Collectively, this implicates nicotine
use as a major biomedical and societal problem, meriting further indepth translational studies.
Acute nicotine exposure evokes positive reinforcing effects, including mild euphoria, heightened arousal, diminished appetite, and
reduced anxiety, while also enhancing aspects of attention and
cognition, in human subjects (Heishman et al., 2010; Pomerleau and
Pomerleau, 1992; Pomerleau et al., 1984). However, whereas chronic
nicotine users report an overall prole of rewarding sensations,
increased comfort and reduced negative mood, such states are
accompanied by negative effects, such as tension and jitteriness
(Cohen and George, 2013; Henningeld and Goldberg, 1983; Rose
et al., 2010; Sofuoglu et al., 2008). Moreover, the perceived calming
effects of continued nicotine exposure may simply represent the
reversal of the negative affect induced by nicotine deprivation, thereby
forming an overall anxiogenic prole (Cohen and George, 2013; Parrott,
1995, 1998).

http://dx.doi.org/10.1016/j.pbb.2015.01.016
0091-3057/ 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Stewart AM, et al, Anxiogenic-like effects of chronic nicotine exposure in zebrash, Pharmacol Biochem Behav (2015),
http://dx.doi.org/10.1016/j.pbb.2015.01.016

A.M. Stewart et al. / Pharmacology, Biochemistry and Behavior xxx (2015) xxxxxx

Addiction

Tranquilizers

Nicotine

High

Nicotine

Depressants

Alcohol
Caffeine

Medium

Alcohol

Caffeine
Low

Psychostimulants

ED50/LD50
0.001

0.01

0.1

Hallucinogens

1.0

Zebrafish publications on nicotine

15
10
5
0

2004

2008

2014

Alcohol (433)
Caffeine (36)
Nicotine (44)

Acute 20-min nicotine exposure (10 mg/L)

0s

Time
360 s

3-D trace reconstructions

Fig. 1. The growing utility of zebrash models for nicotine research. (A) Summary of addictive potential and toxic properties (ED50/LD50 ratio) of acute nicotine, compared to alcohol and
caffeine, according to (Gable, 2006). Right subpanel: classication of main classes of psychotropic drugs. (B) Global tobacco smoking statistics (% of the population) among adults (WHO,
2013), based on graphs freely available in the public domain via Wikipedia. (C) The growing body of zebrash literature on nicotine, based on PubMed search (December 2014).
(D) examples of anxiolytic-like behavior typically produced by acute nicotine (10 mg/L) in the novel tank test (modied from (Cachat et al., 2011), published under the Creative Commons
Attribution license).

Complementing clinical evidence, nicotine's psychopharmacological

prole has been extensively investigated in various animal models
(Matta et al., 2007). For example, acute administration of nicotine at
various doses in rodent models evokes robust anxiolytic-like responses
(Elliott et al., 2004; Irvine et al., 1999); also see similar effects produced
by other cholinergic agonists (Brioni et al., 1994; Turner et al., 2010).
Paralleling clinical ndings, chronic administration induces consistent
anxiogenic-like behavior in rodents treated with nicotine (Caldarone
et al., 2008; Elliott et al., 2004; Mesa-Gresa et al., 2013) and some
other nicotinic agonists (Turner et al., 2010) (see, however, (Turner
et al., 2011)).
Several considerations necessitate novel approaches to studying
nicotine action, as our increased understanding of its molecular effects
and genetic contributions may lead to improved treatment strategies
(Klee et al., 2011, 2012). For example, nicotine research and screening
of drugs with anti-nicotine properties benet markedly from the availability of high-throughput, low-cost alternatives to rodent models.
Being a vertebrate species with high genetic and physiological
homology to mammals, zebrash (Danio rerio) are rapidly emerging
as an excellent model organism for studying pharmacology and
toxicology of various CNS drugs (Grossman et al., 2010; Maximino
et al., 2014; Neelkantan et al., 2013; Tran et al., 2014; Wong et al.,
2010b), including nicotine (Cousin et al., 2014; Eddins et al.,
2010; Klee et al., 2011; Levin, 2011). Moreover, zebrash have a sophisticated molecular genetic tool set, markedly increasing their value and

application for pharmacogenetic research. For example, in addition to

N-ethyl-N-nitrosourea (ENU)-induced (Mullins et al., 1994) and
viral vector-mediated (Amsterdam et al., 1999) mutagenesis or
morpholino-evoked gene silencing, other genetic tools in zebrash include gene-breaking transposon screens (Petzold et al., 2009), clustered regularly interspaced short palindromic repeats (CRISPR)
(Hwang et al., 2013), transcription activator-like effector nuclease
(TALEN) (Zu et al., 2013) and targeting induced local lesions in genomes (TILLING) (Moens et al., 2008). Several genetically modied
zebrash strains, developed using such methods and possessing altered
nicotine responses, have already been reported (Petzold, 2010; Petzold
et al., 2009). Finally, the importance of identifying evolutionarily conserved mechanisms of drug actions further necessitate expanding the
range of model organisms, recognized as a key strategic direction for
drug discovery (Cousin et al., 2014; Klee et al., 2012; Robinson et al.,
2013; Stewart et al., 2014).
Behavioral effects of acute nicotine treatment in zebrash, previously reported in the literature (Klee et al., 2011), include anxiolytic-like
action and potent modulation of cognitive responses in various
memory/learning tasks (Bencan and Levin, 2008; Eddins et al., 2009;
Levin, 2011; Levin et al., 2006, 2007; Levin and Chen, 2004). In addition,
nicotine evokes psychostimulant (Petzold, 2010; Petzold et al., 2009)
and robust reward-like (e.g., preference) behavior in zebrash (Cousin
et al., 2014; Kedikian et al., 2013; Kily et al., 2008; Klee et al., 2012),
making these sh an excellent model organism to study nicotine

Please cite this article as: Stewart AM, et al, Anxiogenic-like effects of chronic nicotine exposure in zebrash, Pharmacol Biochem Behav (2015),
http://dx.doi.org/10.1016/j.pbb.2015.01.016

A.M. Stewart et al. / Pharmacology, Biochemistry and Behavior xxx (2015) xxxxxx

addiction. Initially reported anxiolytic-like effects of acute nicotine in

zebrash (Levin et al., 2007) have been replicated by several laboratories (Cachat et al., 2011; Levine et al., 2011; Sackerman et al., 2010) as
a robust and consistent phenotype in this model species (also see
three-dimensional visualization of locomotor traces in nicotinetreated sh in (Stewart et al., 2014) and Fig. 1). Interestingly, acute nicotine exposure also affects sh behavior in the shoaling test, another
widely used and sensitive zebrash behavioral model, signicantly reducing shoal cohesion and swimming speed (Miller et al., 2013).
While shoaling test reects social behavior in sh species, it is also relevant to anxiety (typically tightening zebrash shoals; Kalueff et al.,
2013). Thus, acute behavioral effects of nicotine in zebrash appear to
be generally anxiolytic in nature.
However, little is known about chronic effects of nicotine exposure
on zebrash anxiety and other related behaviors. This knowledge gap
is surprising, especially because of its increasing usage globally (WHO,
2013), nicotine exposure represents a growing concern for environmental health science, including aquatic biology and toxicology
(Bradley et al., 2007; Buerge et al., 2008). Because both nicotine abuse
and the environmental nicotine effects are more relevant to chronic
exposure protocols, it is therefore important to know what this
compound does in vivo, if given chronically. To address this problem,
here we examined the effects of chronic 4-day nicotine exposure on
zebrash behavior. Utilizing the novel tank test as one of the most
popular behavioral tests in zebrash (Cachat et al., 2011; Levin et al.,
2007; Stewart et al., 2014), here we report anxiogenic-like responses
of zebrash to chronic treatment with nicotine. In addition, chronic
nicotine also increased group cohesion in zebrash shoaling test
but did not cause robust alterations in baseline whole-body cortisol
concentrations.
2. Methods
2.1. Animals and housing
A total of 59 adult (57 months old) wild-type zebrash (~ 50:50
male:female ratio) were obtained from a commercial distributor
(50 Fathoms, Metairie, LA, USA). All sh were given at least 10 days to
acclimate to the laboratory environment and housed in groups of 30
sh per 40-L tank at the Animal Core of the ZENEREI Institute,
as described earlier (Robinson et al., 2013). Tanks were lled with
ltered system water and maintained at 25 C with illumination
(10001100 lx) provided by ceiling-mounted uorescent lights on a
12-h cycle (on: 6.00 h, off: 18.00 h), according to the standards of
zebrash care. All animals used in this study were experimentally
nave and fed Tetramin Tropical Flakes (Tetra USA, Blacks- burg, VA)
twice a day. Following behavioral testing, the animals were euthanized
in 500 mg/L Tricaine (Sigma-Aldrich, St. Louis, MO) buffered to pH 7.0,
as described earlier (Robinson et al., 2013). Animal experimentation in
this study fully adhered to national and institutional guidelines and
regulations and was approved by the ZENEREI Institute.

two equal virtual horizontal portions by a line marking the outside

walls, as in (Egan et al., 2009; Robinson et al., 2013). In this model, the
increase in time spent at top, the number of entries to top and the latency to top are usually attributed to decreased anxiety, whereas increased
immobility/freezing indicates elevated anxiety (Levin et al., 2007).
During testing, zebrash behavior was recorded by 2 trained
observers blind to the treatments, who used the stopwatch and manually scored different behavioral end points (inter- and intra-rater
reliability in all experiments N 0.85), which included the latency to
reach the top half of the tank (s), time spent in top (s), number of transitions to top (top dwelling, behavior no. 1.176 in Zebrash Behavioral
Catalog; Kalueff et al., 2013) as well as the number and duration (s) of
freezing bouts (Robinson et al., 2013). Freezing (behavior no. 1.68 in
zebrash behavioral catalog; Kalueff et al., 2013) was dened as a
total absence of movement, except for the gills and eyes, for N 2 s. Trials
were also recorded to a computer using a USB web-camera (2.0
megapixels, Gigaware, UK) and analyzed by Ethovision XT8.5 (Noldus
IT, Wageningen, Netherlands), assessing swimming bouts, swimming
duration (s), mobility/high mobility activity, turning parameters
(heading, meander, rotation rate), distance traveled (m) and average
velocity (m/s), as described elsewhere (Cachat et al., 2013; Grossman
et al., 2010; Robinson et al., 2013). In order to assess intra-session
(within-trial) habituation, reecting spatial working memory of
zebrash (behavior no. 1.72 in zebrash behavioral catalog; Kalueff
et al., 2013), we examined their responses over a 5-min trial, analyzing
the per-min distribution of manually recorded behavioral end points
mentioned above and comparing the rst vs. last (5th) minute values
for each end point, similar to (Robinson et al., 2013; Stewart et al.,
2013; Wong et al, 2010a).
The shoaling test, performed in separate animal sub-cohorts
(Fig. 2C) according to (Cachat et al., 2013; Grossman et al., 2010), examined the effects of chronic nicotine exposure on sh group
behaviorshoaling (behavior no. 1.141 in zebrash behavioral catalog;
Kalueff et al., 2013), Fig. 3). Briey, groups of 6 zebrash were removed
from control or drug-treated home tanks, transferred to the novel tank
apparatus and group-tested for 5 min (a total of 3 cohorts of 6 sh per
treatment). Following the initial 3-min acclimation period (necessary
to form shoals in novel testing environment), zebrash groups were
photographed from a 10-cm distance for 2 min, taking snapshots
every 5 s. A total of 10 consecutive snapshots per cohort were used for
analyses in this study. Each snapshot was properly calibrated and
analyzed by a trained observer (intra-rater reliability N90%), manually
measuring the distances (cm) between each sh in the group and
then averaging this data to obtain an average inter-sh distance per
snapshot according to Cachat et al. (2013) and Grossman et al. (2010).
In addition, the shoal size (cm) and distances to the farthest and the
nearest neighbors (cm) were analyzed for each snapshot and then
averaged for the respective cohort, as described in (Kyzar et al., 2012);
also see Fig. 3A for visual explanation of the shoaling end points taken.
Final shoaling data for control and experimental cohorts represented
averaged results for all screenshots from each of the three 6-sh groups
per treatment (18 sh per group).

2.2. Behavioral testing

2.3. Pharmacological manipulations
Behavioral testing was performed following a 4-day chronic
exposure to nicotine (Fig. 2). All behavioral tests reported here were
performed in a regular testing laboratory room under standard illumination (~10001100 lx) during the light cycle (between 11.00 am and
2.00 pm), using tanks with water adjusted to the holding room temperature (25 C). On the day of testing, the sh were individually caught
with the net, removed from their home tanks and exposed to the 5min novel tank test, used as a standard procedure to assess zebrash
anxiety and locomotion, as described earlier (Robinson et al., 2013).
Briey, the apparatus used consisted of a 1.5-L trapezoidal tank
(15 cm height 28 cm top 23 cm bottom 7 cm width; Aquatic
Habitats, Apopka, FL) maximally lled with water and divided into

Nicotine for this study was obtained from Sigma-Aldrich (St. Louis,
MO) and added to home tank water housing 10 experimental sh
4 days prior to testing. The dosage for chronic 4-day nicotine treatment
was chosen based on previous studies from our laboratory on acute
nicotine exposure, as well as conversions from rodent literature.
Specically, to avoid the possibility of toxic effects of nicotine, the initial
1 mg/L dose was selected to be signicantly (i.e., 10-fold) lower than the
acutely active 10 mg/L dose reported in our previous studies (Cachat
et al., 2011; Stewart et al., 2014). Because nicotine evokes tolerance, a
dose increment to 2 mg/L was applied during the last 2 days of the 4day chronic treatment protocol. The time course of the protocol is

Please cite this article as: Stewart AM, et al, Anxiogenic-like effects of chronic nicotine exposure in zebrash, Pharmacol Biochem Behav (2015),
http://dx.doi.org/10.1016/j.pbb.2015.01.016

A.M. Stewart et al. / Pharmacology, Biochemistry and Behavior xxx (2015) xxxxxx

Latency to top, s

300

Top transitions

45

200

100

15

Control Nicotine
(n=12) (n=11)

Freezing bouts

75

50

25

30

Control Nicotine
(n=12) (n=11)

*
Control Nicotine
Control

Time freezing, s

100

Time in top, s

60

Nicotine
0

Control Nicotine
(n=12) (n=11)

Top transitions
Water control
Chr Nicotine

1.5

Control Nicotine
(n=12) (n=11)

Time in top, s

15

Water control
Chr Nicotine

10

0.5

Water control
Chr Nicotine

15
10

Time freezing, s

20

2
3
4
Time (min)

5
1

Control fish
Chronic nicotine

3
4
Time (min)

Novel tank
testing
(n=11-12)

Shoaling
testing,
animal
sacrifice
(n=18)

3
4
Time (min)

Cortisol
ELISA
assay
(n=16-17)

Fig. 2. Behavioral effects of chronic 4-day exposure to 12 mg/L nicotine on adult zebrash tested in the novel tank test. Panel A shows manual behavioral end points recorded in standard
5-min novel tank test (n = 1112 per group), as well as representative two-dimensional (2D) traces generated in XY coordinates (side view) by Noldus EthoVision XT8.5 software. In all
experiments, representative traces selected after rating the activity of each subject/cohort from 1 to n based on activity level, and reaching consensus by two highly-trained investigators.
Panel B shows per-minute distribution of selected end points. Data are reported as mean SEM, *P b 0.05 vs. control, #P = 0.050.1, trend (U-test). Within-trial habituation analyses for
manual data are shown as per-minute distribution of activity (*P b 0.05 min 1 vs. min 5, #P = 0.050.1, trend; paired U-test with the Bonferroni correction). Automated data for this
experiment are provided in Table 1. Panel C outlines the overall experimental design of the present study.

based on previous rodent studies showing that tolerance is obtained at

24 days of chronic exposure (Collins et al., 1988; Marks et al., 1985).
The selected dose regimen did not result in overt toxicity, as assessed
by zebrash survival data (100%) during chronic treatment in the
home tank, and by generally unaltered motor responses in the nicotine
cohort in the novel tank test (see Results).
2.4. Cortisol assay
Whole-body cortisol was assessed in zebrash used in the shoaling
test described above (Fig. 2C). Immediately after shoaling testing, the
sh were euthanized and whole-body samples taken. Individual body
samples from experimental and control cohorts were homogenized in
500 l of ice-cold 1 PBS buffer, according to Cachat et al. (2010b)
and Egan et al. (2009). Briey, samples were transferred to glass
extract-O tubes, and cortisol was extracted twice with 5 ml of diethyl

ether (Fisher Scientic, Pittsburgh, PA). After ether evaporation, cortisol

was reconstituted in 1 ml of 1 PBS. To quantify cortisol concentrations,
ELISA was performed using a human salivary cortisol assay kit
(Salimetrics LLC, State College, PA) (Cachat et al., 2010b; Egan et al.,
2009). ELISA plates were measured in a VICTOR-WALLAC plate reader
using the manufacturer's software package. Whole-body cortisol levels
in zebrash were determined here using a 4-parameter sigmoid
minus curve t based on the absorbencies of standardized concentrations and presented as relative concentrations per gram of body weight
for each sh (n = 1617 per group), as described previously (Cachat
et al., 2010b; Egan et al., 2009).
2.5. Statistical analyses
The novel tank behavioral data were analyzed using an unpaired
WilcoxonMannWhitney U-test to compare control vs. nicotine-

Please cite this article as: Stewart AM, et al, Anxiogenic-like effects of chronic nicotine exposure in zebrash, Pharmacol Biochem Behav (2015),
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A
Farthest neighbor distance
Nearest neighbor distance

Shoal size

Control shoal

Chronic nicotine-treated fish

Fig. 3. Behavioral effects of chronic 4-day exposure to 12 mg/L nicotine on adult zebrash tested in the shoaling test. Panel A illustrates manual behavioral end points recorded in standard
5-min shoaling test (6 sh per shoal, 3 shoals per group). Panel B shows representative control and nicotine-treated shoals (see text for data analyses).

treated groups. Inter- and intra-rater reliability for the observers was
determined by Spearman correlation. Data were expressed as
mean SEM. In habituation assays, behavioral data generated for
manual observation were analyzed using a two-sample paired U-test
for signicance between the initial (min 1) and the last (min 5) observation time, followed by the Bonferroni correction. Signicance was set at
P b 0.05 for U-test, but was adjusted accordingly for Bonferroni
corrected post-hoc tests, similar to (Robinson et al., 2013; Wong et al.,
2010a).
3. Results
In the novel tank test, chronic 4-day exposure to 12 mg/L nicotine
evoked a generally anxiogenic-like response, signicantly increasing
the latency to enter the top half of the tank as well as reducing the
number of entries and time spent in top (Fig. 2A). However, there
were no effects on freezing behaviors (frequency and duration), most

Table 1
Behavioral and physiological effects of chronic 4-day exposure to 12 mg/L nicotine
on adult zebrash. Behavioral data were recorded in the standard 5-min novel tank test
(n = 1112 per group) by Noldus EthoVision XT8.5 software. Manual data from this experiment are shown in Fig. 2. Physiological data (whole-body cortisol) were obtained in
a separate cohort of experimentally nave zebrash (n = 1617 per group). Data are reported as mean SEM, *P b 0.05 vs. control, NS no signicance (U-test).
End point
Behavioral data
Distance traveled, cm
Average velocity, cm/s
Mean meander, degree/cm
Mean turn angle, degree
Immobility duration, s

Immobility episodes (number)

Mobility duration, s
Mobility episodes (number)
Number of full 360-degree rotations

Control

Chronic
nicotine

P
(U-test)

1259 263
4 0.9
1.6 0.6
0.4 0.16
238 15
482 104
60 15
487 105
16 4

NS
NS
NS
NS
NS
NS
NS
NS
NS

0.019 0.002

NS

1195 114
4 0.4
0.7 0.1
0.2 0.04
241 7
703 78
56 7
710 79
14 2

Physiological data
Whole-body cortisol, ng/g body weight 0.014 0.001

Calculated as b20% average individual velocity per trial.

Calculated as 2060% average individual velocity per trial.

likely due to low baseline level of this behavior. Finally, automated

computer-generated data (Table 1) show unaltered motor patterns in
zebrash following chronic nicotine exposure, suggesting that motor
activity per se (e.g., distance traveled, velocity and mobility/immobility
indices) was not impaired by chronic nicotine.
Chronic nicotine exposure did not alter zebrash intra-session
(within-trial) habituation in the novel tank test because the rst vs.
last minute values for each manual end point changed in a similar
manner between the control and nicotine groups (Fig. 2B). Intrasession habituation to novelty reects spatial working memory (related
to exploration and cognition) and is observed in various species,
including zebrash (Stewart et al., 2013; Wong et al., 2010a). Generally,
over experimental time, the exploration behaviors (e.g., top dwelling)
of zebrash tend to increase, and their anxiety-like behaviors
(e.g., freezing or bottom dwelling) usually decrease gradually (Wong
et al., 2010a)a normal pattern which was also seen in control sh in
the preset study (Fig. 2B).
In the shoaling test, although the average shoal size was not affected
by chronic nicotine exposure (5.14 0.43 vs. 5.92 0.45 cm in control
zebrash, NS), the drug moderately tightened shoals by reducing the
average nearest neighbor distance (0.76 0.04 vs. 0.93 0.05 cm control, P b 0.05, U-test), but not the average farthest neighbor distance
(3.43 0.30 vs. 4.07 0.29 cm, respectively, NS) or the average
inter-sh distance (1.96 0.14 vs. 2.23 0.19 cm, NS). Finally, there
were no signicant effects on chronic nicotine exposure on zebrash
whole-body cortisol levels (Table 1).
4. Discussion
Because nicotine is one of the most commonly abused drugs, its
pharmacological prole has been extensively investigated in both
humans and rodents (Hambsch et al., 2014; Levin, 2011; Matta et al.,
2007). Such translational studies become particularly important for
cross-species analyses focusing on shared, evolutionarily conserved
mechanisms of nicotine-related modulation of brain function in various
model organisms (Kily et al., 2008; Klee et al., 2011, 2012). As
mentioned earlier, zebrash (D. rerio) have rapidly emerged as an
excellent model organism to study drug abuse, as well as pharmacology
and toxicology of various neuroactive compounds (Kalueff et al., 2014a,
2014b; Neelkantan et al., 2013; Stewart et al., 2014). Zebrash also
posses well-developed cholinergic neural signaling pathways, which

Please cite this article as: Stewart AM, et al, Anxiogenic-like effects of chronic nicotine exposure in zebrash, Pharmacol Biochem Behav (2015),
http://dx.doi.org/10.1016/j.pbb.2015.01.016

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are the main targets of CNS action of nicotine (Ackerman et al., 2009;
Bencan and Levin, 2008; Braida et al., 2014; Edwards et al., 2007;
Menelaou et al, 2014; Papke et al., 2012; Petzold et al., 2009; Ponzoni
et al., 2014).
Not surprisingly, zebrash have also became a popular model
organism for nicotine research (Fig. 1C), including studies of anxiety
(Ackerman et al., 2009; Levin, 2011; Levin et al., 2007), cognitive performance (Braida et al., 2014; Levin, 2011; Levin and Chen, 2004), social
behaviors (Miller et al., 2013) and reward (Kedikian et al., 2013; Kily
et al., 2008; Klee et al., 2012; Petzold, 2010). For example, paralleling
clinical (Pomerleau et al., 1984) and rodent studies (Cheeta et al.,
2001b), anxiolytic-like effects of acute nicotine treatment in zebrash
have been consistently reported in the literature (Bencan and Levin,
2008; Levin et al., 2007; Stewart et al., 2014). These effects were
reversed by 7- and 42 cholinergic receptor antagonists, implicating
both types of nicotinic receptors in the nicotinic modulation of
anxiolysis in zebrash (Bencan and Levin, 2008). Likewise, rewarding
properties of acute nicotine administration have also been reported in
humans (Harvey et al., 2004), rodents (Henningeld and Goldberg,
1983) and zebrash (Kily et al., 2008; Klee et al., 2012), strongly
supporting the possibility of shared and conserved behaviors acrossvarious species. Finally, acute nicotine also affects zebrash social
behaviors, robustly decreasing shoal cohesion, also reducing swimming
speed, moderately disrupting shoaling polarization, but not affecting
the number of sh leaving the shoals (Miller et al., 2013).
Despite the fact that nicotine abuse is more relevant to a chronic
(rather than acute) exposure, little is known about behavioral and
physiological effects of chronic nicotine in zebrash. Mounting
evidence suggests that, unlike acute nicotine, chronic administration
of this drug evokes anxiety-like effects in various rodent models. For
example, chronic nicotine reduced novelty seeking (Aydin et al., 2012)
and exploration (Faraday et al., 1999; Irvine et al, 2001) in rats, as
well as inhibited exploratory activity and increased anxiety in the
elevated plus maze in mice (Mesa-Gresa et al., 2013) (also note sexand age differences in observed anxiogenic responses; Caldarone et al.,
2008; Elliott et al., 2004). Chronic nicotine exposure also has longlasting developmental effects, as assessed in adult mice in the open
eld and elevated plus maze, following a chronic exposure to nicotine
during adolescence (Huang et al., 2007) (also see similar effects in
rats; Slawecki et al., 2004). Furthermore, chronic nicotine exposure
affects cognitive functions (Hambsch et al., 2014), also inducing longlasting synaptic modulation in the amygdala, an important part of
brain's emotional circuits (Huang et al., 2008). In line with this, rat
studies also reveal a modulatory role of chronic nicotine on memoryaversion interplay in various tasks, such as conditioned place preference
(Brielmaier et al., 2008).
Several studies have examined the effects of chronic nicotine
exposure on larva and adult zebrash CNS and physiology. For example,
continuous nicotine exposure evokes neurodevelopmental and neurological decits in larval zebrash (Parker and Connaughton, 2007;
Svoboda et al., 2002). Chronic treatment also evokes stimulant action,
which was altered in several mutant strains (Petzold, 2010; Petzold
et al., 2009). Chronic nicotine exposure to larval zebrash also alters
the expression of endocrine disruptor biomarker genes, suggesting
that nicotine may serve as an anti-estrogenic endocrine disruptor
(Kanungo et al., 2012). In adult zebrash, repeated exposure to nicotine
leads to a robust reward behavior, which persisted for several weeks,
and was accompanied by altered expression of over 1000 genes, many
of which regulate pathways implicated in drug dependence in mammalian models (Kily et al., 2008). Our present study is novel and important
because it extends chronic nicotine exposure model in zebrash to affective (anxiety-related) neurobehavioral domain (Fig. 2).
Nevertheless, there were also several limitations of our study. For
example, we utilized the novel tank test, chosen as one of the most
widely used, sensitive and reliable zebrash behavioral paradigms
(Cachat et al., 2011, 2013; Egan et al., 2009). However, several other

useful anxiety-sensitive paradigms recently developed in zebrash, including open eld and light-dark box tests (Blaser and Rosemberg,
2012; Echevarria et al., 2011; Rosemberg et al., 2011; Stewart et al.,
2011b, 2014), may also be applied to chronic nicotine exposure in future
studies. Another aspect, common for anxiogenic manipulations, relates
to the possibility of altered locomotor activity (which may mask anxiety
responses and therefore complicate data interpretation). For example,
strong anxiety responses evoked by chronic nicotine, might have
masked specic behaviors, such as freezing, the frequency and duration
of which was already low in this study (Fig. 2). In line with this possibility, reduced motor activity following chronic nicotine has also been
reported in some rodent models (Cheeta et al., 2001a; Mesa-Gresa
et al., 2013) (note, however, that other studies showed increased
anxiety but unaltered locomotion; Irvine et al., 2001). Our analyses of
automated computer-generated data (Table 1) revealed unaltered
motor activity in zebrash following chronic 4-day nicotine exposure,
suggesting that motor function (as assessed by distance traveled, velocity and mobility/immobility indices) was not impaired by nicotine. In
contrast, signicant changes in anxiety-related end points, such as top
exploration (Fig. 2A) indicate that anxiogenic-like effects in
zebrash treated with nicotine were rather specic, and most likely
not inuenced by motor-related factors.
The context-dependent effects exhibited by nicotine in humans
(Foulds et al., 1997) and rodents (Cheeta et al., 2001a) also requires
further evaluation of zebrash models. For example, clinically, the
mood enhancing effects of nicotine, and thus its ability to reverse the
mood deterioration caused by withdrawal, are susceptible to situational
factors (Foulds et al., 1997). Rodents are also particularly susceptible to
the mediating effects of context on nicotine responsivity. For example,
social isolation strongly modulates both the anxiolytic and anxiogenic
effects of nicotine, especially in regards to the locomotor activity
(Cheeta et al., 2001a). In line with this, further investigation into
nicotine's effects (or lack thereof) on zebrash motor activity may demonstrate a more complex prole than assessed here.
Another logical question is whether testing the sh in nicotine-free
conditions after chronic drug exposure may cause a withdrawal state,
that itself can have produced anxiety in this study. Notably, zebrash
were removed from their nicotine-containing home tanks and placed
directly into the novel tank for a testing period of 5 min, as described
previously (Egan et al., 2009). Although information on metabolism
rates of chronic nicotine in zebrash is limited, it seems unlikely that
the bioavailability of nicotine after a very short 5-min period in fresh
water would be reduced sharply to a point that it would evoke a withdrawal state. In line with this notion, zebrash chronically treated
with nicotine in their home tanks seem to show increased shoaling, bottom dwelling and overt thigmotaxis (especially corner preference; own
unpublished observations)a behavioral pattern generally consistent
with pharmacogenically elevated stress/anxiety in zebrash reported
here, rather than attributable to withdrawal.
Moreover, zebrash behaviors are known to display sex, strain
(Kalueff et al., 2013; Mahabir et al., 2014; Maximino et al., 2013;
Parker et al., 2013; Sackerman et al., 2010) and individual differences
(Toms and Echevarria, 2014; Tran and Gerlai, 2013) in various tests,
including novel tank-based models. Because sex differences in nicotine
responses have long been established in various rodent behavioral
models (Caldarone et al., 2008; Elliott et al., 2004; Faraday et al., 1999),
future studies may be needed to address sex, strain or individual differences in zebrash responsivity to chronic nicotine (see, for example,
strain differences in zebrash nicotine responses in Sackerman et al.
(2010)). Finally, nicotine is well known to evoke clinical and experimental withdrawal (Cohen and George, 2013; Hughes, 2007; Irvine et al.,
2001), raising the possibility that nicotine withdrawal models can be developed for zebrash as well. For example, zebrash withdrawal models
for other drugs of abuse have been successfully developed already
(Cachat et al., 2010a; Stewart et al., 2011a; Tran et al., 2014), and a similar approach can be applied in future zebrash studies.

Please cite this article as: Stewart AM, et al, Anxiogenic-like effects of chronic nicotine exposure in zebrash, Pharmacol Biochem Behav (2015),
http://dx.doi.org/10.1016/j.pbb.2015.01.016

A.M. Stewart et al. / Pharmacology, Biochemistry and Behavior xxx (2015) xxxxxx

In addition to motor and anxiety responses, the possibility of

nicotine modulation of cognitive phenotypes may also be considered
here. For example, the effects of nicotine on memory and learning
have long been investigated in rodents, implicating nicotine administration in improved memory and learning (Levin, 2011; Schildein et al.,
2002). Similar memory-enhancing responses following acute nicotine
have also been reported in zebrash models (Bencan and Levin, 2008;
Eddins et al., 2009, 2010; Levin, 2011; Levin and Chen, 2004; Levin
et al., 2006). However, reecting zebrash exploration and spatial
working memory, intra-session habituation was clearly seen in the
present study, but was not affected by chronic nicotine treatment
(Fig. 2B). While cognitive abilities of zebrash have not been investigated here in detail, our ndings of unaltered habituation (while showing
elevated anxiety) in sh exposed to nicotine chronically are in line
with earlier reports that zebrash habituation and anxiety most likely
represent distinct behavioral phenomena in novel environments
(Stewart et al., 2013). Clearly, further, more specic memory and
learning tests may be needed in zebrash treated with nicotine
chronically, to parallel rodent and clinical literature on cognitive effects
following chronic nicotine (Amitai and Markou, 2009; Mesa-Gresa et al.,
2013; Slawecki et al., 2004).
As already mentioned, chronic nicotine exposure mildly increased
zebrash shoal cohesion in the shoaling test (Fig. 3), also causing a
similar phenotype in the home tanks during the treatment (data not
shown, own unpublished observations). Interestingly, like nicotine
action on zebrash anxiety, these effects depended on treatment
duration, differing markedly from the shoal-reducing action of
acute nicotine (Miller et al., 2013). Shoaling behavior is robustly
expressed in zebrash and is relevant to both social phenotypes and
anxiety responses (Abaid et al., 2012; Butail et al., 2013; Green et al.,
2012; Polverino et al., 2012). Zebrash shoaling is also sensitive to various pharmacological manipulations, including nicotine (Miller et al.,
2013) and other drugs of abuse (Cachat et al., 2013; Gerlai et al., 2009;
Green et al., 2012; Grossman et al., 2010; Kyzar et al., 2012; Riehl
et al., 2011). Although it is currently unclear whether mildly increased
shoaling in this study was due to higher sociability or anxiety, it did
not conict with the nicotine-induced anxiogenic responses established
in the novel tank (Fig. 2). Representing a potentially interesting nding
for chronic nicotine psychopharmacology, it merits further in-depth
analyses utilizing both anxiety-related and sociability behavioral tests
available for zebrash (Gerlai et al., 2009; Kalueff et al., 2014a, 2014b;
Stewart et al., 2014; Tran and Gerlai, 2013).
Interestingly, unaltered cortisol levels following chronic treatment
(Table 1) differed from the effects of acute 20-min exposure to nicotine
at doses 1 and 10 mg/L (both showing a marked elevation of cortisol
levels in drug-treated zebrash; data not shown). While the latter
effects can be explained by cholinergic control of the sympathetic
nervous system (e.g., activating, via nicotinic cholinoreceptors, the
adrenergic mechanisms that stimulate the adrenal gland and trigger
the production of cortisol), they are consistent with known acute
nicotine action on corticoid hormones in humans and rodents
(Mendelson et al., 2005; Pomerleau and Pomerleau, 1990; Steptoe and
Ussher, 2006; Wilkins et al., 1982). In contrast, although chronic
nicotine effects in humans are less clear (including reports of both
elevated and unaltered cortisol levels in smokers; reviewed by
Tziomalos and Charsoulis (2004)), our zebrash data are in line with recent clinical reports on the lack of elevated cortisol in chronic nicotine
users (al'Absi et al., 2013; Soldin et al., 2011). Notably, chronic exposure
to nicotine also produced conicting results in rodents, including both
elevated (Moon et al, 2014) and unaltered (Seifert et al., 1984) corticosterone levels. Collectively, this indicates that future studies are needed
to address the effects of chronic nicotine exposure on various neuroendocrine end points in zebrash and to dissect the role of central vs.
peripheral mechanisms in their regulation (e.g., note the apparent
dissociation between cortisol levels and the observed behavioral
anxiotropic action for both acute and chronic nicotine treatments).

5. Conclusion
Overall, our present study shows that zebrash, like rodents, respond to chronic administration of nicotine by altering anxiety-like behavior in the novel tank test (Fig. 2). This nding is particularly
interesting because the drug, when administered acutely, evokes
anxiolytic-like prole in both rodents (Elliott et al., 2004; Irvine et al.,
1999) and zebrash (Bencan and Levin, 2008; Levin, 2011; Levin et al.,
2007). Furthermore, our results show high sensitivity of zebrash to
chronically administered nicotine, indicating an anxiogenic-like psychopharmacological prole of this compound, which is also consistent
with its known action in clinical and rodent studies.
A strong behavioral, physiological and genetic similarity between
humans and zebrash (Alderman and Bernier, 2009; Kalueff et al.,
2013, 2014b) makes such aquatic-based models an excellent and inexpensive tool for drug discovery and small molecule screening (Bruni
et al, 2014; Kokel et al., 2010, 2012; Kokel and Peterson, 2008, 2011;
Laggner et al., 2012; Rihel et al., 2010). Our present nicotine data
(Fig. 2) are in line with this notion, demonstrating the developing utility
of adult zebrash based models for in vivo screening of nicotine-related
compounds, to complement the existing rodent experimental paradigms. Paralleling clinical and rodent data, our ndings further support
recent molecular evidence showing evolutionarily conserved cholinergic system and nicotine-related pathways in zebrash and humans
(Kily et al., 2008; Klee et al., 2011, 2012).
Finally, as already mentioned, global increase in nicotine use (WHO,
2013) raises ecological and environmental health concerns about
ecotoxicological effects of long-term exposure to low-dose nicotinecontaining surface water (Bradley et al., 2007; Buerge et al., 2008). For
example, attributed to domestic wastewater discharge, nicotine and
its metabolites have been detected in aquatic ecosystems (Bradley
et al., 2007; Buerge et al., 2008). Nicotine can also be detected in drinking water and, at nanogram concentrations, even in bottled mineral
water (Gonzalez Alonso et al., 2012). Thus, concerns about ecotoxicological risks associated with environmental concentrations of nicotine
and related agents (Buerge et al., 2008; Gonzalez Alonso et al., 2012)
necessitate novel screens to assess the effects of long-term low-dose
nicotine exposure on in vivo systems. Because shes and other aquatic
organisms serve as sensitive neurotoxicological and ecotoxicological
screens (Stewart et al., in press; Sumpter et al., 2014; Sumpter and
Margiotta-Casaluci, 2014), and given our present data, the developing
utility of zebrash for in vivo testing of chronic nicotine effects
becomes clear.
Acknowledgments
This study was supported by the ZENEREI Institute Intramural Research Program and RIMDN, GDOU. AVK is the Director of ZENEREI Institute and Chair of ZNRC.
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Please cite this article as: Stewart AM, et al, Anxiogenic-like effects of chronic nicotine exposure in zebrash, Pharmacol Biochem Behav (2015),
http://dx.doi.org/10.1016/j.pbb.2015.01.016