AbstractNitric oxide (NO) and serotonin (5-HT) interact at the molecular and systems levels to control behavioral variables, including agression, fear, and reactions to novelty. In zebrafish, the 5-HT1B receptor has been implicated in anxiety and reactions to novelty, while the 5-HT1A receptor is associated with anxiety-like behavior; this role of the 5-HT1A receptor is mediated by NO. This work investigated whether NO also participates in the mediation of novelty responses by the 5-HT1B receptor. The 5-HT1B receptor inverse agonist SB 224,289 decreased bottom-dwelling and erratic swimming in zebrafish; the effects on bottom-dwelling, but not on erratic swimming, were blocked by pre-treatment with the nitric oxide synthase inhibitor L-NAME. These effects underline a novel mechanism by which 5-HT controls zebrafish reactivity to novel environments, with implications for the study of neotic reactions, exploratory behavior, and anxiety-like states

Batista, Evander de Jesus Oliveira

Herculano, Anderson Manoel

Lima, Monica Gomes

Maximino, Caio

Oliveira, Karen Renata Herculano Matos

Elsevier - Publisher Connector 

Interaction between 5-HT1B receptors and nitric oxide in zebrafish responses to novelty 

Neuroscience Letters 588 (2015) 54–56
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Research article
Interaction between 5-HT1B receptors and nitric oxide in zebraﬁsh
responses to novelty
Caio Maximinoa,b,∗, Monica Gomes Limaa,b,c, Evander de Jesus Oliveira Batistac,
Karen Renata Herculano Matos Oliveirac, Anderson Manoel Herculanob,c
a Laboratório de Neurociências e Comportamento “Frederico Guilherme Graeff”, Universidade do Estado do Pará, Marabá, PA, Brazil
b International Zebraﬁsh Neuroscience Research Consortium, Brazil
c Laboratório de Neuroendocrinologia, Universidade Federal do Pará, Belém, PA, Brazil
h i g h l i g h t s
• Serotonin and nitric oxide interact at different levels to control behavior.
• 5-HT1B antagonists decrease reactivity to novelty in zebraﬁsh.
• The 5-HT1B inverse agonist SSB224,289 decreased bottom-dwelling and erratic swimming in zebraﬁsh.
• The nitric oxide synthase inhibitor L-NAME blocked the effect of SSB224,289 on bottom-dwelling.
a r t i c l e i n f o
Article history:
Received 15 October 2014
Received in revised form
17 December 2014
Accepted 23 December 2014
Available online 27 December 2014
Keywords:
Serotonin
Anxiety
Novelty
Zebraﬁsh
Nitric oxide
a b s t r a c t
Nitric oxide (NO) and serotonin (5-HT) interact at the molecular and systems levels to control behavioral
variables, including agression, fear, and reactions to novelty. In zebraﬁsh, the 5-HT1B receptor has been
implicated in anxiety and reactions to novelty, while the 5-HT1A receptor is associated with anxiety-like
behavior; this role of the 5-HT1A receptor is mediated by NO. This work investigated whether NO also
participates in the mediation of novelty responses by the 5-HT1B receptor. The 5-HT1B receptor inverse
agonist SB 224,289 decreased bottom-dwelling and erratic swimming in zebraﬁsh; the effects on bottom-
dwelling, but not on erratic swimming, were blocked by pre-treatment with the nitric oxide synthase
inhibitor L-NAME. These effects underline a novelmechanismbywhich 5-HT controls zebraﬁsh reactivity
to novel environments, with implications for the study of neotic reactions, exploratory behavior, and
anxiety-like states.
© 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
An interaction between serotonin (5-HT) and the nitrergic sys-
tem has been observed in the brain at the molecular [1,2], systems
[3,4], andbehavioral [5–9] levels. This complex interaction involves
both nitric oxide (NO) mediation of 5-HT release/uptake [9–12]
as well as the 5-HTergic mediation of NO activity [1,13,14]. In
this sense, chronic treatment with 5-HT1A receptor (5-HT1AR)
agonists has been shown to decrease anxiety-like behavior in a
NO-dependent way [6], and activation of the 5-HT1AR decreases
NMDA receptor-mediated increases in NO production in cortical
∗ Corresponding author at: Laboratório de Neurociências e Comportamento,
“Frederico Guilherme Graeff” – LaNEC, Universidade do Estado do Pará–Campus
VIII, Av. Hiléia, S/N, Agrópolis do INCRA, Bairro Amapá – 68503-120 – Marabá, PA,
Brazil. Tel.: +55 9181850747.
E-mail address: caio.maximino@uepa.br (C. Maximino).
slices [13]. While the role of the 5-HT1AR in anxiety-like behavior
is relatively well-established, the participation of 5-HT1B receptors
(5-HT1BR) is emergent; 5-HT1BR knockout mice show increased
reactivity to novelty and decreased anxiety-like behavior [15],
while zebraﬁsh treated with 5-HT1BR antagonists show decreased
reactivity to novelty [16,17]. Elsewhere, we have shown that the
anxiolytic-like effect of 5-HT1AR antagonists in zebraﬁsh is blocked
by pre-treatment with the nitric oxide synthase (NOS) inhibitor l-
NG-nitroarginine methyl ester (l-NAME) [18]; in the present work,
we analyzed whether the behavioral effects of the 5-HT1BR inverse
agonist SB 224,289 in zebraﬁsh are also mediated by NOS.
2. Materials and methods
47 adult zebraﬁsh from the lof phenotype were acquired in a
local aquarium shop and kept in collective tanks (40 L, 10 ani-
mals/2 L) for at least two weeks before experiments begun. Water
http://dx.doi.org/10.1016/j.neulet.2014.12.049
0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
C. Maximino et al. / Neuroscience Letters 588 (2015) 54–56 55
Table 1
SB224,289 decreases erratic swimming, and this effect is not blocked by L-NAME. Values refer either to mean± S.E.M. or median± interquartile range (IQR). *, p<0.05 vs.
Vehicle.
Vehicle
(n=9)
SB 2.5mg/kg
(n=8)
SB 5.0mg/kg
(n=9)
SB 2.5mg/kg +
NAME (n=12)
SB 5mg/kg +
NAME (n=12)
Squares crossed (median± IQR, N) 141 ± 89 61.5 ± 75.75 175 ± 50 125 ± 75 190 ± 85.5
Erratic swimming (median± IQR, N) 7 ± 4 1 ± 1* 0 ± 3* 1 ± 1 3 ± 1.5
Freezing (mean± S.E.M., s) 23.59 ± 8.25 24.85 ± 15.84 15.80 ± 7.07 14.01 ± 3.52 31.54 ± 9.27
Fig. 1. The effects of SB224,289 on bottom-dwelling are blocked by pre-treatment
with L-NAME. Bars refer to mean± S.E.M. ***, p<0.001 vs. Vehicle; *, p<0.05 vs.
Vehicle; ###, p<0.001 vs. 2.5mg/kg.
conditions, housing, and feeding conditions were standardised as
per recommendations for zebraﬁsh [19]. Animalswere then swiftly
and individually removed fromthehousing tank, cold-anesthetized
(17 ◦C<T<12 ◦C) and injected intraperitoneally [20] with either
vehicle (DMSO 0.5%) or SB 224,289 (2.5 or 5.0mg/kg). Another
cohort of animals was pre-treated with l-NAME (1mg/kg) before
SB 224,289. 30min after injection, animals were subjected to the
novel tank test [21]; behavioral variables were deﬁned as per the
Zebraﬁsh Behavior Catalog [22]. Datawere analyzed using ANOVAs
or Kruskal–Wallis tests whenever appropriate, with either Tukey
or Bonferroni post-tests whenever p-values <0.05. Raw data can be
downloaded at ﬁgshare (doi:10.6084/m9.ﬁgshare.1272823).
3. Results
SB 224,289 dose-dependently decreased bottom-dwelling
(F4,41 = 6.814, p=0.0003; Fig. 1) and erratic swimming
(Hdf=4 = 20.08, p=0.0005; Table 1), without effects on freez-
ing (F4,46 = 0.6668, NS; Table 1) or locomotion (Hdf =4 = 8.55, NS;
Table 1). Pre-treatment with l-NAME blocked the effects of SB
224,289 on bottom-dwelling, but not on erratic swimming (Fig. 1,
Table 1).
4. Discussion
The reduction in bottom-dwelling, the main response of adult
zebraﬁsh to a novel environment [23,24], as well as in erratic
swimming, are indicative of reduced anxiety, stress or fear. The
results from the present experiment provide a novel mechanism
by which serotonin controls behavioral responses to novelty, sug-
gesting an important interaction between the 5-HT1B receptor and
the nitrergic system. The speciﬁc mechanism by which NOS inhi-
bition blocks the neotic effects of 5-HT1BR antagonists in zebraﬁsh
is unclear; this interaction could be due to presynaptic effects of
NO on 5-HT release or uptake [9–12] or to a postsynaptic effect,
with5-HT1B receptor activation increasingNOSactivity [25]. Futher
experiments are needed to clarify the issue.
Acknowledgments
Thisworkwas supportedbyaCNPq/Brazil (U¨niversalg¨rantnum-
ber: 479089/2013-2) and a CAPES/Brazil grant (P¨ró-Amazôniag¨rant
number: 3288/2013). MGL and CM were recipients of a CAPES
scholarship when the experiments for this article were ran. AMH is
the recipient of a CNPq/Brazil productivity grant (304130/2011-7).
The data reported in this article is deposited in the Zebraﬁsh Neu-
rophenome Database (http://www.tulane.edu/-znpindex/search).
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Interaction between 5-HT1B receptors and nitric oxide in zebrafish responses to novelty

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