- Email: [email protected]
Interactions between serotonin and glutamate–nitric oxide pathways in zebrafish scototaxis
Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Interactions between serotonin and glutamate–nitric oxide pathways in zebrafish scototaxis Anderson Manoel Herculano a,b, Bruna Puty a, Vanessa Miranda c, Monica Gomes Lima b,d, Caio Maximino b,d,⁎ a
Laboratório de Neuroendocrinologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil Zebrafish Neuroscience Research Consortium, USA Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil d Departamento de Morfologia e Ciências Fisiológicas, Centro de Ciências Biológicas e da Saúde, Universidade do Estado do Pará, Marabá, PA, Brazil b c
a r t i c l e
i n f o
Article history: Received 26 March 2014 Received in revised form 11 December 2014 Accepted 16 December 2014 Available online 20 December 2014 Keywords: Scototaxis Zebrafish Nitric oxide Glutamate Serotonin 5-HT1A receptor
a b s t r a c t NMDA receptors have been implicated in the acute response to stress, possibly mediated the nitric oxide pathway; serotonin has also been implicated in these responses, and has recently been shown to modulate the nitric oxide pathway via 5-HT1 and 5-HT2 receptors. In this work, we compare the effects of NMDA and a 5-HT1A receptor ligands on light/dark preference in adult zebrafish, and investigate whether nitric oxide mediates the effects of such drugs. The noncompetitive NMDA receptor antagonist MK-801 decreased dark preference (scototaxis), while NMDA increased it; the effects of NMDA were completely blocked by pretreatment with the nitric oxide synthase (NOS) antagonist L-NAME. SNP, a nitric oxide donor, produced a bell-shaped dose–response profile on scototaxis. Treatment with 5-HTP increased scototaxis, an effect which was potentiated by pre-treatment with NMDA, but not MK-801, and partially blocked by L-NAME. The 5-HT1A receptor antagonist WAY 100,635 decreased scototaxis, an effect which was completely blocked by L-NAME. These results suggest that tonic NOS inhibition is an important downstream effector of 5-HT1A receptors in the regulation of dark preference behavior in zebrafish, and that NOS is also under phasic independent control by NMDA receptors. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Glutamate has been implicated in the acute response to stress (Hamane et al., 2009; Harvey, 2006; Lupinsky et al., 2010; Reznikov et al., 2007) as well as in the expression of defensive behavior (Yilmazer-Hanke et al., 2003). Glutamate exerts its actions in the central nervous system through metabotropic and ionotropic receptors; among the latter, the N-methyl-D-aspartate (NMDA) receptor is among the most studied. The NMDA receptor gates inward sodium and calcium currents, and the influx of calcium into the cell leads to the activation of a calcium-dependent isoform of nitric oxide synthase (NOS) and, consequently to the activation of the nitric oxide–cyclic guanosine monophosphate (cGMP) pathway (Garthwaite, 2008). Nitric oxideproducing cells are enriched in regions of the limbic system which organize defensive behavior in mammals (Matsushita et al., 2001; Simpson et al., 2003; Vincent and Kimura, 1992; Wang et al., 1995), amphibians (López et al., 2005) and fish (Bordieri et al., 2005; Giraldez-Perez et al., Abbreviations: 5-HT, 5-hydroxytryptamine, serotonin; cGMP, cyclic guanosine monophosphate; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase. ⁎ Corresponding author at: Departamento de Morfologia e Ciências Fisiológicas, Centro de Ciências Biológicas e da Saúde, Campus de Marabá, Universidade do Estado do Pará, Av. Hiléia do INCRA, S/N, Marabá/PA, Brazil. Tel./fax: +55 94 33243400. E-mail address: [email protected] (C. Maximino).
http://dx.doi.org/10.1016/j.pbb.2014.12.005 0091-3057/© 2014 Elsevier Inc. All rights reserved.
2008; Holmqvist et al., 2007, 2000; Virgili et al., 2001). In rodents, nitrergic neurons are activated after exposure to anxiogenic contexts (Beijamini and Guimarães, 2006), restraint stress (De Oliveira et al., 2000), or ethanol withdrawal (Bonassoli et al., 2011). Systemically, treatment with NOS inhibitors is anxiolytic in the elevated T-maze (Calixto et al., 2001), in the staircase test (Pokk and Väli, 2002) and in the inhibitory avoidance test (Akar et al., 2007). There are some evidences that the nitrergic system interacts with serotonin (5-HT), an important mediator of defensive behavior across species (Maximino, 2012). The raphe nuclei, sites of origin of serotonergic innervation in the central nervous system, project nitrergic fibers to many different sites (Lu et al., 2010; Okere and Waterhouse, 2006; Simpson et al., 2003; Wang et al., 1995). Microinjection of NO inhibitors in the raphe blocks the local NMDA-induced 5-HT release (Smith and Whitton, 2000), prevents the effects of inescapable stress on escape responding and conditioned fear (i.e., learned helplessness) (Grahn et al., 2000) and has a biphasic effect in the Porsolt forced swim test and in the elevated plus-maze (Spiacci Jr et al., 2008). Microinjection of peroxynitrite donors, on the other hand, increases locomotor activity, an effect which is blocked by pre-treatment with the 5-HT1A receptor agonist 8-OH-DPAT (Gualda et al., 2011). The relationship is bilateral, as the activation of 5-HT1A receptors in neocortex (Maura et al., 2000) or hippocampal (Strosznajder et al., 1996) slices reduces glutamateevoked NO production. In cultured hippocampal neurons, treatment
98
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
with 5-HT or 8-OH-DPAT downregulates NOS protein expression, an effect which is blocked by treatment with the 5-HT1AR antagonist NAN-190 (Zhang et al., 2010); in the hippocampus of mice treated with 8-OH-DPAT for 3 days, NOS-1 mRNA is downregulated, and NAN-190 increases NOS-1 mRNA by itself (Zhang et al., 2010). Interestingly, in NOS-1 knockout mice, the anxiolytic effect of chronic 8-OHDPAT treatment is absent, and pre-treatment with the NOS inhibitor 7-NI blocks the anxiogenic effect of NAN-190 microinjected in the hippocampi of wild-type animals (Zhang et al., 2010). Zebrafish (Danio rerio Hamilton 1822) have recently been introduced in the field of behavioral neuroscience and biological psychiatry (Gerlai, 2010; Norton and Bally-cuif, 2010), especially in the field of stress and anxiety research (Jesuthasan, 2011; Kalueff et al., 2012; Maximino et al., 2010b; Steenbergen et al., 2011; Stewart et al., 2011). The serotonergic system of this teleost is relatively conserved (Lillesaar, 2011; Maximino et al., 2013a), although some differences exist such as the duplication genes encoding synthetic enzymes, receptors and transporters (Bellipanni et al., 2002; Norton et al., 2008; Wang et al., 2006), the existence of a single copy of monoamine oxidase (Anichtchik et al., 2006), and nuclei outside the raphe (Kaslin and Panula, 2001). Nonetheless, the pharmacology of the serotonergic system seems to be rather conserved (Maximino and Herculano, 2010; Stewart et al., 2013). We have previously demonstrated that the 5-HT1A partial agonist buspirone and the antagonist WAY 100,635 have anxiolytic-like effects in the light/dark and in the novel tank tests (Maximino et al., 2013b, 2011). In this article, we investigate whether the nitrergic system mediates some of these behavioral effects. 2. Materials and methods 2.1. Animals 247 adult (~ 4 month) unsexed zebrafish from the striped longfin wild-type phenotype were acquired in a local pet shop and used in the present experiments. Animals were group-housed in 40 L tanks, with a maximum density of 25 fish per tank, for at least 2 weeks before experiments begun. Tanks were filled with deionized and reconstituted water at room temperature (28 °C) and a pH of 7.0–8.0. Lighting was provided by fluorescent lamps in a cycle of 14–10 h (LD), according to the standard of care zebrafish (Lawrence, 2007). All manipulations minimized their potential suffering, as per the recommendations of the Canadian Council on Animal Care (Canadian Council on Animal Care, 2005). All procedures complied with the Brazilian Society for Neuroscience and Behavior's (SBNeC) guidelines for the care and use of animals in research.
0.03 mg/kg; n = 8–9 per group) was injected; finally, the mediation of NOS on the effects of WAY 100,635 was analyzed by pre-treating animals with L-NAME (5.0 mg/kg). With the exception of WAY 100,635 and 5HTP, which were tested at these doses in zebrafish (Maximino et al., 2013b; Herculano and Maximino, 2014), all doses were based on rodent literature (Olney et al., 1979; Plaznik et al., 1994; Calixto et al., 2001). 2.3. Light/dark preference The light/dark preference test analyzes zebrafish exploratory behavior under motivational conflict, as opposed to escape as has been proposed in the novel tank test (Maximino et al., 2012). In addition, the effects of serotonergic (Maximino et al., 2013b, 2014) and glutamatergic agents (Maximino et al., 2014) have been described in this test. Thus, determination of drug effects on scototaxis was carried as described elsewhere (Araujo et al., 2012; Maximino et al., 2010a). Briefly, 30 min after drug injection animals were transferred to the central compartment of a black and white tank (15 cm × 10 cm × 45 cm h × d × l) for a 3-min acclimation period, after which the doors which delimit this compartment were removed and the animal was allowed to freely explore the apparatus for 15 min. The following variables were manually recorded by an observer blind to treatment: time on the white compartment: the time spent in the white portion of the tank (percentage of the trial); squares crossed: the number of 10 cm2 squares crossed by the animal in the white compartment; latency to white: the amount of time the animal spends in the black compartment before its first entry in the white compartment(s); entries in white compartment: the number of entries the animal makes in the white compartment in the whole session; erratic swimming: the number of “erratic swimming” events, defined as a zig-zag, fast, and unpredictable course of swimming of short duration; freezing: the proportional duration of freezing events (in % of time in the white compartment), defined as complete cessation of movements with the exception of eye and operculae movements; thigmotaxis: the proportional duration of thigmotaxis events (in % of time in the white compartment), defined as swimming in a distance of 2 cm or less from the white compartment's walls; and risk assessment: the number of “risk assessment” events, defined as a fast (b 1 s) entry in the white compartment followed by re-entry in the black compartment, or as a partial entry in the white compartment (i.e., the pectoral fin does not cross the midline).
2.2. Drugs
2.4. Statistical analysis
All drugs were bought from Sigma-Aldrich (St Louis, MO, USA), dissolved in Cortland's salt solution (Wolf, 1963), and injected intraperitoneally in cold-anesthetized animals (Kinkel et al., 2010). To provide a picture of the role of NMDA receptors in zebrafish anxiety-like behavior, NMDA (0 and 0.011 mg/kg; n = 6 per group) and MK-801 (0, 0.2 and 2.0 mg/kg; n = 10 per group) were used to activate and block the receptor, respectively. The role of NOS as a downstream effector of NMDA was tested by pre-treating animals with the NOS inhibitor L-NAME (0 and 5.0 mg/kg; n = 6 per group) before NMDA injection. The role of NO was further analyzed by injecting animals with the NO donor sodium nitroprusside (SNP, 0.5–10 mg/kg; n = 6–7 per group). Serotonergic tonus was increased by injecting the precursor 5-HTP (0 and 300 mg/kg; n = 9–10 per group); the interaction between 5-HT and the glutamate–NMDA pathway was analyzed by pre-treating 5HTP-injected animals with either NMDA (0 and 0.011 mg/kg; n = 10 per group) or MK-801 (0 and 0.2 mg/kg; n = 10 per group). To analyze the mediation of NOS in the effects of 5-HTP, animals were pre-treated with L-NAME (0 and 5.0 mg/kg; n = 9 per group). To understand the role of 5-HT1A receptors, the antagonist WAY 100,635 (0 and
Drug effects on behavioral parameters were analyzed using one-way parametric or non-parametric (Kruskal–Wallis) analyses of variance followed by Tukey's HSD post-hoc tests whenever appropriate. Results from these statistical analyses were considered significant when p b 0.05. Latency outcomes were analyzed by log-rank Mantel–Cox tests based on survival curves (Jahn-Eimermacher et al., 2011), with α for multiple comparisons adjusted by the Bonferroni method. 3. Results 3.1. NMDA ligands alter behavior in the light/dark test in a NOS-dependent way Treatment with 0.2 mg/kg, but not 2.0 mg/kg, MK-801 increased the time spent on the white compartment (F2, 29 = 11.34, p = 0.0003; Fig. 1A). The highest dose increased the number of entries in the white compartment (F2, 29 = 32.82, p b 0.0001; Fig. 1C) and decreased thigmotaxis (Fig. 1G). No other effects were observed. Treatment with 0.011 mg/kg NMDA decreased the time spent in the white
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
99
Fig. 1. Effects of acute intraperitoneal injection of the NMDA antagonist MK-801 on zebrafish behavior in the light/dark test. (A) Scototaxis; (B) dark preference habituation; (C) entries in the white compartment; (D) number of squares crossed in the white compartment; (E) erratic swimming; (F) freezing; (G) thigmotaxis; and (H) risk assessment. Bar graphs represent mean ± standard error, while boxplots represent data from the 25th to the 75th percentile (boxes) and from minimum to maximum (whiskers). Survival plots represent Kaplan–Meier estimates for the cumulative incidences of first entry latencies. ***, p b 0.001 vs. control; **, p b 0.01 vs. control; *, p b 0.05 vs. control.
compartment, an effect which was abolished by pre-treatment with L-NAME in a dose which was by itself ineffective (F3, 23 = 33.13, p b 0.0001; Fig. 2A); moreover, NMDA impaired white avoidance
habituation, an effect which again was blocked by pre-treatment with L-NAME (F3, 23 = 3.811, p = 0.0261; Fig. 2B). NMDA, L-NAME or both drugs did not alter the latency to enter the white compartment
100
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
Fig. 2. Effects of acute intraperitoneal injection of NMDA and/or L-NAME on zebrafish behavior in the light/dark test. (A) Scototaxis; (B) dark preference habituation; (C) entries in the white compartment; (D) number of squares crossed in the white compartment; (E) erratic swimming; (F) freezing; (G) thigmotaxis; and (H) risk assessment. Bar graphs represent mean ± standard error, while boxplots represent data from the 25th to the 75th percentile (boxes) and from minimum to maximum (whiskers). Survival plots represent Kaplan– Meier estimates for the cumulative incidences of first entry latencies. ***, p b 0.001 vs. control; **, p b 0.01 vs. control; *, p b 0.05 vs. control.
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
101
(χ2[df = 3] = 5.537, p = 0.1365; data not shown). No effects were observed on the number of white compartment entries (H[df = 4] = 2.769, p = 0.4286; Fig. 2C) or on the number of squares crossed in the white compartment (H[df = 4] = 3.894, p = 0.2732; Fig. 2D). NMDA increased the frequency of erratic swimming events (H[df = 4] = 7.896, p = 0.0482; Fig. 2E) and risk assessment (H[df = 4] = 11.95, p = 0.0076; Fig. 2H), and the duration of freezing events (F3, 23 = 9.404, p = 0.0004; Fig. 2F) and thigmotaxis (F3, 23 = 6.5, p = 0.003; Fig. 2G); in all cases, L-NAME pre-treatment blocked the effects of NMDA. 3.2. A nitric oxide donor produces biphasic effects on scototaxis To further understand the role of nitric oxide on the control of anxiety-like behavior, we administered sodium nitroprusside (SNP), a nitric oxide donor, in five doses (from 0.5 to 10 mg/kg) in zebrafish and recorded their behavior in the light/dark test. SNP increased the time on white at 1 and 5 mg/kg, and decreased it at 10 m/kg (F[ 5, 39] = 30.65, p b 0.0001; Fig. 3A); these effects approached a bell-shaped curve when log dose–response was analyzed (unweighted r2 = 0.8526). No effects were observed on the total locomotion in the dose range analyzed (F[5, 40] = 1.224, p = 0.3188; Fig. 3B). 3.3. Synergism between 5-HTP and NMDA treatments in scototaxis Treatment with 5-HTP (300 mg/kg) decreased time on white, an effect which was potentiated by pre-treatment with NMDA (F[2, 54] = 15.78, p b 0.0001 for the interaction term; Fig. 4A). Pre-treatment with MK-801, however, did not alter the effect of 5-HTP on scototaxis — contrary to what happened with pre-treatment with L-NAME, which partially blocked the effect of 5-HTP on scototaxis (F[1, 32] = 3.417, p = 0.05; Fig. 4B). These results suggest that the activation of 5-HT receptors increases scototaxis, an effect which is potentiated, but not
Fig. 4. Effects of 5-HTP, with or without pretreatment with (A) NMDA or MK-801, or (B) L-NAME on scototaxis in the light/dark test. Different colors represent statistically significant differences.
dependent on, activation of the NMDA receptor; moreover, these effects are dependent on the activation of nitric oxide synthase. 3.4. Effects of WAY 100,635 mediated by nitric oxide WAY 100,635 increased the time spent on white, an effect which was blocked by pre-treatment with L-NAME (F4, 39 = 5.614, p = 0.0013; Fig. 5A). No effect was observed on latency to white (χ 2[df = 4] = 2.573, p = 0.6317; data not shown), squares crossed on white (H[df = 5] = 3.23, p = 0.5201; Fig. 5B) or entries on white (H[df = 5] = 6.661, p = 0.1549; Fig. 5C). While WAY 100,635 by itself had no effect on thigmotaxis, pre-treatment with L-NAME plus the highest dose of the 5-HT1AR antagonist decreased it (F4, 39 = 3.093, p = 0.0279; Fig. 5G). No effect was observed on freezing (F4, 39 = 0.1335, p = 0.969; Fig. 5F) nor on erratic swimming (H[df = 5] = 4.077, p = 0.3957; Fig. 5E). The highest dose decreased risk assessment, an effect which was blocked by L-NAME (H[df = 5] = 11.87, p = 0.0183; Fig. 5H). 4. Discussion
Fig. 3. Effects of acute treatment with sodium nitroprusside (SNP) on (A) scototaxis; and (B) number of midline crossings in the light/dark test. Bar graphs and dot-plots represent mean ± standard error. ***, p b 0.001 vs. control; **, p b 0.01 vs. control; *, p b 0.05 vs. control.
In the present work, NMDA was found to increase scototaxis, impaired habituation, and increased erratic swimming, risk assessment, freezing and thigmotaxis in the light/dark tank — effects which were blocked by pre-treatment with a NOS antagonist, L-NAME; treatment with the NMDA non-competitive antagonist MK-801 decreased scototaxis, but did not alter other variables. Similarly, treatment of rodents with MK-801 showed an anti-conflict effect and reduced thigmotaxis in an open-field (Plaznik et al., 1994), while in the plus-maze another non-competitive antagonist is ineffective (Wiley et al., 1995).
102
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
Fig. 5. Effects of WAY 100,635, with or without pretreatment with L-NAME, on (A) scototaxis, (B) latency to white, (C) number of squares crossed in the white compartment, (D) entries on the white compartment, (E) thigmotaxis, (F) freezing, (G) erratic swimming and (H) risk assessment. **, p b 0.01 vs. control (0.0 mg/kg); *, p b 0.05 vs. control.
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
In zebrafish, MK-801 has been shown to not alter light/dark preference (Sison and Gerlai, 2011), although the preference task used differed significantly from that presented here in that the light side is actually a transparent, well-lit portion, while the dark side is covered from the top; in this configuration, animals tend to prefer the light side, instead of the dark one (Blaser and Peñalosa, 2011; Gerlai et al., 2000), and thus comparison of these results with ours is limited. In zebrafish, MK-801 has also been shown to produce hyperactivity (Sison and Gerlai, 2011) and to block habituation of the acoustic startle response (Best et al., 2008; Roberts et al., 2011). Other drugs with antiglutamatergic effects have been shown to affect zebrafish behavior. Most extensively used were drugs with psychotomimetic effects (kynurenic acid, ketamine, MK-801, ibogaine; phencyclidine; Neelkantan et al., 2013) with a polypharmaceutical profile which includes but is not limited to glutamate. In general, these drugs produce an anxiolytic-like profile in different tests that is also accompanied by dissociative-like behavior, suggesting nonspecific effects on anxiety. Nonetheless, MK-801 effects on the light/dark test, as well as the NMDA experiments reported in this paper, suggest that activation of this receptor increases anxiety-like behavior, an effect mediated by nitric oxide production. Previous work showed that freezing and thigmotaxis are higher in the black compartment of a light/dark tank (Blaser et al., 2010; Blaser and Peñalosa, 2011), there is no evidence so far that these variables are increased/decreased by anxiolytic-like and anxiogenic-like treatments. Indeed, Blaser and Peñalosa (2011) did not observe changes in freezing or thigmotaxis in black after ethanol treatment. On the other hand, freezing and/or thigmotaxis on white have been repeatedly shown to be altered by drug treatments (e.g., Maximino et al., 2013b, 2014). The same was observed in the present experiments regarding the effects of NMDA. While this might represent a methodological limitation of the present work, these observations also suggest that freezing and thigmotaxis in the white compartment are qualitatively different from these behaviors in the black compartment and might be controlled by different behavioral states. Further experiments are necessary to disentangle this hypothesis. Nonetheless, stimulation of NO targets with the donor SNP produced a biphasic effect on scototaxis — that is, small and intermediate doses increase scototaxis, while intermediate doses decrease it. Biphasic effects of NO donors were observed on 5-HT release in hypothalamic slices (Kaehler et al., 1999), and 8-Br-cGMP also affects 5-HT uptake in midbrain synaptosomes in a biphasic manner (Ramamoorthy et al., 2007). Low doses of L-NAME increase open arm exploration in the elevated plus-maze and decrease immobility in the forced swim test in rats, while high doses decrease locomotor activity (Spiacci Jr et al., 2008). These results seem to suggest that the mediation of the nitrergic system on behavior or on the serotonergic system is complex, depending on the basal and stimulated levels of nitric oxide. Consistent with these observations, we observed that treatment with the 5-HT precursor 5-HTP increased scototaxis, an effect which was potentiated by pre-treatment with NMDA and partially blocked by pre-treatment with L-NAME. Nonetheless, pre-treatment with MK801 was unable to change this effect, suggesting that either activation of a different site in the NMDA receptor (the orthosteric site) is needed for synergism with 5-HTP, or that the effects of 5-HTP were mediated by nitric oxide downstream of the NMDA receptor. In consonance with this proposal, in cortical or hippocampal slices, the activation of 5-HT1A receptors reduces glutamate-evoked NO production (Maura et al., 2000; Strosznajder et al., 1996). Previous experiments in zebrafish demonstrated that WAY 100,635 by itself produced an anxiolytic-like effect in both the novel tank and the light/dark tests (Maximino et al., 2013b). More interestingly, pretreatment with L-NAME completely blocked the effects of WAY 100,635 on scototaxis and risk assessment, suggesting that nitric oxide might be involved in the mechanism of action of this drug. Combined with the previous observations, these results suggest that 5-HT1A
103
receptors (blocked by WAY 100,635) tonically inhibit nitric oxide production; the removal of this tonic inhibition would increase NO levels in small increments (as observed in rodent cortical or hippocampal slices; Maura et al., 2000; Strosznajder et al., 1996), decreasing scototaxis and risk assessment. NMDA receptor activation, on the other hand, can directly activate NOS, effectively overriding the tonic inhibition and increasing intracellular NO production to levels which increase scototaxis and risk assessment, as well as fear-like behaviors such as erratic swimming, freezing and thigmotaxis. Therefore, NOS could act as a “switch” to signal the balance of serotonergic and glutamatergic input to post-synaptic structures associated with defensive behavior. The present results suggest that tonic NOS-1 inhibition is an important downstream effector of 5-HT1A receptors in the regulation of anxiety-like behavior, and that NOS-1 is also under phasic independent control by NMDA receptors; however, the precise mechanisms by which these receptors interact to regulate anxiety-like behavior is still unclear, and further experiments are necessary to untangle concurrent hypotheses. Acknowledgments Part of this work was financed by CNPq grants (grant numbers 483336/2009-2 and 479089/2013-2) and a CAPES/Pró-Amazônia grant (grant number: 3288/2013). CM, BP and MGL were recipients of CAPES/Brazil studentships. AMH is a recipient of a CNPq/Brazil productivity grant (304130/2011-7). References Akar FY, Ulak G, Tanyeri P, Erden F, Utkan T, Gacar N. 7-Nitroindazole, a neuronal nitric oxide synthase inhibitor, impairs passive-avoidance and elevated plus-maze memory performance in rats. Pharmacol Biochem Behav 2007;87:434–43. Anichtchik O, Sallinen V, Peitsaro N, Panula P. Distinct structure and activity of monoamine oxidase in the brain of zebrafish (Danio rerio). J Comp Neurol 2006; 498:593–610. Araujo J, Maximino C, Brito TM, de, Silva, A.W.B. da, Batista, E. de J.O.Oliveira, K.R.M., Morato S, et al. Behavioral and pharmacological aspects of anxiety in the light/dark preference test. In: Kalueff AV, Stewart AM, editors. Zebrafish protocols for neurobehavioral research. New York: Humana Press; 2012. Beijamini V, Guimarães FS. Activation of neurons containing the enzyme nitric oxide synthase following exposure to an elevated plus maze. Brain Res Bull 2006;69:347–55. Bellipanni G, Rink E, Bally-Cuif L. Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain. Mech Dev 2002;3:S215–20. Best JD, Berghmans S, Hunt JJFG, Clarke SC, Fleming A, Goldsmith P, et al. Non-associative learning in larval zebrafish. Neuropsychopharmacology 2008;33:1206–15. Blaser RE, Peñalosa YM. Stimuli affecting zebrafish (Danio rerio) behavior in the light/dark preference test. Physiol Behav 2011;104:831–7. Blaser RE, Chadwick L, McGinnis GC. Behavioral measures of anxiety in zebrafish (Danio rerio). Behavioural Brain Research 2010;208:56–62. http://dx.doi.org/10.1016/j.bbr. 2009.11.009. Bonassoli VT, Milani H, de Oliveira RMW. Ethanol withdrawal activates nitric oxideproducing neurons in anxiety-related brain areas. Alcohol 2011;45:641–52. Bordieri L, di Patti MCB, Miele R, Cioni C. Partial cloning of neuronal nitric oxide synthase (nNOS) cDNA and regional distribution of nNOS mRNA in the central nervous system of the Nile tilapia Oreochromis niloticus. Mol Brain Res 2005;142:123–33. Calixto AV, Vandresen N, de Nucci G, Moreno Jr H, Faria MS. Nitric oxide may underlie learned fear in the elevated T-maze. Brain Res Bull 2001;55:37–42. Canadian Council on Animal Care. CCAC guidelines on: the care and use of fish in research, teaching and testing; 2005. De Oliveira RMW, Del Bel EA, Mamede-Rosa MLNMLN, Padovan CM, Deakin JFW, Guimarães FS. Expression of neuronal nitric oxide synthase mRNA in stress-related brain areas after restraint in rats. Neuroscience Letters 2000;289:123–6. Garthwaite J. Concepts of neural nitric oxide-mediated transmission. Eur J Neurosci 2008; 27:2783–802. Gerlai R. High-throughput behavioral screens: the first step towards finding genes involved in vertebrate brain function using zebrafish. Molecules 2010;15:2609–22. Gerlai R, Lahav M, Guo S, Rosenthal A. Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects. Pharmacol Biochem Behav 2000;67: 773–82. Giraldez-Perez RM, Gaytan SP, Ruano D, Torres B, Pasaro R. Distribution of NADPHdiaphorase and nitric oxide synthase reactivity in the central nervous system of the goldfish (Carassius auratus). 2008;35:12–32. Grahn RE, Watkins LR, Maier SF. Impaired escape performance and enhanced conditioned fear in rats following exposure to an uncontrollable stressor are mediated by glutamate and nitric oxide in the dorsal raphe nucleus. Behav Brain Res 2000;112:33–41.
104
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
Gualda LB, Martins GG, Müller B, Guimarães FS, de Oliveira RMW. 5-HT1A autoreceptor modulation of locomotor activity induced by nitric oxide in the rat dorsal raphe nucleus. Braz J Med Biol Res 2011;44:332–6. Hamane H, Tsuneyoshi Y, Denbow DM, Furuse M. N-methyl-D-aspartate and alphaamino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks. Amino Acids 2009; 37:733–9. Harvey BH. Adaptive plasticity during stress and depression and the role of glutamate–nitric oxide pathways. S Afr Psychiatry Rev 2006;9:132–9. Herculano AM, Maximino C. Serotonergic modulation of zebrafish behavior: Towards a paradox. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 55; 2014. p. 50–66. http://dx.doi.org/10.1016/j.pnpbp.2014.03.008. Holmqvist B, Ellingsen B, Alm P, Forsell J, Øyan A-M, Goksøyr A, et al. Identification and distribution of nitric oxide synthase in the brain of adult zebrafish. Neurosci Lett 2000;292:119–22. Holmqvist B, Ebbesson L, Alm P. Nitric oxide and the zebrafish (Danio rerio): developmental neurobiology and brain neurogenesis. Advances in experimental biology. Elsevier B. V; 2007. p. 229–74. Jahn-Eimermacher A, Lasarzik I, Raber J. Statistical analysis outcomes in behavioral experiments. Behav Brain Res 2011;221:271–5. Jesuthasan S. Fear, anxiety, and control in the zebrafish. Dev Neurobiol 2011;72:395–403. Kaehler ST, Singewald N, Sinner C, Philippu A. Nitric oxide modulates the release of serotonin in the rat hypothalamus. Brain Res 1999;835:346–9. Kalueff AV, Stewart AM, Kyzar EJ, Cachat J, Gebhardt M, Landsman S, et al. Time to recognize zebrafish “affective” behavior. Behaviour 2012;149:1019–36. Kaslin J, Panula P. Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio). J Comp Neurol 2001;440:342–77. Kinkel MD, Eames SC, Philipson LH, Prince VE. Intraperitoneal injection into adult zebrafish. J of Vis Exp 2010;42:2126. http://dx.doi.org/10.3791/2126. Lawrence C. The husbandry of zebrafish (Danio rerio): a review. Aquaculture 2007;269: 1–20. Lillesaar C. The serotonergic system in fish. Journal of Chemical Neuroanatomy. 2011;41: 294–308. http://dx.doi.org/10.1016/j.jchemneu.2011.05.009. López JM, Moreno N, Morona R, Muñoz M, González A. Colocalization of nitric oxide synthase and monoamines in neurons of the amphibian brain. Brain Res Bull 2005;66: 555–9. Lu Y, Simpson KL, Weaver KJ, Lin RCS. Coexpression of serotonin and nitric oxide in the raphe complex: cortical versus subcortical circuit. Anat Rec 2010;293:1954–65. Lupinsky D, Moquin L, Gratton A. Interhemispheric regulation of the medial prefrontal cortical glutamate stress response in rats. J Neurosci 2010;30:7624–33. Matsushita H, Takeuchi Y, Kawata M, Sawada T. Distribution of NADPH-diaphorasepositive neurons in the mouse brain: differences from previous findings in the rat brain and comparison with the distribution of serotonergic neurons. Acta Histochem Cytochem 2001;34:235–57. Maura G, Marcoli M, Pepicelli O, Rosu C, Viola C, Raiteri M. Serotonin inhibition of the NMDA receptor/nitric oxide/cyclic GMP pathway in human neocortex slices: involvement of 5-HT2C and 5-HT1A receptors. Br J Pharmacol 2000;130:1853–8. Maximino C. Serotonin and anxiety. Neuroanatomical, pharmacological, and functional aspects. New York, NY: Springer; 2012. Maximino C, Herculano AM. A review of monoaminergic neuropsychopharmacology in zebrafish. Zebrafish 2010;7:359–78. Maximino C, da Silva AWB, Araújo J, Lima MG, Miranda V, Puty B, Herculano AM. Fingerprinting of psychoactive drugs in zebrafish anxiety-like behaviors. PLoS ONE 2014;9: e103943. http://dx.doi.org/10.1371/journal.pone.0103943. Maximino C, de Brito TM, da Silva AWB, Herculano AM, Morato S, Gouveia Jr A, et al. Measuring anxiety in zebrafish: a critical review. Behav Brain Res 2010a;214:157–71. Maximino C, de Brito TM, Dias CA, de M, Gouveia Jr A, Morato S. Scototaxis as anxiety-like behavior in fish. Nat. Protoc. 2010b;5:209–16. Maximino C, Silva AWB, Gouveia Jr A, Herculano AM. Pharmacological analysis of zebrafish (Danio rerio) scototaxis. Prog Neuropsychopharmacol Biol Psychiatry 2011;35:624–31. Maximino C, Benzecry R, Oliveira KRM, Batista EJO, Herculano AM, Rosemberg DB, et al. A comparison of the light/dark and novel tank tests in zebrafish. Behaviour 2012;149: 1099–123. Maximino C, Lima MG, Araujo J, Oliveira KRM, Herculano AM, Stewart AM, et al. The serotonergic system of zebrafish: genomics, neuroanatomy and neuropharmacology. In: Hall FS, editor. Serotonin: biosynthesis. New York, NY: Regulation and Health Implications. Nova Science; 2013a. p. 53–67. Maximino C, Puty B, Benzecry R, Araújo J, Lima MG, Batista, E. de J.O., et al. Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect
of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine in two behavioral models. Neuropharmacology 2013b;71:83–97. Neelkantan N, Mikhaylova A, Stewart AM, Arnold R, Gjeloshi V, Kondaveeti D, et al. Perspectives on zebrafish models of hallucinogenic drugs and related psychotropic compounds. ACS Chem Neurosci 2013;4:1137–50. Norton W, Bally-cuif L. Adult zebrafish as a model organism for behavioural genetics. BMC Neurosci 2010;11:90. Norton WHJ, Folchert A, Bally-Cuif L. Comparative analysis of serotonin receptor (HTR1A/ HTR1B families) and transporter (slc6a4a/b) gene expression in the zebrafish brain. J Comp Neurol 2008;511:521–42. Okere CO, Waterhouse BD. Activity-dependent heterogeneous populations of nitric oxide synthase neurons in the rat dorsal raphe nucleus 6; 2006. Olney JW, de Gubareff T, Labruyere J. α-Aminoadipate blocks the neurotoxic action of N-methyl aspartate. Life Sci 1979;25:537–40. Plaznik A, Palejko W, Nazar M, Jessa M. Effects of antagonists at the NMDA receptor complex in two models of anxiety. Eur Neuropsychopharmacol 1994;4:503–12. Pokk P, Väli M. Effects of nitric oxide synthase inhibitors 7-NI, L-NAME, and L-NOARG in staircase test. Arch Med Res 2002;33:265–8. Ramamoorthy S, Samuvel DJ, Buck ER, Rudnick G, Jayanthi LD. Phosphorylation of threonine residue 276 is required for acute regulation of serotonin transporter by cyclic GMP. J Biol Chem 2007;282:11639–47. Reznikov LR, Grillo CA, Piroli GG, Pasumarthi RK, Reagan LP, Fadel J. Acute stressmediated increases in extracellular glutamate levels in the rat amygdala: differential effects of antidepressant treatment. Eur J Neurosci 2007;25:3109–14. Roberts AC, Reichl J, Song MY, Dearinger AD, Moridzadeh N, Lu ED, et al. Habituation of the C-start response in larval zebrafish exhibits several distinct phases and sensitivity to NMDA receptor blockade. PLoS ONE 2011;6:e29132. Simpson KL, Waterhouse BD, Lin RCS. Differential expression of nitric oxide in serotonergic projection neurons: neurochemical identification of dorsal raphe inputs to rodent trigeminal somatosensory targets. J Comp Neurol 2003;512:495–512. Sison M, Gerlai R. Behavioral performance altering effects of MK-801 in zebrafish (Danio rerio). Behav Brain Res 2011;220:331–7. Smith JCE, Whitton PS. Nitric oxide modulates N-methyl-D-aspartate-evoked serotonin release in the raphe nuclei and frontal cortex of the freely moving rat. Neurosci Lett 2000;291:5–8. Spiacci Jr A, Kanamaru F, Guimarães FS, Oliveira RMW. Nitric oxide-mediated anxiolyticlike and antidepressant-like effects in animal models of anxiety and depression. 2008;88:247–55. Steenbergen PJ, Richardson MK, Champagne DL. The use of the zebrafish model in stress research. Prog Neuropsychopharmacol Biol Psychiatry 2011;35:1432–51. Stewart A, Gaikwad S, Kyzar E, Green J, Roth A, Kalueff AV. Modeling anxiety using adult zebrafish: a conceptual review. Neuropharmacology 2011;62:135–43. Stewart AM, Cachat J, Gaikwad S, Robinson K, Gebhardt M, Kalueff AV. Perspectives on experimental models of serotonin syndrome in zebrafish. Neurochemistry International 2013;62:893–902. http://dx.doi.org/10.1016/j.neuint.2013.02.018. Strosznajder J, Chalimoniuk M, Samochocki M. Activation of serotonergic 5-HT1A receptor reduces Ca2+- and glutamatergic receptor-evoked arachidonic acid and NO/cGMP release in adult hippocampus. Neurochem Int 1996;28:439–44. Vincent SR, Kimura H. Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 1992;46:755–84. Virgili M, Poli A, Beraudi A, Giuliani A, Villani L. Regional distribution of nitric oxide synthase and NADPH-diaphorase activities in the central nervous system of teleosts. Brain Res 2001;901:202–7. Wang Q-P, Guan J-L, Nakai Y. Distribution and synaptic relations of NOS neurons in the dorsal raphe nucleus: a comparison to 5-HT neurons. Brain Res Bull 1995;37:177–87. Wang Y, Takai R, Yoshioka H, Shirabe K. Characterization and expression of serotonin transporter genes in zebrafish. Tohoku J Exp Med 2006;208:267–74. Wiley JL, Cristello AF, Balster RL. Effects of site-selective NMDA receptor antagonists in an elevated plus-maze model of anxiety in mice. Eur J Pharmacol 1995;294:101–7. Wolf K. Physiological salines for freshwater teleosts. Prog Fish Cult 1963;25:135–40. Yilmazer-Hanke DM, Roskoden T, Zilles K, Schwegler H. Anxiety-related behavior and densities of glutamate, GABA A, acetylcholine and serotonin receptors in the amygdala of seven inbred mouse strains. Behav Brain Res 2003;145:145–59. Zhang J, Huang X-Y, Ye M-L, Luo C, Wu H-Y, Hu Y, et al. Neuronal nitric oxide synthase alteration accounts for the role of 5-HT1A receptor in modulating anxiety-related behaviors. J Neurosci 2010;30:2433–41.
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Interactions between serotonin and glutamate–nitric oxide pathways in zebrafish scototaxis Anderson Manoel Herculano a,b, Bruna Puty a, Vanessa Miranda c, Monica Gomes Lima b,d, Caio Maximino b,d,⁎ a
Laboratório de Neuroendocrinologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil Zebrafish Neuroscience Research Consortium, USA Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil d Departamento de Morfologia e Ciências Fisiológicas, Centro de Ciências Biológicas e da Saúde, Universidade do Estado do Pará, Marabá, PA, Brazil b c
a r t i c l e
i n f o
Article history: Received 26 March 2014 Received in revised form 11 December 2014 Accepted 16 December 2014 Available online 20 December 2014 Keywords: Scototaxis Zebrafish Nitric oxide Glutamate Serotonin 5-HT1A receptor
a b s t r a c t NMDA receptors have been implicated in the acute response to stress, possibly mediated the nitric oxide pathway; serotonin has also been implicated in these responses, and has recently been shown to modulate the nitric oxide pathway via 5-HT1 and 5-HT2 receptors. In this work, we compare the effects of NMDA and a 5-HT1A receptor ligands on light/dark preference in adult zebrafish, and investigate whether nitric oxide mediates the effects of such drugs. The noncompetitive NMDA receptor antagonist MK-801 decreased dark preference (scototaxis), while NMDA increased it; the effects of NMDA were completely blocked by pretreatment with the nitric oxide synthase (NOS) antagonist L-NAME. SNP, a nitric oxide donor, produced a bell-shaped dose–response profile on scototaxis. Treatment with 5-HTP increased scototaxis, an effect which was potentiated by pre-treatment with NMDA, but not MK-801, and partially blocked by L-NAME. The 5-HT1A receptor antagonist WAY 100,635 decreased scototaxis, an effect which was completely blocked by L-NAME. These results suggest that tonic NOS inhibition is an important downstream effector of 5-HT1A receptors in the regulation of dark preference behavior in zebrafish, and that NOS is also under phasic independent control by NMDA receptors. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Glutamate has been implicated in the acute response to stress (Hamane et al., 2009; Harvey, 2006; Lupinsky et al., 2010; Reznikov et al., 2007) as well as in the expression of defensive behavior (Yilmazer-Hanke et al., 2003). Glutamate exerts its actions in the central nervous system through metabotropic and ionotropic receptors; among the latter, the N-methyl-D-aspartate (NMDA) receptor is among the most studied. The NMDA receptor gates inward sodium and calcium currents, and the influx of calcium into the cell leads to the activation of a calcium-dependent isoform of nitric oxide synthase (NOS) and, consequently to the activation of the nitric oxide–cyclic guanosine monophosphate (cGMP) pathway (Garthwaite, 2008). Nitric oxideproducing cells are enriched in regions of the limbic system which organize defensive behavior in mammals (Matsushita et al., 2001; Simpson et al., 2003; Vincent and Kimura, 1992; Wang et al., 1995), amphibians (López et al., 2005) and fish (Bordieri et al., 2005; Giraldez-Perez et al., Abbreviations: 5-HT, 5-hydroxytryptamine, serotonin; cGMP, cyclic guanosine monophosphate; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase. ⁎ Corresponding author at: Departamento de Morfologia e Ciências Fisiológicas, Centro de Ciências Biológicas e da Saúde, Campus de Marabá, Universidade do Estado do Pará, Av. Hiléia do INCRA, S/N, Marabá/PA, Brazil. Tel./fax: +55 94 33243400. E-mail address: [email protected] (C. Maximino).
http://dx.doi.org/10.1016/j.pbb.2014.12.005 0091-3057/© 2014 Elsevier Inc. All rights reserved.
2008; Holmqvist et al., 2007, 2000; Virgili et al., 2001). In rodents, nitrergic neurons are activated after exposure to anxiogenic contexts (Beijamini and Guimarães, 2006), restraint stress (De Oliveira et al., 2000), or ethanol withdrawal (Bonassoli et al., 2011). Systemically, treatment with NOS inhibitors is anxiolytic in the elevated T-maze (Calixto et al., 2001), in the staircase test (Pokk and Väli, 2002) and in the inhibitory avoidance test (Akar et al., 2007). There are some evidences that the nitrergic system interacts with serotonin (5-HT), an important mediator of defensive behavior across species (Maximino, 2012). The raphe nuclei, sites of origin of serotonergic innervation in the central nervous system, project nitrergic fibers to many different sites (Lu et al., 2010; Okere and Waterhouse, 2006; Simpson et al., 2003; Wang et al., 1995). Microinjection of NO inhibitors in the raphe blocks the local NMDA-induced 5-HT release (Smith and Whitton, 2000), prevents the effects of inescapable stress on escape responding and conditioned fear (i.e., learned helplessness) (Grahn et al., 2000) and has a biphasic effect in the Porsolt forced swim test and in the elevated plus-maze (Spiacci Jr et al., 2008). Microinjection of peroxynitrite donors, on the other hand, increases locomotor activity, an effect which is blocked by pre-treatment with the 5-HT1A receptor agonist 8-OH-DPAT (Gualda et al., 2011). The relationship is bilateral, as the activation of 5-HT1A receptors in neocortex (Maura et al., 2000) or hippocampal (Strosznajder et al., 1996) slices reduces glutamateevoked NO production. In cultured hippocampal neurons, treatment
98
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
with 5-HT or 8-OH-DPAT downregulates NOS protein expression, an effect which is blocked by treatment with the 5-HT1AR antagonist NAN-190 (Zhang et al., 2010); in the hippocampus of mice treated with 8-OH-DPAT for 3 days, NOS-1 mRNA is downregulated, and NAN-190 increases NOS-1 mRNA by itself (Zhang et al., 2010). Interestingly, in NOS-1 knockout mice, the anxiolytic effect of chronic 8-OHDPAT treatment is absent, and pre-treatment with the NOS inhibitor 7-NI blocks the anxiogenic effect of NAN-190 microinjected in the hippocampi of wild-type animals (Zhang et al., 2010). Zebrafish (Danio rerio Hamilton 1822) have recently been introduced in the field of behavioral neuroscience and biological psychiatry (Gerlai, 2010; Norton and Bally-cuif, 2010), especially in the field of stress and anxiety research (Jesuthasan, 2011; Kalueff et al., 2012; Maximino et al., 2010b; Steenbergen et al., 2011; Stewart et al., 2011). The serotonergic system of this teleost is relatively conserved (Lillesaar, 2011; Maximino et al., 2013a), although some differences exist such as the duplication genes encoding synthetic enzymes, receptors and transporters (Bellipanni et al., 2002; Norton et al., 2008; Wang et al., 2006), the existence of a single copy of monoamine oxidase (Anichtchik et al., 2006), and nuclei outside the raphe (Kaslin and Panula, 2001). Nonetheless, the pharmacology of the serotonergic system seems to be rather conserved (Maximino and Herculano, 2010; Stewart et al., 2013). We have previously demonstrated that the 5-HT1A partial agonist buspirone and the antagonist WAY 100,635 have anxiolytic-like effects in the light/dark and in the novel tank tests (Maximino et al., 2013b, 2011). In this article, we investigate whether the nitrergic system mediates some of these behavioral effects. 2. Materials and methods 2.1. Animals 247 adult (~ 4 month) unsexed zebrafish from the striped longfin wild-type phenotype were acquired in a local pet shop and used in the present experiments. Animals were group-housed in 40 L tanks, with a maximum density of 25 fish per tank, for at least 2 weeks before experiments begun. Tanks were filled with deionized and reconstituted water at room temperature (28 °C) and a pH of 7.0–8.0. Lighting was provided by fluorescent lamps in a cycle of 14–10 h (LD), according to the standard of care zebrafish (Lawrence, 2007). All manipulations minimized their potential suffering, as per the recommendations of the Canadian Council on Animal Care (Canadian Council on Animal Care, 2005). All procedures complied with the Brazilian Society for Neuroscience and Behavior's (SBNeC) guidelines for the care and use of animals in research.
0.03 mg/kg; n = 8–9 per group) was injected; finally, the mediation of NOS on the effects of WAY 100,635 was analyzed by pre-treating animals with L-NAME (5.0 mg/kg). With the exception of WAY 100,635 and 5HTP, which were tested at these doses in zebrafish (Maximino et al., 2013b; Herculano and Maximino, 2014), all doses were based on rodent literature (Olney et al., 1979; Plaznik et al., 1994; Calixto et al., 2001). 2.3. Light/dark preference The light/dark preference test analyzes zebrafish exploratory behavior under motivational conflict, as opposed to escape as has been proposed in the novel tank test (Maximino et al., 2012). In addition, the effects of serotonergic (Maximino et al., 2013b, 2014) and glutamatergic agents (Maximino et al., 2014) have been described in this test. Thus, determination of drug effects on scototaxis was carried as described elsewhere (Araujo et al., 2012; Maximino et al., 2010a). Briefly, 30 min after drug injection animals were transferred to the central compartment of a black and white tank (15 cm × 10 cm × 45 cm h × d × l) for a 3-min acclimation period, after which the doors which delimit this compartment were removed and the animal was allowed to freely explore the apparatus for 15 min. The following variables were manually recorded by an observer blind to treatment: time on the white compartment: the time spent in the white portion of the tank (percentage of the trial); squares crossed: the number of 10 cm2 squares crossed by the animal in the white compartment; latency to white: the amount of time the animal spends in the black compartment before its first entry in the white compartment(s); entries in white compartment: the number of entries the animal makes in the white compartment in the whole session; erratic swimming: the number of “erratic swimming” events, defined as a zig-zag, fast, and unpredictable course of swimming of short duration; freezing: the proportional duration of freezing events (in % of time in the white compartment), defined as complete cessation of movements with the exception of eye and operculae movements; thigmotaxis: the proportional duration of thigmotaxis events (in % of time in the white compartment), defined as swimming in a distance of 2 cm or less from the white compartment's walls; and risk assessment: the number of “risk assessment” events, defined as a fast (b 1 s) entry in the white compartment followed by re-entry in the black compartment, or as a partial entry in the white compartment (i.e., the pectoral fin does not cross the midline).
2.2. Drugs
2.4. Statistical analysis
All drugs were bought from Sigma-Aldrich (St Louis, MO, USA), dissolved in Cortland's salt solution (Wolf, 1963), and injected intraperitoneally in cold-anesthetized animals (Kinkel et al., 2010). To provide a picture of the role of NMDA receptors in zebrafish anxiety-like behavior, NMDA (0 and 0.011 mg/kg; n = 6 per group) and MK-801 (0, 0.2 and 2.0 mg/kg; n = 10 per group) were used to activate and block the receptor, respectively. The role of NOS as a downstream effector of NMDA was tested by pre-treating animals with the NOS inhibitor L-NAME (0 and 5.0 mg/kg; n = 6 per group) before NMDA injection. The role of NO was further analyzed by injecting animals with the NO donor sodium nitroprusside (SNP, 0.5–10 mg/kg; n = 6–7 per group). Serotonergic tonus was increased by injecting the precursor 5-HTP (0 and 300 mg/kg; n = 9–10 per group); the interaction between 5-HT and the glutamate–NMDA pathway was analyzed by pre-treating 5HTP-injected animals with either NMDA (0 and 0.011 mg/kg; n = 10 per group) or MK-801 (0 and 0.2 mg/kg; n = 10 per group). To analyze the mediation of NOS in the effects of 5-HTP, animals were pre-treated with L-NAME (0 and 5.0 mg/kg; n = 9 per group). To understand the role of 5-HT1A receptors, the antagonist WAY 100,635 (0 and
Drug effects on behavioral parameters were analyzed using one-way parametric or non-parametric (Kruskal–Wallis) analyses of variance followed by Tukey's HSD post-hoc tests whenever appropriate. Results from these statistical analyses were considered significant when p b 0.05. Latency outcomes were analyzed by log-rank Mantel–Cox tests based on survival curves (Jahn-Eimermacher et al., 2011), with α for multiple comparisons adjusted by the Bonferroni method. 3. Results 3.1. NMDA ligands alter behavior in the light/dark test in a NOS-dependent way Treatment with 0.2 mg/kg, but not 2.0 mg/kg, MK-801 increased the time spent on the white compartment (F2, 29 = 11.34, p = 0.0003; Fig. 1A). The highest dose increased the number of entries in the white compartment (F2, 29 = 32.82, p b 0.0001; Fig. 1C) and decreased thigmotaxis (Fig. 1G). No other effects were observed. Treatment with 0.011 mg/kg NMDA decreased the time spent in the white
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
99
Fig. 1. Effects of acute intraperitoneal injection of the NMDA antagonist MK-801 on zebrafish behavior in the light/dark test. (A) Scototaxis; (B) dark preference habituation; (C) entries in the white compartment; (D) number of squares crossed in the white compartment; (E) erratic swimming; (F) freezing; (G) thigmotaxis; and (H) risk assessment. Bar graphs represent mean ± standard error, while boxplots represent data from the 25th to the 75th percentile (boxes) and from minimum to maximum (whiskers). Survival plots represent Kaplan–Meier estimates for the cumulative incidences of first entry latencies. ***, p b 0.001 vs. control; **, p b 0.01 vs. control; *, p b 0.05 vs. control.
compartment, an effect which was abolished by pre-treatment with L-NAME in a dose which was by itself ineffective (F3, 23 = 33.13, p b 0.0001; Fig. 2A); moreover, NMDA impaired white avoidance
habituation, an effect which again was blocked by pre-treatment with L-NAME (F3, 23 = 3.811, p = 0.0261; Fig. 2B). NMDA, L-NAME or both drugs did not alter the latency to enter the white compartment
100
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
Fig. 2. Effects of acute intraperitoneal injection of NMDA and/or L-NAME on zebrafish behavior in the light/dark test. (A) Scototaxis; (B) dark preference habituation; (C) entries in the white compartment; (D) number of squares crossed in the white compartment; (E) erratic swimming; (F) freezing; (G) thigmotaxis; and (H) risk assessment. Bar graphs represent mean ± standard error, while boxplots represent data from the 25th to the 75th percentile (boxes) and from minimum to maximum (whiskers). Survival plots represent Kaplan– Meier estimates for the cumulative incidences of first entry latencies. ***, p b 0.001 vs. control; **, p b 0.01 vs. control; *, p b 0.05 vs. control.
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
101
(χ2[df = 3] = 5.537, p = 0.1365; data not shown). No effects were observed on the number of white compartment entries (H[df = 4] = 2.769, p = 0.4286; Fig. 2C) or on the number of squares crossed in the white compartment (H[df = 4] = 3.894, p = 0.2732; Fig. 2D). NMDA increased the frequency of erratic swimming events (H[df = 4] = 7.896, p = 0.0482; Fig. 2E) and risk assessment (H[df = 4] = 11.95, p = 0.0076; Fig. 2H), and the duration of freezing events (F3, 23 = 9.404, p = 0.0004; Fig. 2F) and thigmotaxis (F3, 23 = 6.5, p = 0.003; Fig. 2G); in all cases, L-NAME pre-treatment blocked the effects of NMDA. 3.2. A nitric oxide donor produces biphasic effects on scototaxis To further understand the role of nitric oxide on the control of anxiety-like behavior, we administered sodium nitroprusside (SNP), a nitric oxide donor, in five doses (from 0.5 to 10 mg/kg) in zebrafish and recorded their behavior in the light/dark test. SNP increased the time on white at 1 and 5 mg/kg, and decreased it at 10 m/kg (F[ 5, 39] = 30.65, p b 0.0001; Fig. 3A); these effects approached a bell-shaped curve when log dose–response was analyzed (unweighted r2 = 0.8526). No effects were observed on the total locomotion in the dose range analyzed (F[5, 40] = 1.224, p = 0.3188; Fig. 3B). 3.3. Synergism between 5-HTP and NMDA treatments in scototaxis Treatment with 5-HTP (300 mg/kg) decreased time on white, an effect which was potentiated by pre-treatment with NMDA (F[2, 54] = 15.78, p b 0.0001 for the interaction term; Fig. 4A). Pre-treatment with MK-801, however, did not alter the effect of 5-HTP on scototaxis — contrary to what happened with pre-treatment with L-NAME, which partially blocked the effect of 5-HTP on scototaxis (F[1, 32] = 3.417, p = 0.05; Fig. 4B). These results suggest that the activation of 5-HT receptors increases scototaxis, an effect which is potentiated, but not
Fig. 4. Effects of 5-HTP, with or without pretreatment with (A) NMDA or MK-801, or (B) L-NAME on scototaxis in the light/dark test. Different colors represent statistically significant differences.
dependent on, activation of the NMDA receptor; moreover, these effects are dependent on the activation of nitric oxide synthase. 3.4. Effects of WAY 100,635 mediated by nitric oxide WAY 100,635 increased the time spent on white, an effect which was blocked by pre-treatment with L-NAME (F4, 39 = 5.614, p = 0.0013; Fig. 5A). No effect was observed on latency to white (χ 2[df = 4] = 2.573, p = 0.6317; data not shown), squares crossed on white (H[df = 5] = 3.23, p = 0.5201; Fig. 5B) or entries on white (H[df = 5] = 6.661, p = 0.1549; Fig. 5C). While WAY 100,635 by itself had no effect on thigmotaxis, pre-treatment with L-NAME plus the highest dose of the 5-HT1AR antagonist decreased it (F4, 39 = 3.093, p = 0.0279; Fig. 5G). No effect was observed on freezing (F4, 39 = 0.1335, p = 0.969; Fig. 5F) nor on erratic swimming (H[df = 5] = 4.077, p = 0.3957; Fig. 5E). The highest dose decreased risk assessment, an effect which was blocked by L-NAME (H[df = 5] = 11.87, p = 0.0183; Fig. 5H). 4. Discussion
Fig. 3. Effects of acute treatment with sodium nitroprusside (SNP) on (A) scototaxis; and (B) number of midline crossings in the light/dark test. Bar graphs and dot-plots represent mean ± standard error. ***, p b 0.001 vs. control; **, p b 0.01 vs. control; *, p b 0.05 vs. control.
In the present work, NMDA was found to increase scototaxis, impaired habituation, and increased erratic swimming, risk assessment, freezing and thigmotaxis in the light/dark tank — effects which were blocked by pre-treatment with a NOS antagonist, L-NAME; treatment with the NMDA non-competitive antagonist MK-801 decreased scototaxis, but did not alter other variables. Similarly, treatment of rodents with MK-801 showed an anti-conflict effect and reduced thigmotaxis in an open-field (Plaznik et al., 1994), while in the plus-maze another non-competitive antagonist is ineffective (Wiley et al., 1995).
102
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
Fig. 5. Effects of WAY 100,635, with or without pretreatment with L-NAME, on (A) scototaxis, (B) latency to white, (C) number of squares crossed in the white compartment, (D) entries on the white compartment, (E) thigmotaxis, (F) freezing, (G) erratic swimming and (H) risk assessment. **, p b 0.01 vs. control (0.0 mg/kg); *, p b 0.05 vs. control.
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
In zebrafish, MK-801 has been shown to not alter light/dark preference (Sison and Gerlai, 2011), although the preference task used differed significantly from that presented here in that the light side is actually a transparent, well-lit portion, while the dark side is covered from the top; in this configuration, animals tend to prefer the light side, instead of the dark one (Blaser and Peñalosa, 2011; Gerlai et al., 2000), and thus comparison of these results with ours is limited. In zebrafish, MK-801 has also been shown to produce hyperactivity (Sison and Gerlai, 2011) and to block habituation of the acoustic startle response (Best et al., 2008; Roberts et al., 2011). Other drugs with antiglutamatergic effects have been shown to affect zebrafish behavior. Most extensively used were drugs with psychotomimetic effects (kynurenic acid, ketamine, MK-801, ibogaine; phencyclidine; Neelkantan et al., 2013) with a polypharmaceutical profile which includes but is not limited to glutamate. In general, these drugs produce an anxiolytic-like profile in different tests that is also accompanied by dissociative-like behavior, suggesting nonspecific effects on anxiety. Nonetheless, MK-801 effects on the light/dark test, as well as the NMDA experiments reported in this paper, suggest that activation of this receptor increases anxiety-like behavior, an effect mediated by nitric oxide production. Previous work showed that freezing and thigmotaxis are higher in the black compartment of a light/dark tank (Blaser et al., 2010; Blaser and Peñalosa, 2011), there is no evidence so far that these variables are increased/decreased by anxiolytic-like and anxiogenic-like treatments. Indeed, Blaser and Peñalosa (2011) did not observe changes in freezing or thigmotaxis in black after ethanol treatment. On the other hand, freezing and/or thigmotaxis on white have been repeatedly shown to be altered by drug treatments (e.g., Maximino et al., 2013b, 2014). The same was observed in the present experiments regarding the effects of NMDA. While this might represent a methodological limitation of the present work, these observations also suggest that freezing and thigmotaxis in the white compartment are qualitatively different from these behaviors in the black compartment and might be controlled by different behavioral states. Further experiments are necessary to disentangle this hypothesis. Nonetheless, stimulation of NO targets with the donor SNP produced a biphasic effect on scototaxis — that is, small and intermediate doses increase scototaxis, while intermediate doses decrease it. Biphasic effects of NO donors were observed on 5-HT release in hypothalamic slices (Kaehler et al., 1999), and 8-Br-cGMP also affects 5-HT uptake in midbrain synaptosomes in a biphasic manner (Ramamoorthy et al., 2007). Low doses of L-NAME increase open arm exploration in the elevated plus-maze and decrease immobility in the forced swim test in rats, while high doses decrease locomotor activity (Spiacci Jr et al., 2008). These results seem to suggest that the mediation of the nitrergic system on behavior or on the serotonergic system is complex, depending on the basal and stimulated levels of nitric oxide. Consistent with these observations, we observed that treatment with the 5-HT precursor 5-HTP increased scototaxis, an effect which was potentiated by pre-treatment with NMDA and partially blocked by pre-treatment with L-NAME. Nonetheless, pre-treatment with MK801 was unable to change this effect, suggesting that either activation of a different site in the NMDA receptor (the orthosteric site) is needed for synergism with 5-HTP, or that the effects of 5-HTP were mediated by nitric oxide downstream of the NMDA receptor. In consonance with this proposal, in cortical or hippocampal slices, the activation of 5-HT1A receptors reduces glutamate-evoked NO production (Maura et al., 2000; Strosznajder et al., 1996). Previous experiments in zebrafish demonstrated that WAY 100,635 by itself produced an anxiolytic-like effect in both the novel tank and the light/dark tests (Maximino et al., 2013b). More interestingly, pretreatment with L-NAME completely blocked the effects of WAY 100,635 on scototaxis and risk assessment, suggesting that nitric oxide might be involved in the mechanism of action of this drug. Combined with the previous observations, these results suggest that 5-HT1A
103
receptors (blocked by WAY 100,635) tonically inhibit nitric oxide production; the removal of this tonic inhibition would increase NO levels in small increments (as observed in rodent cortical or hippocampal slices; Maura et al., 2000; Strosznajder et al., 1996), decreasing scototaxis and risk assessment. NMDA receptor activation, on the other hand, can directly activate NOS, effectively overriding the tonic inhibition and increasing intracellular NO production to levels which increase scototaxis and risk assessment, as well as fear-like behaviors such as erratic swimming, freezing and thigmotaxis. Therefore, NOS could act as a “switch” to signal the balance of serotonergic and glutamatergic input to post-synaptic structures associated with defensive behavior. The present results suggest that tonic NOS-1 inhibition is an important downstream effector of 5-HT1A receptors in the regulation of anxiety-like behavior, and that NOS-1 is also under phasic independent control by NMDA receptors; however, the precise mechanisms by which these receptors interact to regulate anxiety-like behavior is still unclear, and further experiments are necessary to untangle concurrent hypotheses. Acknowledgments Part of this work was financed by CNPq grants (grant numbers 483336/2009-2 and 479089/2013-2) and a CAPES/Pró-Amazônia grant (grant number: 3288/2013). CM, BP and MGL were recipients of CAPES/Brazil studentships. AMH is a recipient of a CNPq/Brazil productivity grant (304130/2011-7). References Akar FY, Ulak G, Tanyeri P, Erden F, Utkan T, Gacar N. 7-Nitroindazole, a neuronal nitric oxide synthase inhibitor, impairs passive-avoidance and elevated plus-maze memory performance in rats. Pharmacol Biochem Behav 2007;87:434–43. Anichtchik O, Sallinen V, Peitsaro N, Panula P. Distinct structure and activity of monoamine oxidase in the brain of zebrafish (Danio rerio). J Comp Neurol 2006; 498:593–610. Araujo J, Maximino C, Brito TM, de, Silva, A.W.B. da, Batista, E. de J.O.Oliveira, K.R.M., Morato S, et al. Behavioral and pharmacological aspects of anxiety in the light/dark preference test. In: Kalueff AV, Stewart AM, editors. Zebrafish protocols for neurobehavioral research. New York: Humana Press; 2012. Beijamini V, Guimarães FS. Activation of neurons containing the enzyme nitric oxide synthase following exposure to an elevated plus maze. Brain Res Bull 2006;69:347–55. Bellipanni G, Rink E, Bally-Cuif L. Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain. Mech Dev 2002;3:S215–20. Best JD, Berghmans S, Hunt JJFG, Clarke SC, Fleming A, Goldsmith P, et al. Non-associative learning in larval zebrafish. Neuropsychopharmacology 2008;33:1206–15. Blaser RE, Peñalosa YM. Stimuli affecting zebrafish (Danio rerio) behavior in the light/dark preference test. Physiol Behav 2011;104:831–7. Blaser RE, Chadwick L, McGinnis GC. Behavioral measures of anxiety in zebrafish (Danio rerio). Behavioural Brain Research 2010;208:56–62. http://dx.doi.org/10.1016/j.bbr. 2009.11.009. Bonassoli VT, Milani H, de Oliveira RMW. Ethanol withdrawal activates nitric oxideproducing neurons in anxiety-related brain areas. Alcohol 2011;45:641–52. Bordieri L, di Patti MCB, Miele R, Cioni C. Partial cloning of neuronal nitric oxide synthase (nNOS) cDNA and regional distribution of nNOS mRNA in the central nervous system of the Nile tilapia Oreochromis niloticus. Mol Brain Res 2005;142:123–33. Calixto AV, Vandresen N, de Nucci G, Moreno Jr H, Faria MS. Nitric oxide may underlie learned fear in the elevated T-maze. Brain Res Bull 2001;55:37–42. Canadian Council on Animal Care. CCAC guidelines on: the care and use of fish in research, teaching and testing; 2005. De Oliveira RMW, Del Bel EA, Mamede-Rosa MLNMLN, Padovan CM, Deakin JFW, Guimarães FS. Expression of neuronal nitric oxide synthase mRNA in stress-related brain areas after restraint in rats. Neuroscience Letters 2000;289:123–6. Garthwaite J. Concepts of neural nitric oxide-mediated transmission. Eur J Neurosci 2008; 27:2783–802. Gerlai R. High-throughput behavioral screens: the first step towards finding genes involved in vertebrate brain function using zebrafish. Molecules 2010;15:2609–22. Gerlai R, Lahav M, Guo S, Rosenthal A. Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects. Pharmacol Biochem Behav 2000;67: 773–82. Giraldez-Perez RM, Gaytan SP, Ruano D, Torres B, Pasaro R. Distribution of NADPHdiaphorase and nitric oxide synthase reactivity in the central nervous system of the goldfish (Carassius auratus). 2008;35:12–32. Grahn RE, Watkins LR, Maier SF. Impaired escape performance and enhanced conditioned fear in rats following exposure to an uncontrollable stressor are mediated by glutamate and nitric oxide in the dorsal raphe nucleus. Behav Brain Res 2000;112:33–41.
104
A.M. Herculano et al. / Pharmacology, Biochemistry and Behavior 129 (2015) 97–104
Gualda LB, Martins GG, Müller B, Guimarães FS, de Oliveira RMW. 5-HT1A autoreceptor modulation of locomotor activity induced by nitric oxide in the rat dorsal raphe nucleus. Braz J Med Biol Res 2011;44:332–6. Hamane H, Tsuneyoshi Y, Denbow DM, Furuse M. N-methyl-D-aspartate and alphaamino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks. Amino Acids 2009; 37:733–9. Harvey BH. Adaptive plasticity during stress and depression and the role of glutamate–nitric oxide pathways. S Afr Psychiatry Rev 2006;9:132–9. Herculano AM, Maximino C. Serotonergic modulation of zebrafish behavior: Towards a paradox. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 55; 2014. p. 50–66. http://dx.doi.org/10.1016/j.pnpbp.2014.03.008. Holmqvist B, Ellingsen B, Alm P, Forsell J, Øyan A-M, Goksøyr A, et al. Identification and distribution of nitric oxide synthase in the brain of adult zebrafish. Neurosci Lett 2000;292:119–22. Holmqvist B, Ebbesson L, Alm P. Nitric oxide and the zebrafish (Danio rerio): developmental neurobiology and brain neurogenesis. Advances in experimental biology. Elsevier B. V; 2007. p. 229–74. Jahn-Eimermacher A, Lasarzik I, Raber J. Statistical analysis outcomes in behavioral experiments. Behav Brain Res 2011;221:271–5. Jesuthasan S. Fear, anxiety, and control in the zebrafish. Dev Neurobiol 2011;72:395–403. Kaehler ST, Singewald N, Sinner C, Philippu A. Nitric oxide modulates the release of serotonin in the rat hypothalamus. Brain Res 1999;835:346–9. Kalueff AV, Stewart AM, Kyzar EJ, Cachat J, Gebhardt M, Landsman S, et al. Time to recognize zebrafish “affective” behavior. Behaviour 2012;149:1019–36. Kaslin J, Panula P. Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio). J Comp Neurol 2001;440:342–77. Kinkel MD, Eames SC, Philipson LH, Prince VE. Intraperitoneal injection into adult zebrafish. J of Vis Exp 2010;42:2126. http://dx.doi.org/10.3791/2126. Lawrence C. The husbandry of zebrafish (Danio rerio): a review. Aquaculture 2007;269: 1–20. Lillesaar C. The serotonergic system in fish. Journal of Chemical Neuroanatomy. 2011;41: 294–308. http://dx.doi.org/10.1016/j.jchemneu.2011.05.009. López JM, Moreno N, Morona R, Muñoz M, González A. Colocalization of nitric oxide synthase and monoamines in neurons of the amphibian brain. Brain Res Bull 2005;66: 555–9. Lu Y, Simpson KL, Weaver KJ, Lin RCS. Coexpression of serotonin and nitric oxide in the raphe complex: cortical versus subcortical circuit. Anat Rec 2010;293:1954–65. Lupinsky D, Moquin L, Gratton A. Interhemispheric regulation of the medial prefrontal cortical glutamate stress response in rats. J Neurosci 2010;30:7624–33. Matsushita H, Takeuchi Y, Kawata M, Sawada T. Distribution of NADPH-diaphorasepositive neurons in the mouse brain: differences from previous findings in the rat brain and comparison with the distribution of serotonergic neurons. Acta Histochem Cytochem 2001;34:235–57. Maura G, Marcoli M, Pepicelli O, Rosu C, Viola C, Raiteri M. Serotonin inhibition of the NMDA receptor/nitric oxide/cyclic GMP pathway in human neocortex slices: involvement of 5-HT2C and 5-HT1A receptors. Br J Pharmacol 2000;130:1853–8. Maximino C. Serotonin and anxiety. Neuroanatomical, pharmacological, and functional aspects. New York, NY: Springer; 2012. Maximino C, Herculano AM. A review of monoaminergic neuropsychopharmacology in zebrafish. Zebrafish 2010;7:359–78. Maximino C, da Silva AWB, Araújo J, Lima MG, Miranda V, Puty B, Herculano AM. Fingerprinting of psychoactive drugs in zebrafish anxiety-like behaviors. PLoS ONE 2014;9: e103943. http://dx.doi.org/10.1371/journal.pone.0103943. Maximino C, de Brito TM, da Silva AWB, Herculano AM, Morato S, Gouveia Jr A, et al. Measuring anxiety in zebrafish: a critical review. Behav Brain Res 2010a;214:157–71. Maximino C, de Brito TM, Dias CA, de M, Gouveia Jr A, Morato S. Scototaxis as anxiety-like behavior in fish. Nat. Protoc. 2010b;5:209–16. Maximino C, Silva AWB, Gouveia Jr A, Herculano AM. Pharmacological analysis of zebrafish (Danio rerio) scototaxis. Prog Neuropsychopharmacol Biol Psychiatry 2011;35:624–31. Maximino C, Benzecry R, Oliveira KRM, Batista EJO, Herculano AM, Rosemberg DB, et al. A comparison of the light/dark and novel tank tests in zebrafish. Behaviour 2012;149: 1099–123. Maximino C, Lima MG, Araujo J, Oliveira KRM, Herculano AM, Stewart AM, et al. The serotonergic system of zebrafish: genomics, neuroanatomy and neuropharmacology. In: Hall FS, editor. Serotonin: biosynthesis. New York, NY: Regulation and Health Implications. Nova Science; 2013a. p. 53–67. Maximino C, Puty B, Benzecry R, Araújo J, Lima MG, Batista, E. de J.O., et al. Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect
of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine in two behavioral models. Neuropharmacology 2013b;71:83–97. Neelkantan N, Mikhaylova A, Stewart AM, Arnold R, Gjeloshi V, Kondaveeti D, et al. Perspectives on zebrafish models of hallucinogenic drugs and related psychotropic compounds. ACS Chem Neurosci 2013;4:1137–50. Norton W, Bally-cuif L. Adult zebrafish as a model organism for behavioural genetics. BMC Neurosci 2010;11:90. Norton WHJ, Folchert A, Bally-Cuif L. Comparative analysis of serotonin receptor (HTR1A/ HTR1B families) and transporter (slc6a4a/b) gene expression in the zebrafish brain. J Comp Neurol 2008;511:521–42. Okere CO, Waterhouse BD. Activity-dependent heterogeneous populations of nitric oxide synthase neurons in the rat dorsal raphe nucleus 6; 2006. Olney JW, de Gubareff T, Labruyere J. α-Aminoadipate blocks the neurotoxic action of N-methyl aspartate. Life Sci 1979;25:537–40. Plaznik A, Palejko W, Nazar M, Jessa M. Effects of antagonists at the NMDA receptor complex in two models of anxiety. Eur Neuropsychopharmacol 1994;4:503–12. Pokk P, Väli M. Effects of nitric oxide synthase inhibitors 7-NI, L-NAME, and L-NOARG in staircase test. Arch Med Res 2002;33:265–8. Ramamoorthy S, Samuvel DJ, Buck ER, Rudnick G, Jayanthi LD. Phosphorylation of threonine residue 276 is required for acute regulation of serotonin transporter by cyclic GMP. J Biol Chem 2007;282:11639–47. Reznikov LR, Grillo CA, Piroli GG, Pasumarthi RK, Reagan LP, Fadel J. Acute stressmediated increases in extracellular glutamate levels in the rat amygdala: differential effects of antidepressant treatment. Eur J Neurosci 2007;25:3109–14. Roberts AC, Reichl J, Song MY, Dearinger AD, Moridzadeh N, Lu ED, et al. Habituation of the C-start response in larval zebrafish exhibits several distinct phases and sensitivity to NMDA receptor blockade. PLoS ONE 2011;6:e29132. Simpson KL, Waterhouse BD, Lin RCS. Differential expression of nitric oxide in serotonergic projection neurons: neurochemical identification of dorsal raphe inputs to rodent trigeminal somatosensory targets. J Comp Neurol 2003;512:495–512. Sison M, Gerlai R. Behavioral performance altering effects of MK-801 in zebrafish (Danio rerio). Behav Brain Res 2011;220:331–7. Smith JCE, Whitton PS. Nitric oxide modulates N-methyl-D-aspartate-evoked serotonin release in the raphe nuclei and frontal cortex of the freely moving rat. Neurosci Lett 2000;291:5–8. Spiacci Jr A, Kanamaru F, Guimarães FS, Oliveira RMW. Nitric oxide-mediated anxiolyticlike and antidepressant-like effects in animal models of anxiety and depression. 2008;88:247–55. Steenbergen PJ, Richardson MK, Champagne DL. The use of the zebrafish model in stress research. Prog Neuropsychopharmacol Biol Psychiatry 2011;35:1432–51. Stewart A, Gaikwad S, Kyzar E, Green J, Roth A, Kalueff AV. Modeling anxiety using adult zebrafish: a conceptual review. Neuropharmacology 2011;62:135–43. Stewart AM, Cachat J, Gaikwad S, Robinson K, Gebhardt M, Kalueff AV. Perspectives on experimental models of serotonin syndrome in zebrafish. Neurochemistry International 2013;62:893–902. http://dx.doi.org/10.1016/j.neuint.2013.02.018. Strosznajder J, Chalimoniuk M, Samochocki M. Activation of serotonergic 5-HT1A receptor reduces Ca2+- and glutamatergic receptor-evoked arachidonic acid and NO/cGMP release in adult hippocampus. Neurochem Int 1996;28:439–44. Vincent SR, Kimura H. Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 1992;46:755–84. Virgili M, Poli A, Beraudi A, Giuliani A, Villani L. Regional distribution of nitric oxide synthase and NADPH-diaphorase activities in the central nervous system of teleosts. Brain Res 2001;901:202–7. Wang Q-P, Guan J-L, Nakai Y. Distribution and synaptic relations of NOS neurons in the dorsal raphe nucleus: a comparison to 5-HT neurons. Brain Res Bull 1995;37:177–87. Wang Y, Takai R, Yoshioka H, Shirabe K. Characterization and expression of serotonin transporter genes in zebrafish. Tohoku J Exp Med 2006;208:267–74. Wiley JL, Cristello AF, Balster RL. Effects of site-selective NMDA receptor antagonists in an elevated plus-maze model of anxiety in mice. Eur J Pharmacol 1995;294:101–7. Wolf K. Physiological salines for freshwater teleosts. Prog Fish Cult 1963;25:135–40. Yilmazer-Hanke DM, Roskoden T, Zilles K, Schwegler H. Anxiety-related behavior and densities of glutamate, GABA A, acetylcholine and serotonin receptors in the amygdala of seven inbred mouse strains. Behav Brain Res 2003;145:145–59. Zhang J, Huang X-Y, Ye M-L, Luo C, Wu H-Y, Hu Y, et al. Neuronal nitric oxide synthase alteration accounts for the role of 5-HT1A receptor in modulating anxiety-related behaviors. J Neurosci 2010;30:2433–41.