- Email: [email protected]
Distribution and chronotropic effects of serotonin in the zebrafish heart
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Distribution and chronotropic effects of serotonin in the zebrafish heart Matthew R. Stoyeka,b, Michael G. Jonzc, Frank M. Smitha,1, Roger P. Crollb,⁎,1 a b c
Department of Medical Neuroscience, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Department of Physiology & Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Department of Biology, University of Ottawa, Ottawa, Ontario, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords: Serotonin 5-Hydroxytryptamine Zebrafish Heart Intracardiac nervous system Chronotropy
Several lines of evidence suggest that serotonin (5-HT) has a regulatory role in cardiovascular function from embryogenesis through adulthood. However, the reported actions of 5-HT are often contradictory and include bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction. Clarifying such cardiac effects requires further research and may benefit from utilizing a model simpler than the mammalian hearts traditionally used in these studies. In the present study, we describe the cardiac distribution and chronotropic responses of 5-HT in the zebrafish heart. A combined anatomical, electrophysiological, and pharmacological approach was used to investigate the involvement of 5-HT pathways, and to compare neural and direct myocardial pathways of biological action. Immunohistochemical methods revealed 5-HT in endocardial cells, glial-like cells, and intracardiac neurons in the atrium. Electrocardiogram (ECG) recordings combined with the administration of pharmacological agents demonstrated that 5-HT acted predominantly through direct myocardial pathways resulting in a reduction of heart rate. Overall, the results of this study contribute significant advances in the establishment of the zebrafish as a new model for studies of the role of 5HT in autonomic cardiac control.
1. Introduction Serotonin (5-hydroxytryptamine; 5-HT) was first isolated from blood and defined as a vasoconstrictor (Nebigil and Maroteaux, 2001), but has also been shown more recently to have diverse cardiovascular effects (Frishman and Grewall, 2000). Described effects include bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction, and these effects vary greatly depending on route of administration and experimental parameters (see review by Nebigil and Maroteaux, 2001). Clarification of these complex and seemingly conflicting effects requires further research and may benefit from the employment of simpler models than the mammalian hearts traditionally used in these studies. The zebrafish heart has been proposed as a powerful tool for studying cardiac electrophysiology, with the potential to provide broad insights into cardiovascular function (Briggs, 2002; Chi et al., 2008; Dvornikov et al., 2014; Nemtsas et al., 2010; Rider et al., 2012; Tessadori et al., 2012). Stoyek et al. (2015) previously described the basic structure of the intracardiac nervous system in zebrafish, which is consistent with that of humans and other mammalian models (Irisawa, 1978; Mangoni and Nargeot, 2008; Pauza et al., 2013; Pauza et al.,
⁎
1
2014; Li et al., 2015). In addition, the zebrafish appears to have genes encoding for autonomic receptors, including 5-HT receptors, (5-HT: Airhart et al., 2007; Prieto et al., 2012; Maximino et al., 2013; Stewart et al., 2013), that are homologous to those that affect heart rate (HR) in mammals (β-adrenergic/catecholaminergic: Steele et al., 2011; nicotinic, muscarinic and β-adrenergic; Stoyek et al., 2016). The study of serotonergic processes in zebrafish is well established. Previous reports have described behavioral responses (predominantly a decrease in spontaneous locomotor activity) to 5-HT exposure that are similar to those observed in mammalian studies, indicating the potential validity of the zebrafish model in such research (Airhart et al., 2007; Prieto et al., 2012; Maximino et al., 2013; Stewart et al., 2013). To date investigations of the organ-specific physiological effects of 5-HT in zebrafish have been limited, focusing primarily on the role of pathways involved in release of 5-HT from chromaffin cells in the head kidney (Steele et al., 2011) or chemosensory neuroepithelial cells in the gills (Jonz et al., 2004). While 5-HT is known to be present in the circulatory system of the zebrafish (Steele et al., 2011), information on the cardiac effects of 5-HT is lacking, with only a single report alluding to chronotropic effects of high doses of fluoxetine, a commonly used 5-HT selective reuptake inhibitor (SSRI; Airhart et al., 2007).
Corresponding author at: Department of Physiology & Biophysics, Faculty of Medicine, Dalhousie University, 5850 College Street, PO Box 15000, Halifax, NS B3H 4R2, Canada. E-mail address: [email protected] (R.P. Croll). Both authors contributed equally.
http://dx.doi.org/10.1016/j.autneu.2017.07.004 Received 1 November 2016; Received in revised form 3 May 2017; Accepted 17 July 2017 1566-0702/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Stoyek, M.R., Autonomic Neuroscience: Basic and Clinical (2017), http://dx.doi.org/10.1016/j.autneu.2017.07.004
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
PBS-T). After incubation in primary antibodies for 3–5 d with agitation at 4 °C, the tissues were rinsed in PBS-T, and then transferred to a solution of PBS-T containing the appropriate secondary antibodies, raised in mouse or rabbit, and conjugated to AlexaFluor 488, 555, or 647 fluorophores (Life Technologies, Burlington, ON, Canada). Incubation time with secondary antibodies was also 3–5 d with agitation at 4 °C. Final rinsing was done in PBS, and then specimens were placed in Scale CUBIC-1 clearing solution (Susaki et al., 2014) overnight at room temperature with gentle agitation. Tissues were mounted on glass slides in CUBIC-1 for confocal microscopy.
The aim of the present study was to investigate the distribution of 5HT and determine the chronotropic responses to 5-HT application in isolated zebrafish hearts. A combined anatomical, electrophysiological, and pharmacological approach was used to investigate the role of 5-HT, and to define the neural- and tissue-level actions of this agent. We used immunohistochemical methods to characterize the distribution of 5-HT and tryptophan hydroxylase (TPH, the rate-limiting enzyme in the production of 5-HT; Levy, 2006) within intracardiac neurons (ICNs) and myocardial cells. Electrocardiogram (ECG) recordings combined with bath-applied pharmacological agents were used to demonstrate in the isolated zebrafish heart that 5-HT acted predominantly through direct myocardial pathways to modulate heart rate (HR). The results of this study establish this preparation as a tractable model to investigate serotonergic effects on the heart.
2.5. Antibodies Serotonergic elements were detected with an anti-5-HT antibody (dilution 1:100; 20080, Immunostar). This antibody has been used in previous studies in zebrafish (Uyttebroek et al., 2010; Jackson et al., 2013). Antibodies against tryptophan hydroxylase (TPH; dilution 1:100; P21961, Life Technologies), the rate-limiting enzyme in the synthesis of 5-HT, and tyrosine hydroxylase, the rate limiting enzyme in the synthesis of norepeinephrine (TH; dilution 1:100; 22941; Immunostar) were used to investigate potential synthetic pathways for monoamines. As the TPH antibody was raised in the same host as the 5HT antibody, it was not possible to co-label for 5-HT in the same heart. To compare 5-HT elements to the general innervation of the heart, antibodies against acetylated tubulin (AcT, axons; dilution 1:250; T6793, Sigma Aldrich) and human neuronal protein C/D (Hu, neuronal somata; dilution 1:250; A21271, Life Technologies) were combined as previously described (Stoyek et al., 2015). In a separate trial (n = 8) hearts were processed as outlined above, except that either the primary or secondary antibody was omitted, both of which eliminated detection of histofluorescence in all specimens.
2. Methods 2.1. Animals A total of 35 AB strain adult zebrafish (12–18 month post fertilization; 33 ± 5 mm standard body length) was used in this study. Procedures for animal care and use followed the “Guidelines on the Care and Use of Fish in Research, Teaching and Testing” document issued by the Canadian Council of Animal Care. Institutional approval (Protocol #15-006) for animal use in this study was obtained from the Dalhousie University Committee on Laboratory Animals. 2.2. Animal husbandry Animals were acquired from breeding stocks in the Faculty of Medicine Zebrafish Facility at Dalhousie University. Fish were maintained in standard 3–10 L tanks (Aquatic Habitats, Apopka, FL, USA) at 28.5 °C, supplied continuously with conditioned water from a recirculating water system, and subjected to a 14 hour light:10 hour dark illumination cycle. Zebrafish were fed commercial, dry fish food (Golden Pearl pellets, Brine Shrimp Direct, Ogden, UT, USA) and live Artemia (raised in-house) twice a day.
2.6. Myocardial labelling In order to determine how immunohistochemically labelled neuronal elements were related to the regional structure of the myocardium, some specimens were double-labelled either with AcT-Hu or 5-HT antibodies in combination with the F-actin marker phalloidin (77418, Sigma Aldrich; Stoyek et al., 2015), conjugated with tetramethyl rhodamine isothiocyanate to show cardiac myocytes.
2.3. Heart isolation Zebrafish hearts were isolated following procedures described in Stoyek et al. (2016). Briefly, fish were anaesthetised in a buffered solution (pH 7.2) of tricaine (MS-222; 1.5 mM; Sigma–Aldrich, Oakville, ON, Canada) in tank water (28.5 °C) until opercular movements ceased and the animals lacked response to a fin pinch with forceps. A ventral midline incision was made through the body wall to expose the heart, and a block of tissue encompassing the ventral aorta, ventricle, atrium, sinus venosus and ducts of Cuvier was then removed for whole-mount immunohistochemistry or in vitro recordings.
2.7. Imaging Tissues were viewed using a Zeiss LSM510 confocal microscope operating with Zen2009 software (Zeiss Canada, Mississauga, ON). Preparations were epi-illuminated with a 488 nm argon laser, and 543 nm and 605 nm helium‑neon lasers, reflected by a 488/543/ 633 nm dichroic mirror (HFT 488/543/633; Carl Zeiss AG). Emitted fluorescence was collected from the specimens, using 480–520 nm and 500–615 nm band-pass filters (BP565-615; Carl Zeiss AG) and a 565–615 nm long pass filter (LP565-615; Carl Zeiss AG) through a 10×, 0.45 NA objective (Plan-Apochromat SF25; Carl Zeiss AG), a 25×, 0.80 NA objective (LCI Plan-Neofluar; Carl Zeiss AG), or a 40 ×, 0.95 NA objective (Plan-Apochromat M27; Carl Zeiss AG). Z-stacks were taken from regions of interest surrounding immunoreactive tissues, and ranged from 20 to 100 μm in depth. Z-stack scans also encompassed 5–25 μm above and below the regions of interest to ensure that all structures were captured, while limiting issues of light scattering associated with deeper tissue scans. Image stacks were processed with Zeiss Zen2009 software. Figure plates were constructed from images processed with Photoshop CS6 (Adobe Systems Inc., San Jose, CA, USA). Brightness and contrast of some images were adjusted to ensure panel-to-panel consistency in each figure.
2.4. Tissue preparation for immunohistochemistry Tissues were fixed overnight in 4% paraformaldehyde (PFA; RT15710, Electron Microscopy Sciences, Hatfield, PA, USA) in phosphatebuffered saline (PBS, composition in mM: 50 Na2HPO4, 140 NaCl, pH 7.2). To visualize the overall spatial relationship of serotonergic elements within the heart of the zebrafish, the whole-mount procedures used for immunohistochemistry in this study were similar to those described in previous publications (Newton et al., 2014; Stoyek et al., 2015; Stoyek et al., 2016). Briefly, fixed tissues were rinsed in PBS and then transferred to a PBS solution containing 2% Triton X-100 (X100, Sigma Aldrich), 1% bovine serum albumin (BSA; A9576, Sigma Aldrich) and 1% normal goat serum (NGS; G9023, Sigma Aldrich) for 48 h at 4 °C with gentle agitation. Tissues were then incubated with primary antibodies (see Section 2.5), which were diluted in a solution containing 0.25% Triton X-100, 1% BSA and 1% NGS in PBS (designated 2
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
Fig. 1. 5-HT-immunoreactivity in the atrial wall. A: Schematic showing the cardiac regions: atrium, a; bulbus arteriosus, ba; sinoatrial region, SAR; sinus venosus, sv; ventricle, v. Box indicates the approximate location of image in panel B. B: Acetylated tubulin and human neuronal protein C/D (AcT-Hu; white) and 5-HT (green) immunoreactivity in the atrial myocardium. Musculature is labelled with phalloidin (Phal; magenta). 5-HT-like immunoreactive (-LIR) cells were localized to the luminal edges of atrial trabeculae. Dashed box indicates the approximate region of panels C–E. C–D: Single confocal images enlarged to show details of the variety of 5-HT-LIR cell morphologies from panel A. 5-HT-LIR cells within the atrial wall were restricted to the endocardial region. Most 5-HT-LIR cells were round to ovoid in shape (C, arrows) with some elongated spindle-shaped cells also observed (D, arrows). In all preparations AcT-Hu-IR somata and axons were observed in close proximity to 5-HT-LIR cells but axonal contacts with these cells were not observed. E: In all preparations tyrosine hydroxylase (TH)-LIR axons were observed in close proximity to 5-HT-LIR cells but neither axonal contacts with these cells, nor coexpression of TH with 5-HT, was observed. Scale bars: 150 μm in B; 35 μm in C–E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
from light, and delivered to the bath at the chamber inlet in 200 μL boluses through a micropipette via a calibrated syringe attached to a screw-drive microinjector (IM-4B; Narishige, East Meadow, NY, USA). The peak concentration within the constant flow bath was calculated to be 20 μM. Based on visual dye experiments a homogeneous distribution within the bath was reached in approximately 0.5–1 min, with much of the washout occurring between 1.5 and 2 min. As a control, 200 μL boluses of saline were delivered to the tissue in the same manner and did not elicit chronotropic responses. Ketanserin (5-HT2-receptor blocker; 10 μM; S006, Sigma Aldrich), spiperone (5-HT1-receptor blocker; 10 μM; S7395, Sigma Aldrich), and fluoxetine (selective 5-HT reuptake inhibitor at presynaptic receptors; 5 μM; F132, Sigma Aldrich) were dissolved in saline and the tissue was superfused continuously with these agents for 15 min prior to recording. Concentrations of serotonergic agents were chosen based upon previous studies reporting effective blockade with minimal direct HR effects (see Section 4.2). Autonomic antagonists atropine (post-junctional muscarinic receptor blocker; 10 μM; A0132, Sigma Aldrich) and timolol (post-junctional βadrenergic receptor blocker; 100 μM; T6394, Sigma Aldrich) were dissolved in saline and superfused continuously over the tissue for 15 min prior to recording. Responses to 5-HT were then recorded in the presence of either serotonergic (ketanserin, spiperone, or fluoxetine) or
2.8. Measurement of heart rate Isolated, beating hearts were pinned through the ventral aorta and walls of the ducts of Cuvier to the Sylgard rubber (Dow Corning, Midland, MI, USA) bottom of a 5 mL chamber which was superperfused with zebrafish saline (composition in mM: 124.1 NaCl, 5.1 KCl, 2.9 Na2HPO4, 1.9 MgSO4-7H2O, 1.4 CaCl2-2H2O, 11.9 NaHCO3; aerated with room air; pH 7.2; 25 °C) at a constant rate of 10 mL·min− 1 (Stoyek et al., 2016). Hearts were allowed to equilibrate in the bath for 30 min prior to testing. Electrocardiogram (ECG) signals were recorded from the surface of the atrium with a bipolar suction electrode, differentially amplified (sample rate 2 kHz; total gain ×1000–10,000), and stored on a personal computer after analog-digital conversion (Digidata 1322A, Axon Instruments, Foster City, CA, USA). To quantify HR responses, the time between adjacent R-waves of the ECG (R-R interval) was measured using Axoscope software (Axon Instruments). 2.9. Pharmacological agents Basal R-R intervals (“control”) were first recorded prior to the delivery of any pharmacological agents. 5-HT (1 mM; H7752, Sigma Aldrich) was dissolved in saline on the day of the experiment, protected 3
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
of any heart. In hearts labelled for TPH (n = 12), a population of ICNs with similar spatial distribution to 5-HT-LIR ICNs (Fig. 2E) was observed in the dorsal SAR (see Table 1). TPH-LIR ICNs showed co-labelling with AcT-Hu (n = 6) in all hearts examined, while in preparations co-labelled for TH (n = 6) no co-labelling was observed. As colabelling of 5-HT and TPH could not be performed, however, it was assumed that 5-HT-LIR ICNs were also TPH-LIR, based upon similar numbers of immunoreactive ICNs, their morphology and location within the SAR.
autonomic blockade (atropine/timolol) to investigate the pathways of 5-HT action. After recording responses to 5-HT in the presence of serotonergic agents or autonomic antagonists, the bath supply was switched to fresh saline and these chemicals were washed (“washout”) from the chamber and responses to 5-HT alone were again recorded (“recovery”) to control for toxicity or long-lasting effects of the pharmacological agents. 2.10. Data analyses
3.2. Effects of 5-HT on R-R interval
Numerical values are expressed as mean ± 1 standard error (SE). Statistical analyses (two-way ANOVA with Tukey's post-hoc, Student's ttest and regression) were performed using SPSS (IBM Canada, Markham, ON) to detect significant differences among means with significance set at P ≤ 0.05.
In the current study the initial HR of the isolated hearts in the bath was 112 ± 5 beats·min− 1 (n = 40; range 88–144 beats per minute). Chronotropic changes during trials are shown as proportional changes in R-R interval relative to values obtained prior to each experimental trial (“controls”). The effects of 5-HT and serotonergic agents are summarized in Fig. 3. Application of 5-HT alone resulted in a significant increase in RR interval (Fig. 3A, B). Exposure to ketanserin (KET; a 5-HT2 receptor family antagonist, Fig. 3A, C) and spiperone (SPIP; a general 5-HT and dopamine D2 receptor antagonist, Fig. 3A) prevented the elongation of the R-R interval that occurred during initial exposure to 5-HT, and R-R interval was found to be not significantly different from control. In hearts exposed to fluoxetine (FLX; 5-HT reuptake inhibitor at presynaptic membrane, Fig. 3A, D) there was a significant increase in 5HT-induced R-R elongation compared with control hearts and those hearts receiving 5-HT alone. In hearts exposed to 5-HT during autonomic blockade (AB, Fig. 3A) a significant elongation of the R-R interval was maintained. In order to control for possible toxicity or other long lasting effects of serotonergic agents or of AB, each heart was exposed again to 5-HT after the agents were washed from the chamber. In all cases, the post-washout responses to 5-HT were found to not be significantly different than those observed in control preparations exposed to 5-HT alone. It should also be noted that the total duration of each experiment (< 1 h) was less than the period in which we have previously described the zebrafish heart as having a stable R-R interval (6 h; Stoyek et al., 2016).
3. Results 3.1. Cardiac distribution of 5-HT In this study 5-HT-like-immunoreactive (-LIR) cells were observed solely within the atrium. Within the ventricle, only AcT-Hu and TH immunoreactivities were observed as we have previously described (Stoyek et al., 2015), and therefore will not be dealt with in this study. Three distinct types of cells containing 5-HT were observed within the atrium, which for the purposes of this study have been classified based upon their morphology, location, and immunoreactivity. The most numerous of the 5-HT-LIR cell types within the atrium was associated with the lumenal surfaces of the atrium along the trabeculae. These cells did not co-label with phalloidin (Fig. 1B), thus indicating that they were not myocytes. These cells appeared to be part of the endocardial cell layer, thus we have termed them “endocardial” in this study (size range 10-30 μm). These cells were also neither TPH-LIR, nor TH-LIR (Fig. 1E), thus suggesting that they were likely sequestering 5HT but not synthesizing it or catecholamines. The endocardial 5-HT-LIR cells were typically within 5–15 μm of axons labelled with AcT-Hu, but direct axonal terminations on these cells were not observed. To control for the possibility that some of the observed 5-HT immunoreactivity in the heart was due to 5-HT contained in platelets or erythrocytes that adhered to the trabeculae, blood samples were collected from the cardiac vessels and then fixed and incubated with antibodies against 5-HT (n = 3) and TPH (n = 3). No immunoreactive erythrocytes were found in any samples, but 5-HT-LIR platelets were observed in all samples. The size of the platelets (< 3 μm) was inconsistent with any of the 5HT-LIR cells observed in the atrial wall, and it is therefore unlikely that they contributed to our descriptions of cells reported here. A second population of 5-HT-LIR cells was detected within the sinoatrial valve region (SAR) at the junction of the sinus venosus and atrial myocardium (n = 12; Fig. 2B–C). These cells often had numerous small projections from the somata and given this morphology these cells are here referred to as “multipolar” cells (size range 25-50 μm). No immunoreactivity was co-localized to AcT-Hu and 5-HT (n = 6; Fig. 2B) or TH and 5-HT (n = 6; Fig. 2C) within the multipolar 5-HT-LIR cells. These cells were closely associated with varicose axons (within 5–15 μm), but did not themselves appear to receive direct innervation. In hearts labelled for TPH (n = 6), no multipolar cells within this region exhibited immunoreactivity, suggesting that, as with the endocardial cells, these cells were likely sequestering rather than synthesizing 5-HT. A third population of 5-HT cells in the atrium appear to be intracardiac neurons (ICNs). Within the sinoatrial plexus a number of 5HT-LIR ICNs was identified based on morphology and co-localized immunoreactivity with AcT-Hu (Fig. 2); their occurrence was variable among the hearts examined (see Table 1) and was restricted to the dorsal SAR region (see Stoyek et al., 2015). In hearts in which 5-HT-LIR ICNs were observed, all showed co-labelling with AcT-Hu (n = 6). There was, however, no co-expression of 5-HT and TH (n = 6) in ICNs
4. Discussion Several lines of evidence now suggest that 5-HT has a regulatory role in cardiovascular function throughout life (Nebigil and Maroteaux, 2001). Taken together these reports indicate a need to better understand the distribution and basic actions of 5-HT in the heart. 4.1. Distribution of 5-HT within the zebrafish heart 5-HT has previously been identified in the hearts of rats (Beauvallet et al., 1968; Berkowitz et al., 1974), cats (Votavova et al., 1971), dogs (Madan et al., 1970), and humans (Singh et al., 1999). Although previous studies have described the distribution of 5-HT in peripheral tissues of zebrafish (gills: Jonz et al., 2004; Qin et al., 2010; gut: Olsson et al., 2008), the current study represents the first description of its distribution within the zebrafish heart. Endocardial cells within the atrium represented the most plentiful source of 5-HT immunoreactivity identified in the current study. These cells were found to be associated with atrial trabeculae, and did not extend into the working myocardium. Final determination of the type of cell observed here may eventually require the use of a combination of markers for 5-HT and for endothelial markers but it should also be noted that 5-HT has previously been reported in endocardial cells in mice (Yavarone et al., 1993). The endocardial cells in zebrafish did not express TPH or TH, indicating that 5-HT content was most likely the result of uptake, and that these cells could not synthesize either 5-HT or catecholamines. In support of this, Sari and Zhou (2003) showed in 4
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
Fig. 2. Organization of 5-HT-immunoreactivity in the sinoatrial region (SAR). A: Schematic showing the cardiac regions (labels as in Fig. 1). Boxes indicate the locations of images in panels B-F. B: AcT-Hu and 5-HT immunoreactivity at the junctions of the left vagosympathetic trunk with the SAP. Dashed box indicates the region of panel C. C: Cells in this region showed 5-HT-LIR most strongly in somata with short projections. These cells did not express AcT-Hu, or TH, immunoreactivity. D: Within the SAR, some neuronal somata displayed 5-HTLIR, and co-expressed AcT-Hu (somata indicated by arrowheads). E–F: In a separate group of hearts neuronal somata with the same morphology as 5-HT-LIR somata in panel D coexpressed AcT-Hu and tryptophan hydroxylase (E; TPH, indicated by arrowheads). TH-LIR (F) was observed in axons distinct from 5-HT-LIR neuronal somata and axons. Scale bars: 75 μm in B; 40 μm in C, 40 μm in D–F.
tempting to speculate that the 5-HT-LIR multipolar cells we observed in the heart of the zebrafish represent a peripheral variant of the cells in the CNS in other species. In the zebrafish heart 5-HT-LIR multipolar cells did not express immunoreactivity for the general neuronal markers AcT-Hu. Though AcT-Hu has previously been established as a general neuronal marker in the zebrafish (Olsson, 2009; Stoyek et al., 2015), it is not currently known how efficacious these markers are for detecting all neuron types. That the multipolar cells shown in this study were Hunegative, and displayed glial- or astrocyte-like morphology, does not necessarily eliminate the possibility that such cells may represent a class of neurons, thus further investigation into their cellular identity must be performed. The third cell type appeared to be neuronal and thus represent a
cultured rat myocytes that 5-HT was detected in the absence of TPH, indicating that 5-HT was present in these cells due to uptake and not synthesis (Yavarone et al., 1993). In the tissue at the sinoatrial junction, there existed a second population of 5-HT-LIR cells, which given their morphology, we have termed multipolar cells. These cells were smaller than other 5-HT-LIR cell types in the heart, with numerous short projections from the cell somata, imparting a glial- or astrocyte-like appearance. These cells, like the endocardial cells, did not appear to show immunoreactivity for the synthesizing enzyme TPH, indicating that 5-HT within these cells is also likely the result of uptake rather than synthesis. In the CNS, glia and astrocytes can actively sequester neurotransmitter substances including NE, dopamine, and 5-HT (Henn and Hamberger, 1971). It is thus Table 1 Details of 5-HT- and TPH-LIR in ICNs. 5-HT-LIR
TPH-LIR
ICN #
n/N
AcT-Hu +
TH +
Size range
ICN #
n/N
AcT-Hu +
TH +
Size range
8 5 0
9/12 2/12 1/12
Y
N
40–75 μm
6 4 0
5/12 5/12 2/12
Y
N
40–75 μm
5
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
Fig. 3. A: Chronotropic responses to 5-HT and serotonergic agents. Exposure to 5-HT significantly (*) increased R-R interval. Effects of 5-HT were eliminated during exposure to ketanserin (KET) and spiperone (SPIP) but not during combined fluoxetine (FLX) and 5-HT exposure or during autonomic input blockade (atropine and timolol combined with 5-HT). In all cases, normal responses to 5-HT alone were observed after each agent was washed from the chamber (Post KET, Post SPIP, Post FLX, Post AB). B–D: Representative electrocardiograms from experiments in panel A showing cardiac responses. R-R interval during exogenous 5-HT application was elongated in comparison with pre-treatment (B). 5-HT initially evoked R-R interval increases that were prevented when 5-HT was re-applied in the presence of ketanserin (KET; C). 5-HT evoked R-R interval increases were enhanced when 5-HT was re-applied in the presence of fluoxetine (FLX; D). *P < 0.05 vs. control by pair-wise t-test; n = 8 for each group.
current study, however, no evidence for co-localization of either 5-HT or TPH with TH in ICNs was not observed. It has also been suggested that inconsistencies in the literature regarding 5-HT-immunoreactivity could be due to the sensitivity of the technique used to detect 5-HT, the state and source of the tissues examined, or the species examined (Singh et al., 1999). This is supported by reports that pre-treatment with serotonergic agents greatly altered the pattern and density of 5-HT detected by immunohistochemical methods (see Yavarone et al., 1993). Therefore, it is possible that, in the zebrafish heart, pretreatment with serotonergic compounds could potentially enhance the content of 5-HT contained within ICNs, which may reveal such colocalization of these markers.
subset of the diverse, intracardiac neurons (ICNs) previously described in the zebrafish using antibodies to AcT-Hu (Stoyek et al., 2015). Cardiac ganglia have traditionally been thought to contain only cholinergic post-ganglionic neurons, though there is accumulating evidence that cardiac ganglia contain a heterogeneous population of neurons that synthesize and respond to numerous neurotransmitters and neuromodulators (Singh et al., 1999; Pauza et al., 2014; Stoyek et al., 2015). Singh et al. (1999) previously reported that a subset of ICNs in the human heart were the only cells that contained TPH, and that they were capable of synthesizing 5-HT. In the zebrafish heart, a population of TPH-LIR ICNs was observed with similar morphology and distribution to 5-HT-LIR ICNs, thus indicating that in zebrafish these neurons have the potential to synthesize 5-HT, and this may be a feature of serotonergic ICNs that is conserved from zebrafish to mammals. Final confirmation, however, will require direct double labelling experiments using different 5–HT and TPH antibodies. 5-HT is known to be taken up by sympathetic neurons for later corelease with norepinephrine and other aminergic transmitters and peptides (Nebigil and Maroteaux, 2001; Jaffré et al., 2009). In the
4.2. 5-HT effects on heart rate Application of 5-HT to the isolated zebrafish heart presumably mimicked the endogenous release of 5-HT from one or more of the cell types identified in this study. Although the absolute concentration of the exogenously applied 5-HT was not known at the cellular level in our 6
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
have been shown to have minimal effects on cardiac conduction, there exists a growing number of reports which implicate them in fibrillation and arrhythmic events (Pacher et al., 1999). Compounding this are reports implicating psychosocial factors, such as mood and anxiety disorders, as having a significant role in the prognosis of adverse cardiac events during SSRI usage. In other words, a population of patients already potentially susceptible to cardiac dysfunction could be taking drugs which themselves are known to potentially adversely affect cardiac function (Roose and Miyazaki, 2005; Gorman and Sloan, 2000), emphasizing the need to better understand the detrimental cardiac effects of serotonergic agents on cardiac performance. Taken together the results of this study lay the foundation for the use of zebrafish in future studies investigating the pharmacology of 5-HT in the heart. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.autneu.2017.07.004.
preparation, bolus applications of 5-HT reliably resulted in a degree of bradycardia similar to that observed during 5-HT exposure in the hearts of rats (Yamano et al., 1994; Chuang et al., 1993), cats (Schneider and Yonkman, 1953; Jacobs and Comroe, 1971), and dogs (MacCanon and Horvath, 1954). In the current study the serotonergic antagonists ketanserin and spiperone prevented 5-HT-induced bradycardia, supporting the concept that 5-HT receptor activation was likely responsible for the negative chronotropic effects of this agent. Spiperone is known to act at both 5HT1 and 5-HT2 receptor types, while ketanserin, a selective antagonist of 5-HT2 receptors, blocks the physiologic effects of 5-HT on the chemosensory afferent fibres of the gills in the heart (Shakarchi et al., 2013). That both spiperone and ketanserin blocked 5-HT-induced bradycardia in the present study suggests a role for the 5-HT2 receptor in the zebrafish heart. That the effects of 5-HT were not affected by autonomic blockade with atropine and timolol suggests that 5-HT acted by a direct myocardial mechanism rather than indirectly through modulation of classical parasympathetic and sympathetic pathways. Further studies are necessary to clarify this issue. The potential for a serotonergic tonus was taken into account during initial experiments. The concentrations of ketanserin, spiperone, and fluoxetine were chosen based upon levels which effectively blocked a response to exogenous 5-HT, but which did not alter basal heart rate. From this it was assumed that if a 5-HT tonus existed within the isolated heart, its level was minimal. It was also therefore assumed that the enhanced bradycardic effects of 5-HT observed in the current study during exposure to fluoxetine were the result of increased duration of myocardial 5-HT exposure, due to the prevention of 5-HT reuptake, and not a direct myocardial action of fluoxetine itself. In preliminary experiments, however, exposure to fluoxetine or ketanserin alone at much higher concentrations of between 20 and 100 μM, was found to induce bradycardia and arrhythmias. Furthermore, at the highest concentration of fluoxetine, there was frequent initiation of dysrhythmia within 1 min of exposure, and which frequently led to subsequent, sustained periods of highly irregular and uncoordinated contractions, resembling fibrillation (Supplemental Fig. 1). These results are consistent with previous reports of an increase in the probability of arrhythmogenesis at high concentrations of either ketanserin or fluoxetine (Saman et al., 1985; Rajamani et al., 2006). The electrophysiologic effects of serotonergic compounds such as fluoxetine are known to include alterations in ion channel function, L-type calcium currents, and transient outward potassium currents (Park et al., 1999; Sala et al., 2006). It is possible that such changes could represent the underlying cause for the fluoxetine-induced bradycardia observed in this study. The presence of arrhythmogenic effects of serotonergic compounds in the zebrafish heart, similar to those observed in mammalian hearts, thus further supports the potential utility of this model for studying the effects of 5HT in pathological states.
Disclosure In relation to the research contained within this manuscript the authors declare no conflicts of interest, financial or otherwise. Author contributions M.R.S.: conception and design of experiments, collection, analysis and interpretation of data, drafting of manuscript. M.G.J.: conception and design of experiments; collection and interpretation of data; drafting of manuscript. R.P.C., F.M.S.: design of experiments; interpretation of data, drafting of manuscript. Funding The Natural Sciences and Engineering Research Council of Canada (NSERC) supported this work through a Discovery Grant to R.P.C. (Grant 327140) and M.G.J. (Grant 342303). M.R.S. was a funded by a NSERC Post-Graduate Research Scholarship. This work was also supported in part by the Dalhousie University Faculty of Medicine Research Fund through a grant to F.M.S. (Grant DAL48596). Acknowledgements None. References Airhart, M.J., Lee, D.H., Wilson, T.D., Miller, B.E., Miller, M.N., Skalko, R.G., 2007. Movement disorders and neurochemical changes in zebrafish larvae after bath exposure to fluoxetine (PROZAC). Neurotoxicol. Teratol. 29 (6), 652–664. Beauvallet, M., Godefroy, F., Weil-Fugazza, J., 1968. Modification of the heart 5-hydroxytryptamine level during a diet high in sodium chloride. C. R. Seances Soc. Biol. Fil. 162 (12), 2085–2088. Berkowitz, B.A., Lee, C.H., Spector, S., 1974. Disposition of serotonin in the rat blood vessels and heart. Clin. Exp. Pharmacol. Physiol. 1 (5), 397–400. Bianchi, P., Pimentel, D.R., Murphy, M.P., Colucci, W.S., Parini, A., 2005. A new hypertrophic mechanism of serotonin in cardiac myocytes: receptor-independent ROS generation. FASEB J. 19 (6), 641–643. Briggs, J.P., 2002. The zebrafish: a new model organism for integrative physiology. Am. J. Phys. Regul. Integr. Comp. Phys. 282 (1), R3–R9. Chi, N.C., Shaw, R.M., Jungblut, B., Huisken, J., Ferrer, T., Arnaout, R., Scott, I., Beis, D., Xiao, T., Baier, H., Jan, L.Y., 2008. Genetic and physiologic dissection of the vertebrate cardiac conduction system. PLoS Biol. 6 (5), e109. Chuang, J.I., Chen, S.S., Lin, M.T., 1993. Melatonin decreases brain serotonin release, arterial pressure and heart rate in rats. Pharmacology 47 (2), 91–97. Dawood, T., Lambert, E.A., Barton, D.A., Laude, D., Elghozi, J., Esler, M.D., Haikerwal, D., Kaye, D.M., Hotchkin, E.J., Lambert, G.W., 2007. Specific serotonin reuptake inhibition in major depressive disorder adversely affects novel markers of cardiac risk. Hypertens. Res. 30 (4), 285. Dvornikov, A.V., Dewan, S., Alekhina, O.V., Pickett, F.B., Tombe, P.P., 2014. Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte. J. Physiol. 592 (9), 1949–1956. Fishman, A.P., 1999. Aminorex to fen/phen an epidemic foretold. Circulation 99 (1), 156–161.
4.3. Clinical perspectives 5-HT-related mechanisms have been implicated in normal cardiac physiology (Xu et al., 2002; Sari and Zhou, 2003; Yavarone et al., 1993; Yabanoglu et al., 2009), as well as in the initiation and progression of pathological states including cardiac hypertrophy (Bianchi et al., 2005; Jaffré et al., 2009), heart failure (Sole et al., 1979), coronary and pulmonary hypertension (Vikenes et al., 1999; Fishman, 1999), and cardiac arrhythmias (Saman et al., 1985; Fishman, 1999; Nebigil and Maroteaux, 2003). As a result 5-HT-related pathways, and possible side effects, have been widely explored for development of pharmaceuticals for the treatment of pathologies ranging from antidepressants to appetite suppressants (Levy, 2006). Perhaps the best studied of these are the interactions of 5-HT-based antidepressants with the cardiovascular system (see Levy, 2006; Gorman and Sloan, 2000; Dawood et al., 2007). The most commonly prescribed medications for depression are 5-HT selective reuptake inhibitors (SSRIs) (Prieto et al., 2012). While SSRIs 7
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
Qin, Z., Lewis, J.E., Perry, S.F., 2010. Zebrafish (Danio rerio) gill neuroepithelial cells are sensitive chemoreceptors for environmental CO2. J. Physiol. 588 (5), 861–872. Rajamani, S., Eckhardt, L.L., Valdivia, C.R., Klemens, C.A., Gillman, B.M., Anderson, C.L., Holzem, K.M., Delisle, B.P., Anson, B.D., Makielski, J.C., January, C.T., 2006. Druginduced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine. Br. J. Pharmacol. 149 (5), 481–489. Rider, S.A., Tucker, C.S., del- Pozo, J., Rose, K.N., CA, MacRae, Bailey, M.A., Mullins, J.J., 2012. Techniques for the in vivo assessment of cardio-renal function in zebrafish (Danio rerio) larvae. J. Physiol. 590 (8), 1803–1809. Roose, S.P., Miyazaki, M., 2005. Pharmacologic treatment of depression in patients with heart disease. Psychosom. Med. 67, S54–S57. Sala, M., Coppa, F., Cappucciati, C., Brambilla, P., d'Allio, G., Caverzasi, E., Barale, F., De Ferrari, G.M., 2006. Antidepressants: their effects on cardiac channels, QT prolongation and Torsade de Pointes. Curr. Opin. Investig. Drugs 7 (3), 256–263. Saman, S., Thandroyen, F., Opie, L.H., 1985. Serotonin and the heart: effects of ketanserin on myocardial function, heart rate, and arrhythmias. J. Cardiovasc. Pharmacol. 7, 70–75. Sari, Y., Zhou, F.C., 2003. Serotonin and its transporter on proliferation of fetal heart cells. Int. J. Dev. Neurosci. 21 (8), 417–424. Schneider, J.A., Yonkman, F.F., 1953. Action of serotonin (5-hydroxytryptamine) on vagal afferent impulses in the cat. Am. J. Physiol.—Legacy Content 174 (1), 127–134. Shakarchi, K., Zachar, P.C., Jonz, M.G., 2013. Serotonergic and cholinergic elements of the hypoxic ventilatory response in developing zebrafish. J. Exp. Biol. 216 (5), 869–880. Singh, S., Johnson, P.I., Javed, A., Gray, T.S., Lonchyna, V.A., Wurster, R.D., 1999. Monoamine-and histamine-synthesizing enzymes and neurotransmitters within neurons of adult human cardiac ganglia. Circulation 99 (3), 411–419. Sole, M.J., Shum, A., Van Loon, G.R., 1979. Serotonin metabolism in the normal and failing hamster heart. Circ. Res. 45 (5), 629–634. Steele, S.L., Ekker, M., Perry, S.F., 2011. Interactive effects of development and hypoxia on catecholamine synthesis and cardiac function in zebrafish (Danio rerio). J. Comp. Physiol. B. 181 (4), 527–538. Stewart, A.M., Cachat, J., Gaikwad, S., Robinson, K.S., Gebhardt, M., Kalueff, A.V., 2013. Perspectives on experimental models of serotonin syndrome in zebrafish. Neurochem. Int. 62 (6), 893–902. Stoyek, M.R., Croll, R.P., Smith, F.M., 2015. Intrinsic and extrinsic innervation of the heart in zebrafish (Danio rerio). J. Comp. Neurol. 523 (11), 1683–1700. Stoyek, M.R., Croll, R.P., Smith, F.M., 2016. Zebrafish heart as a model to study the integrative autonomic control of pacemaker function. Am. J. Physiol. Heart Circ. Physiol. 311, H676–H688. Susaki, E.A., Tainaka, K., Perrin, D., Kishino, F., Tawara, T., Watanabe, T.M., Yokoyama, C., Onoe, H., Eguchi, M., Yamaguchi, S., Abe, T., 2014. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157 (3), 726–739. Tessadori, F., van Weerd, J.H., Burkhard, S.B., Verkerk, A.O., de Pater, E., Boukens, B.J., Vink, A., Christoffels, V.M., Bakkers, J., 2012. Identification and functional characterization of cardiac pacemaker cells in zebrafish. PLoS One 7 (10), e47644. Uyttebroek, L., Shepherd, I.T., Harrisson, F., Hubens, G., Blust, R., Timmermans, J.P., Van Nassauw, L., 2010. Neurochemical coding of enteric neurons in adult and embryonic zebrafish (Danio rerio). J. Comp. Neurol. 518 (21), 4419–4438. Vikenes, K., Farstad, M., Nordrehaug, J.E., 1999. Serotonin is associated with coronary artery disease and cardiac events. Circulation 100 (5), 483–489. Votavova, M., Boullin, D.J., Costa, E., 1971. Specificity of action of 6-hydroxydopamine in peripheral cat tissues: depletion of noradrenaline without depletion of 5-hydroxytryptamine. Life Sci. 10 (2), 87–91. Xu, J., Jian, B., Chu, R., Lu, Z., Li, Q., Dunlop, J., Rosenzweig-Lipson, S., McGonigle, P., Levy, R.J., Liang, B., 2002. Serotonin mechanisms in heart valve disease II: the 5-HT2 receptor and its signaling pathway in aortic valve interstitial cells. Am. J. Pathol. 161 (6), 2209–2218. Yabanoglu, S., Akkiki, M., Seguelas, M.H., Mialet-Perez, J., Parini, A., Pizzinat, N., 2009. Platelet derived serotonin drives the activation of rat cardiac fibroblasts by 5-HT2A receptors. J. Mol. Cell. Cardiol. 46 (4), 518–525. Yamano, M., Kamato, T., Nishida, A., Ito, H., Yuki, H., Tsutsumi, R., Honda, K., Miyata, K., 1994. Serotonin (5-HT) 3-receptor antagonism of 4,5,6,7-tetrahydrobenzimidazole derivatives against 5-HT-induced bradycardia in anesthetized rats. Jpn. J. Pharmacol. 65 (3), 241–248. Yavarone, M.S., Shuey, D.L., Tamir, H., Sadler, T.W., Lauder, J.M., 1993. Serotonin and cardiac morphogenesis in the mouse embryo. Teratology 47 (6), 573–584.
Frishman, W.H., Grewall, P., 2000. Serotonin and the heart. Ann. Med. 32 (3), 195–209. Gorman, J.M., Sloan, R.P., 2000. Heart rate variability in depressive and anxiety disorders. Am. Heart J. 140 (4), S77–S83. Henn, F.A., Hamberger, A., 1971. Glial cell function: uptake of transmitter substances. Proc. Natl. Acad. Sci. 68 (11), 2686–2690. Irisawa, H., 1978. Comparative physiology of the cardiac pacemaker mechanism. Physiol. Rev. 58 (2), 461–498. Jackson, R., Braubach, O.R., Bilkey, J., Zhang, J., Akimenko, M.A., Fine, A., Croll, R.P., Jonz, M.G., 2013. Expression of sall4 in taste buds of zebrafish. Dev. Neurobiol. 73 (7), 543–558. Jacobs, L., Comroe, J.H., 1971. Reflex apnea, bradycardia, and hypotension produced by serotonin and phenyldiguanidecting on the nodose ganglia of the cat. Circ. Res. 29 (2), 145–155. Jaffré, F., Bonnin, P., Callebert, J., Debbabi, H., Setola, V., Doly, S., Monassier, L., Mettauer, B., Blaxall, B.C., Launay, J.M., Maroteaux, L., 2009. Serotonin and angiotensin receptors in cardiac fibroblasts coregulate adrenergic-dependent cardiac hypertrophy. Circ. Res. 104 (1), 113–123. Jonz, M.G., Fearon, I.M., Nurse, C.A., 2004. Neuroepithelial oxygen chemoreceptors of the zebrafish gill. J. Physiol. 560 (3), 737–752. Levy, R.J., 2006. Serotonin transporter mechanisms and cardiac disease. Circulation 113 (1), 2–4. Li, N., Csepe, T.A., Hansen, B.J., Dobrzynski, H., Higgins, R.S., Kilic, A., Mohler, P.J., Janssen, P.M., Rosen, M.R., Biesiadecki, B.J., Fedorov, V.V., 2015. Molecular mapping of sinoatrial node HCN channel expression in the human heart. Circ. Arrhythm. Electrophysiol. 8 (5), 1219–1227. MacCanon, D.M., Horvath, S.M., 1954. Some effects of serotonin in pentobarbital anesthetized dogs. Am. J. Physiol.—Legacy Content 179 (1), 131–134. Madan, B.R., Khanna, N.K., Godhwani, J.L., Pendse, V.K., 1970. Changes in the 5-hydroxytryptamine (5-HT) content of the heart during ectopic ventricular arrhythmia and consequent to its reversion by quinidine. Indian J. Med. Res. 58 (1), 130. Mangoni, M.E., Nargeot, J., 2008. Genesis and regulation of the heart automaticity. Physiol. Rev. 88 (3), 919–982. Maximino, C., Puty, B., Benzecry, R., Araújo, J., Lima, M.G., Batista, E.D.J.O., de Matos Oliveira, K.R., Crespo-Lopez, M.E., Herculano, A.M., 2013. Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models. Neuropharmacology 71, 83–97. Nebigil, C.G., Maroteaux, L., 2001. A novel role for serotonin in heart. Trends Cardiovasc. Med. 11 (8), 329–335. Nebigil, C.G., Maroteaux, L., 2003. Functional consequence of serotonin/5-HT2B receptor signaling in heart role of mitochondria in transition between hypertrophy and heart failure? Circulation 108 (7), 902–908. Nemtsas, P., Wettwer, E., Christ, T., Weidinger, G., Ravens, U., 2010. Adult zebrafish heart as a model for human heart? An electrophysiological study. J. Mol. Cell. Cardiol. 48 (1), 161–171. Newton, C.M., Stoyek, M.R., Croll, R.P., Smith, F.M., 2014. Regional innervation of the heart in the goldfish, Carassius auratus: a confocal microscopy study. J. Comp. Neurol. 522 (2), 456–478. Olsson, C., 2009. Autonomic innervation of the fish gut. Acta Histochem. 111 (3), 185–195. Olsson, C., Holmberg, A., Holmgren, S., 2008. Development of enteric and vagal innervation of the zebrafish (Danio rerio) gut. J. Comp. Neurol. 508 (5), 756–770. Pacher, P., Ungvari, Z., Nanasi, P.P., Furst, S., Kecskemeti, V., 1999. Speculations on difference between tricyclic and selective serotonin reuptake inhibitor antidepressants on their cardiac effects. Is there any? Curr. Med. Chem. 6 (6), 469–480. Park, K.S., Kong, I.D., Park, K.C., Lee, J.W., 1999. Fluoxetine inhibits L-type Ca2 + and transient outward K+ currents in rat ventricular myocytes. Yonsei Med. J. 40 (2), 144–151. Pauza, D.H., Saburkina, I., Rysevaite, K., Inokaitis, H., Jokubauskas, M., Jalife, J., Pauziene, N., 2013. Neuroanatomy of the murine cardiac conduction system: a combined stereomicroscopic and fluorescence immunohistochemical study. Auton. Neurosci. 176 (1), 32–47. Pauza, D.H., Rysevaite, K., Inokaitis, H., Jokubauskas, M., Pauza, A.G., Brack, K.E., Pauziene, N., 2014. Innervation of sinoatrial nodal cardiomyocytes in mouse. A combined approach using immunofluorescent and electron microscopy. J. Mol. Cell. Cardiol. 75, 188–197. Prieto, M.J., Gutierrez, H.C., Arévalo, R.A., Chiaramoni, N.S., del Valle, Alonso S., 2012. Effect of risperidone and fluoxetine on the movement and neurochemical changes of zebrafish. Open J. Med. Chem. 2 (4), 129–138.
8
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Distribution and chronotropic effects of serotonin in the zebrafish heart Matthew R. Stoyeka,b, Michael G. Jonzc, Frank M. Smitha,1, Roger P. Crollb,⁎,1 a b c
Department of Medical Neuroscience, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Department of Physiology & Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Department of Biology, University of Ottawa, Ottawa, Ontario, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords: Serotonin 5-Hydroxytryptamine Zebrafish Heart Intracardiac nervous system Chronotropy
Several lines of evidence suggest that serotonin (5-HT) has a regulatory role in cardiovascular function from embryogenesis through adulthood. However, the reported actions of 5-HT are often contradictory and include bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction. Clarifying such cardiac effects requires further research and may benefit from utilizing a model simpler than the mammalian hearts traditionally used in these studies. In the present study, we describe the cardiac distribution and chronotropic responses of 5-HT in the zebrafish heart. A combined anatomical, electrophysiological, and pharmacological approach was used to investigate the involvement of 5-HT pathways, and to compare neural and direct myocardial pathways of biological action. Immunohistochemical methods revealed 5-HT in endocardial cells, glial-like cells, and intracardiac neurons in the atrium. Electrocardiogram (ECG) recordings combined with the administration of pharmacological agents demonstrated that 5-HT acted predominantly through direct myocardial pathways resulting in a reduction of heart rate. Overall, the results of this study contribute significant advances in the establishment of the zebrafish as a new model for studies of the role of 5HT in autonomic cardiac control.
1. Introduction Serotonin (5-hydroxytryptamine; 5-HT) was first isolated from blood and defined as a vasoconstrictor (Nebigil and Maroteaux, 2001), but has also been shown more recently to have diverse cardiovascular effects (Frishman and Grewall, 2000). Described effects include bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction, and these effects vary greatly depending on route of administration and experimental parameters (see review by Nebigil and Maroteaux, 2001). Clarification of these complex and seemingly conflicting effects requires further research and may benefit from the employment of simpler models than the mammalian hearts traditionally used in these studies. The zebrafish heart has been proposed as a powerful tool for studying cardiac electrophysiology, with the potential to provide broad insights into cardiovascular function (Briggs, 2002; Chi et al., 2008; Dvornikov et al., 2014; Nemtsas et al., 2010; Rider et al., 2012; Tessadori et al., 2012). Stoyek et al. (2015) previously described the basic structure of the intracardiac nervous system in zebrafish, which is consistent with that of humans and other mammalian models (Irisawa, 1978; Mangoni and Nargeot, 2008; Pauza et al., 2013; Pauza et al.,
⁎
1
2014; Li et al., 2015). In addition, the zebrafish appears to have genes encoding for autonomic receptors, including 5-HT receptors, (5-HT: Airhart et al., 2007; Prieto et al., 2012; Maximino et al., 2013; Stewart et al., 2013), that are homologous to those that affect heart rate (HR) in mammals (β-adrenergic/catecholaminergic: Steele et al., 2011; nicotinic, muscarinic and β-adrenergic; Stoyek et al., 2016). The study of serotonergic processes in zebrafish is well established. Previous reports have described behavioral responses (predominantly a decrease in spontaneous locomotor activity) to 5-HT exposure that are similar to those observed in mammalian studies, indicating the potential validity of the zebrafish model in such research (Airhart et al., 2007; Prieto et al., 2012; Maximino et al., 2013; Stewart et al., 2013). To date investigations of the organ-specific physiological effects of 5-HT in zebrafish have been limited, focusing primarily on the role of pathways involved in release of 5-HT from chromaffin cells in the head kidney (Steele et al., 2011) or chemosensory neuroepithelial cells in the gills (Jonz et al., 2004). While 5-HT is known to be present in the circulatory system of the zebrafish (Steele et al., 2011), information on the cardiac effects of 5-HT is lacking, with only a single report alluding to chronotropic effects of high doses of fluoxetine, a commonly used 5-HT selective reuptake inhibitor (SSRI; Airhart et al., 2007).
Corresponding author at: Department of Physiology & Biophysics, Faculty of Medicine, Dalhousie University, 5850 College Street, PO Box 15000, Halifax, NS B3H 4R2, Canada. E-mail address: [email protected] (R.P. Croll). Both authors contributed equally.
http://dx.doi.org/10.1016/j.autneu.2017.07.004 Received 1 November 2016; Received in revised form 3 May 2017; Accepted 17 July 2017 1566-0702/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Stoyek, M.R., Autonomic Neuroscience: Basic and Clinical (2017), http://dx.doi.org/10.1016/j.autneu.2017.07.004
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
PBS-T). After incubation in primary antibodies for 3–5 d with agitation at 4 °C, the tissues were rinsed in PBS-T, and then transferred to a solution of PBS-T containing the appropriate secondary antibodies, raised in mouse or rabbit, and conjugated to AlexaFluor 488, 555, or 647 fluorophores (Life Technologies, Burlington, ON, Canada). Incubation time with secondary antibodies was also 3–5 d with agitation at 4 °C. Final rinsing was done in PBS, and then specimens were placed in Scale CUBIC-1 clearing solution (Susaki et al., 2014) overnight at room temperature with gentle agitation. Tissues were mounted on glass slides in CUBIC-1 for confocal microscopy.
The aim of the present study was to investigate the distribution of 5HT and determine the chronotropic responses to 5-HT application in isolated zebrafish hearts. A combined anatomical, electrophysiological, and pharmacological approach was used to investigate the role of 5-HT, and to define the neural- and tissue-level actions of this agent. We used immunohistochemical methods to characterize the distribution of 5-HT and tryptophan hydroxylase (TPH, the rate-limiting enzyme in the production of 5-HT; Levy, 2006) within intracardiac neurons (ICNs) and myocardial cells. Electrocardiogram (ECG) recordings combined with bath-applied pharmacological agents were used to demonstrate in the isolated zebrafish heart that 5-HT acted predominantly through direct myocardial pathways to modulate heart rate (HR). The results of this study establish this preparation as a tractable model to investigate serotonergic effects on the heart.
2.5. Antibodies Serotonergic elements were detected with an anti-5-HT antibody (dilution 1:100; 20080, Immunostar). This antibody has been used in previous studies in zebrafish (Uyttebroek et al., 2010; Jackson et al., 2013). Antibodies against tryptophan hydroxylase (TPH; dilution 1:100; P21961, Life Technologies), the rate-limiting enzyme in the synthesis of 5-HT, and tyrosine hydroxylase, the rate limiting enzyme in the synthesis of norepeinephrine (TH; dilution 1:100; 22941; Immunostar) were used to investigate potential synthetic pathways for monoamines. As the TPH antibody was raised in the same host as the 5HT antibody, it was not possible to co-label for 5-HT in the same heart. To compare 5-HT elements to the general innervation of the heart, antibodies against acetylated tubulin (AcT, axons; dilution 1:250; T6793, Sigma Aldrich) and human neuronal protein C/D (Hu, neuronal somata; dilution 1:250; A21271, Life Technologies) were combined as previously described (Stoyek et al., 2015). In a separate trial (n = 8) hearts were processed as outlined above, except that either the primary or secondary antibody was omitted, both of which eliminated detection of histofluorescence in all specimens.
2. Methods 2.1. Animals A total of 35 AB strain adult zebrafish (12–18 month post fertilization; 33 ± 5 mm standard body length) was used in this study. Procedures for animal care and use followed the “Guidelines on the Care and Use of Fish in Research, Teaching and Testing” document issued by the Canadian Council of Animal Care. Institutional approval (Protocol #15-006) for animal use in this study was obtained from the Dalhousie University Committee on Laboratory Animals. 2.2. Animal husbandry Animals were acquired from breeding stocks in the Faculty of Medicine Zebrafish Facility at Dalhousie University. Fish were maintained in standard 3–10 L tanks (Aquatic Habitats, Apopka, FL, USA) at 28.5 °C, supplied continuously with conditioned water from a recirculating water system, and subjected to a 14 hour light:10 hour dark illumination cycle. Zebrafish were fed commercial, dry fish food (Golden Pearl pellets, Brine Shrimp Direct, Ogden, UT, USA) and live Artemia (raised in-house) twice a day.
2.6. Myocardial labelling In order to determine how immunohistochemically labelled neuronal elements were related to the regional structure of the myocardium, some specimens were double-labelled either with AcT-Hu or 5-HT antibodies in combination with the F-actin marker phalloidin (77418, Sigma Aldrich; Stoyek et al., 2015), conjugated with tetramethyl rhodamine isothiocyanate to show cardiac myocytes.
2.3. Heart isolation Zebrafish hearts were isolated following procedures described in Stoyek et al. (2016). Briefly, fish were anaesthetised in a buffered solution (pH 7.2) of tricaine (MS-222; 1.5 mM; Sigma–Aldrich, Oakville, ON, Canada) in tank water (28.5 °C) until opercular movements ceased and the animals lacked response to a fin pinch with forceps. A ventral midline incision was made through the body wall to expose the heart, and a block of tissue encompassing the ventral aorta, ventricle, atrium, sinus venosus and ducts of Cuvier was then removed for whole-mount immunohistochemistry or in vitro recordings.
2.7. Imaging Tissues were viewed using a Zeiss LSM510 confocal microscope operating with Zen2009 software (Zeiss Canada, Mississauga, ON). Preparations were epi-illuminated with a 488 nm argon laser, and 543 nm and 605 nm helium‑neon lasers, reflected by a 488/543/ 633 nm dichroic mirror (HFT 488/543/633; Carl Zeiss AG). Emitted fluorescence was collected from the specimens, using 480–520 nm and 500–615 nm band-pass filters (BP565-615; Carl Zeiss AG) and a 565–615 nm long pass filter (LP565-615; Carl Zeiss AG) through a 10×, 0.45 NA objective (Plan-Apochromat SF25; Carl Zeiss AG), a 25×, 0.80 NA objective (LCI Plan-Neofluar; Carl Zeiss AG), or a 40 ×, 0.95 NA objective (Plan-Apochromat M27; Carl Zeiss AG). Z-stacks were taken from regions of interest surrounding immunoreactive tissues, and ranged from 20 to 100 μm in depth. Z-stack scans also encompassed 5–25 μm above and below the regions of interest to ensure that all structures were captured, while limiting issues of light scattering associated with deeper tissue scans. Image stacks were processed with Zeiss Zen2009 software. Figure plates were constructed from images processed with Photoshop CS6 (Adobe Systems Inc., San Jose, CA, USA). Brightness and contrast of some images were adjusted to ensure panel-to-panel consistency in each figure.
2.4. Tissue preparation for immunohistochemistry Tissues were fixed overnight in 4% paraformaldehyde (PFA; RT15710, Electron Microscopy Sciences, Hatfield, PA, USA) in phosphatebuffered saline (PBS, composition in mM: 50 Na2HPO4, 140 NaCl, pH 7.2). To visualize the overall spatial relationship of serotonergic elements within the heart of the zebrafish, the whole-mount procedures used for immunohistochemistry in this study were similar to those described in previous publications (Newton et al., 2014; Stoyek et al., 2015; Stoyek et al., 2016). Briefly, fixed tissues were rinsed in PBS and then transferred to a PBS solution containing 2% Triton X-100 (X100, Sigma Aldrich), 1% bovine serum albumin (BSA; A9576, Sigma Aldrich) and 1% normal goat serum (NGS; G9023, Sigma Aldrich) for 48 h at 4 °C with gentle agitation. Tissues were then incubated with primary antibodies (see Section 2.5), which were diluted in a solution containing 0.25% Triton X-100, 1% BSA and 1% NGS in PBS (designated 2
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
Fig. 1. 5-HT-immunoreactivity in the atrial wall. A: Schematic showing the cardiac regions: atrium, a; bulbus arteriosus, ba; sinoatrial region, SAR; sinus venosus, sv; ventricle, v. Box indicates the approximate location of image in panel B. B: Acetylated tubulin and human neuronal protein C/D (AcT-Hu; white) and 5-HT (green) immunoreactivity in the atrial myocardium. Musculature is labelled with phalloidin (Phal; magenta). 5-HT-like immunoreactive (-LIR) cells were localized to the luminal edges of atrial trabeculae. Dashed box indicates the approximate region of panels C–E. C–D: Single confocal images enlarged to show details of the variety of 5-HT-LIR cell morphologies from panel A. 5-HT-LIR cells within the atrial wall were restricted to the endocardial region. Most 5-HT-LIR cells were round to ovoid in shape (C, arrows) with some elongated spindle-shaped cells also observed (D, arrows). In all preparations AcT-Hu-IR somata and axons were observed in close proximity to 5-HT-LIR cells but axonal contacts with these cells were not observed. E: In all preparations tyrosine hydroxylase (TH)-LIR axons were observed in close proximity to 5-HT-LIR cells but neither axonal contacts with these cells, nor coexpression of TH with 5-HT, was observed. Scale bars: 150 μm in B; 35 μm in C–E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
from light, and delivered to the bath at the chamber inlet in 200 μL boluses through a micropipette via a calibrated syringe attached to a screw-drive microinjector (IM-4B; Narishige, East Meadow, NY, USA). The peak concentration within the constant flow bath was calculated to be 20 μM. Based on visual dye experiments a homogeneous distribution within the bath was reached in approximately 0.5–1 min, with much of the washout occurring between 1.5 and 2 min. As a control, 200 μL boluses of saline were delivered to the tissue in the same manner and did not elicit chronotropic responses. Ketanserin (5-HT2-receptor blocker; 10 μM; S006, Sigma Aldrich), spiperone (5-HT1-receptor blocker; 10 μM; S7395, Sigma Aldrich), and fluoxetine (selective 5-HT reuptake inhibitor at presynaptic receptors; 5 μM; F132, Sigma Aldrich) were dissolved in saline and the tissue was superfused continuously with these agents for 15 min prior to recording. Concentrations of serotonergic agents were chosen based upon previous studies reporting effective blockade with minimal direct HR effects (see Section 4.2). Autonomic antagonists atropine (post-junctional muscarinic receptor blocker; 10 μM; A0132, Sigma Aldrich) and timolol (post-junctional βadrenergic receptor blocker; 100 μM; T6394, Sigma Aldrich) were dissolved in saline and superfused continuously over the tissue for 15 min prior to recording. Responses to 5-HT were then recorded in the presence of either serotonergic (ketanserin, spiperone, or fluoxetine) or
2.8. Measurement of heart rate Isolated, beating hearts were pinned through the ventral aorta and walls of the ducts of Cuvier to the Sylgard rubber (Dow Corning, Midland, MI, USA) bottom of a 5 mL chamber which was superperfused with zebrafish saline (composition in mM: 124.1 NaCl, 5.1 KCl, 2.9 Na2HPO4, 1.9 MgSO4-7H2O, 1.4 CaCl2-2H2O, 11.9 NaHCO3; aerated with room air; pH 7.2; 25 °C) at a constant rate of 10 mL·min− 1 (Stoyek et al., 2016). Hearts were allowed to equilibrate in the bath for 30 min prior to testing. Electrocardiogram (ECG) signals were recorded from the surface of the atrium with a bipolar suction electrode, differentially amplified (sample rate 2 kHz; total gain ×1000–10,000), and stored on a personal computer after analog-digital conversion (Digidata 1322A, Axon Instruments, Foster City, CA, USA). To quantify HR responses, the time between adjacent R-waves of the ECG (R-R interval) was measured using Axoscope software (Axon Instruments). 2.9. Pharmacological agents Basal R-R intervals (“control”) were first recorded prior to the delivery of any pharmacological agents. 5-HT (1 mM; H7752, Sigma Aldrich) was dissolved in saline on the day of the experiment, protected 3
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
of any heart. In hearts labelled for TPH (n = 12), a population of ICNs with similar spatial distribution to 5-HT-LIR ICNs (Fig. 2E) was observed in the dorsal SAR (see Table 1). TPH-LIR ICNs showed co-labelling with AcT-Hu (n = 6) in all hearts examined, while in preparations co-labelled for TH (n = 6) no co-labelling was observed. As colabelling of 5-HT and TPH could not be performed, however, it was assumed that 5-HT-LIR ICNs were also TPH-LIR, based upon similar numbers of immunoreactive ICNs, their morphology and location within the SAR.
autonomic blockade (atropine/timolol) to investigate the pathways of 5-HT action. After recording responses to 5-HT in the presence of serotonergic agents or autonomic antagonists, the bath supply was switched to fresh saline and these chemicals were washed (“washout”) from the chamber and responses to 5-HT alone were again recorded (“recovery”) to control for toxicity or long-lasting effects of the pharmacological agents. 2.10. Data analyses
3.2. Effects of 5-HT on R-R interval
Numerical values are expressed as mean ± 1 standard error (SE). Statistical analyses (two-way ANOVA with Tukey's post-hoc, Student's ttest and regression) were performed using SPSS (IBM Canada, Markham, ON) to detect significant differences among means with significance set at P ≤ 0.05.
In the current study the initial HR of the isolated hearts in the bath was 112 ± 5 beats·min− 1 (n = 40; range 88–144 beats per minute). Chronotropic changes during trials are shown as proportional changes in R-R interval relative to values obtained prior to each experimental trial (“controls”). The effects of 5-HT and serotonergic agents are summarized in Fig. 3. Application of 5-HT alone resulted in a significant increase in RR interval (Fig. 3A, B). Exposure to ketanserin (KET; a 5-HT2 receptor family antagonist, Fig. 3A, C) and spiperone (SPIP; a general 5-HT and dopamine D2 receptor antagonist, Fig. 3A) prevented the elongation of the R-R interval that occurred during initial exposure to 5-HT, and R-R interval was found to be not significantly different from control. In hearts exposed to fluoxetine (FLX; 5-HT reuptake inhibitor at presynaptic membrane, Fig. 3A, D) there was a significant increase in 5HT-induced R-R elongation compared with control hearts and those hearts receiving 5-HT alone. In hearts exposed to 5-HT during autonomic blockade (AB, Fig. 3A) a significant elongation of the R-R interval was maintained. In order to control for possible toxicity or other long lasting effects of serotonergic agents or of AB, each heart was exposed again to 5-HT after the agents were washed from the chamber. In all cases, the post-washout responses to 5-HT were found to not be significantly different than those observed in control preparations exposed to 5-HT alone. It should also be noted that the total duration of each experiment (< 1 h) was less than the period in which we have previously described the zebrafish heart as having a stable R-R interval (6 h; Stoyek et al., 2016).
3. Results 3.1. Cardiac distribution of 5-HT In this study 5-HT-like-immunoreactive (-LIR) cells were observed solely within the atrium. Within the ventricle, only AcT-Hu and TH immunoreactivities were observed as we have previously described (Stoyek et al., 2015), and therefore will not be dealt with in this study. Three distinct types of cells containing 5-HT were observed within the atrium, which for the purposes of this study have been classified based upon their morphology, location, and immunoreactivity. The most numerous of the 5-HT-LIR cell types within the atrium was associated with the lumenal surfaces of the atrium along the trabeculae. These cells did not co-label with phalloidin (Fig. 1B), thus indicating that they were not myocytes. These cells appeared to be part of the endocardial cell layer, thus we have termed them “endocardial” in this study (size range 10-30 μm). These cells were also neither TPH-LIR, nor TH-LIR (Fig. 1E), thus suggesting that they were likely sequestering 5HT but not synthesizing it or catecholamines. The endocardial 5-HT-LIR cells were typically within 5–15 μm of axons labelled with AcT-Hu, but direct axonal terminations on these cells were not observed. To control for the possibility that some of the observed 5-HT immunoreactivity in the heart was due to 5-HT contained in platelets or erythrocytes that adhered to the trabeculae, blood samples were collected from the cardiac vessels and then fixed and incubated with antibodies against 5-HT (n = 3) and TPH (n = 3). No immunoreactive erythrocytes were found in any samples, but 5-HT-LIR platelets were observed in all samples. The size of the platelets (< 3 μm) was inconsistent with any of the 5HT-LIR cells observed in the atrial wall, and it is therefore unlikely that they contributed to our descriptions of cells reported here. A second population of 5-HT-LIR cells was detected within the sinoatrial valve region (SAR) at the junction of the sinus venosus and atrial myocardium (n = 12; Fig. 2B–C). These cells often had numerous small projections from the somata and given this morphology these cells are here referred to as “multipolar” cells (size range 25-50 μm). No immunoreactivity was co-localized to AcT-Hu and 5-HT (n = 6; Fig. 2B) or TH and 5-HT (n = 6; Fig. 2C) within the multipolar 5-HT-LIR cells. These cells were closely associated with varicose axons (within 5–15 μm), but did not themselves appear to receive direct innervation. In hearts labelled for TPH (n = 6), no multipolar cells within this region exhibited immunoreactivity, suggesting that, as with the endocardial cells, these cells were likely sequestering rather than synthesizing 5-HT. A third population of 5-HT cells in the atrium appear to be intracardiac neurons (ICNs). Within the sinoatrial plexus a number of 5HT-LIR ICNs was identified based on morphology and co-localized immunoreactivity with AcT-Hu (Fig. 2); their occurrence was variable among the hearts examined (see Table 1) and was restricted to the dorsal SAR region (see Stoyek et al., 2015). In hearts in which 5-HT-LIR ICNs were observed, all showed co-labelling with AcT-Hu (n = 6). There was, however, no co-expression of 5-HT and TH (n = 6) in ICNs
4. Discussion Several lines of evidence now suggest that 5-HT has a regulatory role in cardiovascular function throughout life (Nebigil and Maroteaux, 2001). Taken together these reports indicate a need to better understand the distribution and basic actions of 5-HT in the heart. 4.1. Distribution of 5-HT within the zebrafish heart 5-HT has previously been identified in the hearts of rats (Beauvallet et al., 1968; Berkowitz et al., 1974), cats (Votavova et al., 1971), dogs (Madan et al., 1970), and humans (Singh et al., 1999). Although previous studies have described the distribution of 5-HT in peripheral tissues of zebrafish (gills: Jonz et al., 2004; Qin et al., 2010; gut: Olsson et al., 2008), the current study represents the first description of its distribution within the zebrafish heart. Endocardial cells within the atrium represented the most plentiful source of 5-HT immunoreactivity identified in the current study. These cells were found to be associated with atrial trabeculae, and did not extend into the working myocardium. Final determination of the type of cell observed here may eventually require the use of a combination of markers for 5-HT and for endothelial markers but it should also be noted that 5-HT has previously been reported in endocardial cells in mice (Yavarone et al., 1993). The endocardial cells in zebrafish did not express TPH or TH, indicating that 5-HT content was most likely the result of uptake, and that these cells could not synthesize either 5-HT or catecholamines. In support of this, Sari and Zhou (2003) showed in 4
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
Fig. 2. Organization of 5-HT-immunoreactivity in the sinoatrial region (SAR). A: Schematic showing the cardiac regions (labels as in Fig. 1). Boxes indicate the locations of images in panels B-F. B: AcT-Hu and 5-HT immunoreactivity at the junctions of the left vagosympathetic trunk with the SAP. Dashed box indicates the region of panel C. C: Cells in this region showed 5-HT-LIR most strongly in somata with short projections. These cells did not express AcT-Hu, or TH, immunoreactivity. D: Within the SAR, some neuronal somata displayed 5-HTLIR, and co-expressed AcT-Hu (somata indicated by arrowheads). E–F: In a separate group of hearts neuronal somata with the same morphology as 5-HT-LIR somata in panel D coexpressed AcT-Hu and tryptophan hydroxylase (E; TPH, indicated by arrowheads). TH-LIR (F) was observed in axons distinct from 5-HT-LIR neuronal somata and axons. Scale bars: 75 μm in B; 40 μm in C, 40 μm in D–F.
tempting to speculate that the 5-HT-LIR multipolar cells we observed in the heart of the zebrafish represent a peripheral variant of the cells in the CNS in other species. In the zebrafish heart 5-HT-LIR multipolar cells did not express immunoreactivity for the general neuronal markers AcT-Hu. Though AcT-Hu has previously been established as a general neuronal marker in the zebrafish (Olsson, 2009; Stoyek et al., 2015), it is not currently known how efficacious these markers are for detecting all neuron types. That the multipolar cells shown in this study were Hunegative, and displayed glial- or astrocyte-like morphology, does not necessarily eliminate the possibility that such cells may represent a class of neurons, thus further investigation into their cellular identity must be performed. The third cell type appeared to be neuronal and thus represent a
cultured rat myocytes that 5-HT was detected in the absence of TPH, indicating that 5-HT was present in these cells due to uptake and not synthesis (Yavarone et al., 1993). In the tissue at the sinoatrial junction, there existed a second population of 5-HT-LIR cells, which given their morphology, we have termed multipolar cells. These cells were smaller than other 5-HT-LIR cell types in the heart, with numerous short projections from the cell somata, imparting a glial- or astrocyte-like appearance. These cells, like the endocardial cells, did not appear to show immunoreactivity for the synthesizing enzyme TPH, indicating that 5-HT within these cells is also likely the result of uptake rather than synthesis. In the CNS, glia and astrocytes can actively sequester neurotransmitter substances including NE, dopamine, and 5-HT (Henn and Hamberger, 1971). It is thus Table 1 Details of 5-HT- and TPH-LIR in ICNs. 5-HT-LIR
TPH-LIR
ICN #
n/N
AcT-Hu +
TH +
Size range
ICN #
n/N
AcT-Hu +
TH +
Size range
8 5 0
9/12 2/12 1/12
Y
N
40–75 μm
6 4 0
5/12 5/12 2/12
Y
N
40–75 μm
5
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
Fig. 3. A: Chronotropic responses to 5-HT and serotonergic agents. Exposure to 5-HT significantly (*) increased R-R interval. Effects of 5-HT were eliminated during exposure to ketanserin (KET) and spiperone (SPIP) but not during combined fluoxetine (FLX) and 5-HT exposure or during autonomic input blockade (atropine and timolol combined with 5-HT). In all cases, normal responses to 5-HT alone were observed after each agent was washed from the chamber (Post KET, Post SPIP, Post FLX, Post AB). B–D: Representative electrocardiograms from experiments in panel A showing cardiac responses. R-R interval during exogenous 5-HT application was elongated in comparison with pre-treatment (B). 5-HT initially evoked R-R interval increases that were prevented when 5-HT was re-applied in the presence of ketanserin (KET; C). 5-HT evoked R-R interval increases were enhanced when 5-HT was re-applied in the presence of fluoxetine (FLX; D). *P < 0.05 vs. control by pair-wise t-test; n = 8 for each group.
current study, however, no evidence for co-localization of either 5-HT or TPH with TH in ICNs was not observed. It has also been suggested that inconsistencies in the literature regarding 5-HT-immunoreactivity could be due to the sensitivity of the technique used to detect 5-HT, the state and source of the tissues examined, or the species examined (Singh et al., 1999). This is supported by reports that pre-treatment with serotonergic agents greatly altered the pattern and density of 5-HT detected by immunohistochemical methods (see Yavarone et al., 1993). Therefore, it is possible that, in the zebrafish heart, pretreatment with serotonergic compounds could potentially enhance the content of 5-HT contained within ICNs, which may reveal such colocalization of these markers.
subset of the diverse, intracardiac neurons (ICNs) previously described in the zebrafish using antibodies to AcT-Hu (Stoyek et al., 2015). Cardiac ganglia have traditionally been thought to contain only cholinergic post-ganglionic neurons, though there is accumulating evidence that cardiac ganglia contain a heterogeneous population of neurons that synthesize and respond to numerous neurotransmitters and neuromodulators (Singh et al., 1999; Pauza et al., 2014; Stoyek et al., 2015). Singh et al. (1999) previously reported that a subset of ICNs in the human heart were the only cells that contained TPH, and that they were capable of synthesizing 5-HT. In the zebrafish heart, a population of TPH-LIR ICNs was observed with similar morphology and distribution to 5-HT-LIR ICNs, thus indicating that in zebrafish these neurons have the potential to synthesize 5-HT, and this may be a feature of serotonergic ICNs that is conserved from zebrafish to mammals. Final confirmation, however, will require direct double labelling experiments using different 5–HT and TPH antibodies. 5-HT is known to be taken up by sympathetic neurons for later corelease with norepinephrine and other aminergic transmitters and peptides (Nebigil and Maroteaux, 2001; Jaffré et al., 2009). In the
4.2. 5-HT effects on heart rate Application of 5-HT to the isolated zebrafish heart presumably mimicked the endogenous release of 5-HT from one or more of the cell types identified in this study. Although the absolute concentration of the exogenously applied 5-HT was not known at the cellular level in our 6
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
have been shown to have minimal effects on cardiac conduction, there exists a growing number of reports which implicate them in fibrillation and arrhythmic events (Pacher et al., 1999). Compounding this are reports implicating psychosocial factors, such as mood and anxiety disorders, as having a significant role in the prognosis of adverse cardiac events during SSRI usage. In other words, a population of patients already potentially susceptible to cardiac dysfunction could be taking drugs which themselves are known to potentially adversely affect cardiac function (Roose and Miyazaki, 2005; Gorman and Sloan, 2000), emphasizing the need to better understand the detrimental cardiac effects of serotonergic agents on cardiac performance. Taken together the results of this study lay the foundation for the use of zebrafish in future studies investigating the pharmacology of 5-HT in the heart. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.autneu.2017.07.004.
preparation, bolus applications of 5-HT reliably resulted in a degree of bradycardia similar to that observed during 5-HT exposure in the hearts of rats (Yamano et al., 1994; Chuang et al., 1993), cats (Schneider and Yonkman, 1953; Jacobs and Comroe, 1971), and dogs (MacCanon and Horvath, 1954). In the current study the serotonergic antagonists ketanserin and spiperone prevented 5-HT-induced bradycardia, supporting the concept that 5-HT receptor activation was likely responsible for the negative chronotropic effects of this agent. Spiperone is known to act at both 5HT1 and 5-HT2 receptor types, while ketanserin, a selective antagonist of 5-HT2 receptors, blocks the physiologic effects of 5-HT on the chemosensory afferent fibres of the gills in the heart (Shakarchi et al., 2013). That both spiperone and ketanserin blocked 5-HT-induced bradycardia in the present study suggests a role for the 5-HT2 receptor in the zebrafish heart. That the effects of 5-HT were not affected by autonomic blockade with atropine and timolol suggests that 5-HT acted by a direct myocardial mechanism rather than indirectly through modulation of classical parasympathetic and sympathetic pathways. Further studies are necessary to clarify this issue. The potential for a serotonergic tonus was taken into account during initial experiments. The concentrations of ketanserin, spiperone, and fluoxetine were chosen based upon levels which effectively blocked a response to exogenous 5-HT, but which did not alter basal heart rate. From this it was assumed that if a 5-HT tonus existed within the isolated heart, its level was minimal. It was also therefore assumed that the enhanced bradycardic effects of 5-HT observed in the current study during exposure to fluoxetine were the result of increased duration of myocardial 5-HT exposure, due to the prevention of 5-HT reuptake, and not a direct myocardial action of fluoxetine itself. In preliminary experiments, however, exposure to fluoxetine or ketanserin alone at much higher concentrations of between 20 and 100 μM, was found to induce bradycardia and arrhythmias. Furthermore, at the highest concentration of fluoxetine, there was frequent initiation of dysrhythmia within 1 min of exposure, and which frequently led to subsequent, sustained periods of highly irregular and uncoordinated contractions, resembling fibrillation (Supplemental Fig. 1). These results are consistent with previous reports of an increase in the probability of arrhythmogenesis at high concentrations of either ketanserin or fluoxetine (Saman et al., 1985; Rajamani et al., 2006). The electrophysiologic effects of serotonergic compounds such as fluoxetine are known to include alterations in ion channel function, L-type calcium currents, and transient outward potassium currents (Park et al., 1999; Sala et al., 2006). It is possible that such changes could represent the underlying cause for the fluoxetine-induced bradycardia observed in this study. The presence of arrhythmogenic effects of serotonergic compounds in the zebrafish heart, similar to those observed in mammalian hearts, thus further supports the potential utility of this model for studying the effects of 5HT in pathological states.
Disclosure In relation to the research contained within this manuscript the authors declare no conflicts of interest, financial or otherwise. Author contributions M.R.S.: conception and design of experiments, collection, analysis and interpretation of data, drafting of manuscript. M.G.J.: conception and design of experiments; collection and interpretation of data; drafting of manuscript. R.P.C., F.M.S.: design of experiments; interpretation of data, drafting of manuscript. Funding The Natural Sciences and Engineering Research Council of Canada (NSERC) supported this work through a Discovery Grant to R.P.C. (Grant 327140) and M.G.J. (Grant 342303). M.R.S. was a funded by a NSERC Post-Graduate Research Scholarship. This work was also supported in part by the Dalhousie University Faculty of Medicine Research Fund through a grant to F.M.S. (Grant DAL48596). Acknowledgements None. References Airhart, M.J., Lee, D.H., Wilson, T.D., Miller, B.E., Miller, M.N., Skalko, R.G., 2007. Movement disorders and neurochemical changes in zebrafish larvae after bath exposure to fluoxetine (PROZAC). Neurotoxicol. Teratol. 29 (6), 652–664. Beauvallet, M., Godefroy, F., Weil-Fugazza, J., 1968. Modification of the heart 5-hydroxytryptamine level during a diet high in sodium chloride. C. R. Seances Soc. Biol. Fil. 162 (12), 2085–2088. Berkowitz, B.A., Lee, C.H., Spector, S., 1974. Disposition of serotonin in the rat blood vessels and heart. Clin. Exp. Pharmacol. Physiol. 1 (5), 397–400. Bianchi, P., Pimentel, D.R., Murphy, M.P., Colucci, W.S., Parini, A., 2005. A new hypertrophic mechanism of serotonin in cardiac myocytes: receptor-independent ROS generation. FASEB J. 19 (6), 641–643. Briggs, J.P., 2002. The zebrafish: a new model organism for integrative physiology. Am. J. Phys. Regul. Integr. Comp. Phys. 282 (1), R3–R9. Chi, N.C., Shaw, R.M., Jungblut, B., Huisken, J., Ferrer, T., Arnaout, R., Scott, I., Beis, D., Xiao, T., Baier, H., Jan, L.Y., 2008. Genetic and physiologic dissection of the vertebrate cardiac conduction system. PLoS Biol. 6 (5), e109. Chuang, J.I., Chen, S.S., Lin, M.T., 1993. Melatonin decreases brain serotonin release, arterial pressure and heart rate in rats. Pharmacology 47 (2), 91–97. Dawood, T., Lambert, E.A., Barton, D.A., Laude, D., Elghozi, J., Esler, M.D., Haikerwal, D., Kaye, D.M., Hotchkin, E.J., Lambert, G.W., 2007. Specific serotonin reuptake inhibition in major depressive disorder adversely affects novel markers of cardiac risk. Hypertens. Res. 30 (4), 285. Dvornikov, A.V., Dewan, S., Alekhina, O.V., Pickett, F.B., Tombe, P.P., 2014. Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte. J. Physiol. 592 (9), 1949–1956. Fishman, A.P., 1999. Aminorex to fen/phen an epidemic foretold. Circulation 99 (1), 156–161.
4.3. Clinical perspectives 5-HT-related mechanisms have been implicated in normal cardiac physiology (Xu et al., 2002; Sari and Zhou, 2003; Yavarone et al., 1993; Yabanoglu et al., 2009), as well as in the initiation and progression of pathological states including cardiac hypertrophy (Bianchi et al., 2005; Jaffré et al., 2009), heart failure (Sole et al., 1979), coronary and pulmonary hypertension (Vikenes et al., 1999; Fishman, 1999), and cardiac arrhythmias (Saman et al., 1985; Fishman, 1999; Nebigil and Maroteaux, 2003). As a result 5-HT-related pathways, and possible side effects, have been widely explored for development of pharmaceuticals for the treatment of pathologies ranging from antidepressants to appetite suppressants (Levy, 2006). Perhaps the best studied of these are the interactions of 5-HT-based antidepressants with the cardiovascular system (see Levy, 2006; Gorman and Sloan, 2000; Dawood et al., 2007). The most commonly prescribed medications for depression are 5-HT selective reuptake inhibitors (SSRIs) (Prieto et al., 2012). While SSRIs 7
Autonomic Neuroscience: Basic and Clinical xxx (xxxx) xxx–xxx
M.R. Stoyek et al.
Qin, Z., Lewis, J.E., Perry, S.F., 2010. Zebrafish (Danio rerio) gill neuroepithelial cells are sensitive chemoreceptors for environmental CO2. J. Physiol. 588 (5), 861–872. Rajamani, S., Eckhardt, L.L., Valdivia, C.R., Klemens, C.A., Gillman, B.M., Anderson, C.L., Holzem, K.M., Delisle, B.P., Anson, B.D., Makielski, J.C., January, C.T., 2006. Druginduced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine. Br. J. Pharmacol. 149 (5), 481–489. Rider, S.A., Tucker, C.S., del- Pozo, J., Rose, K.N., CA, MacRae, Bailey, M.A., Mullins, J.J., 2012. Techniques for the in vivo assessment of cardio-renal function in zebrafish (Danio rerio) larvae. J. Physiol. 590 (8), 1803–1809. Roose, S.P., Miyazaki, M., 2005. Pharmacologic treatment of depression in patients with heart disease. Psychosom. Med. 67, S54–S57. Sala, M., Coppa, F., Cappucciati, C., Brambilla, P., d'Allio, G., Caverzasi, E., Barale, F., De Ferrari, G.M., 2006. Antidepressants: their effects on cardiac channels, QT prolongation and Torsade de Pointes. Curr. Opin. Investig. Drugs 7 (3), 256–263. Saman, S., Thandroyen, F., Opie, L.H., 1985. Serotonin and the heart: effects of ketanserin on myocardial function, heart rate, and arrhythmias. J. Cardiovasc. Pharmacol. 7, 70–75. Sari, Y., Zhou, F.C., 2003. Serotonin and its transporter on proliferation of fetal heart cells. Int. J. Dev. Neurosci. 21 (8), 417–424. Schneider, J.A., Yonkman, F.F., 1953. Action of serotonin (5-hydroxytryptamine) on vagal afferent impulses in the cat. Am. J. Physiol.—Legacy Content 174 (1), 127–134. Shakarchi, K., Zachar, P.C., Jonz, M.G., 2013. Serotonergic and cholinergic elements of the hypoxic ventilatory response in developing zebrafish. J. Exp. Biol. 216 (5), 869–880. Singh, S., Johnson, P.I., Javed, A., Gray, T.S., Lonchyna, V.A., Wurster, R.D., 1999. Monoamine-and histamine-synthesizing enzymes and neurotransmitters within neurons of adult human cardiac ganglia. Circulation 99 (3), 411–419. Sole, M.J., Shum, A., Van Loon, G.R., 1979. Serotonin metabolism in the normal and failing hamster heart. Circ. Res. 45 (5), 629–634. Steele, S.L., Ekker, M., Perry, S.F., 2011. Interactive effects of development and hypoxia on catecholamine synthesis and cardiac function in zebrafish (Danio rerio). J. Comp. Physiol. B. 181 (4), 527–538. Stewart, A.M., Cachat, J., Gaikwad, S., Robinson, K.S., Gebhardt, M., Kalueff, A.V., 2013. Perspectives on experimental models of serotonin syndrome in zebrafish. Neurochem. Int. 62 (6), 893–902. Stoyek, M.R., Croll, R.P., Smith, F.M., 2015. Intrinsic and extrinsic innervation of the heart in zebrafish (Danio rerio). J. Comp. Neurol. 523 (11), 1683–1700. Stoyek, M.R., Croll, R.P., Smith, F.M., 2016. Zebrafish heart as a model to study the integrative autonomic control of pacemaker function. Am. J. Physiol. Heart Circ. Physiol. 311, H676–H688. Susaki, E.A., Tainaka, K., Perrin, D., Kishino, F., Tawara, T., Watanabe, T.M., Yokoyama, C., Onoe, H., Eguchi, M., Yamaguchi, S., Abe, T., 2014. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157 (3), 726–739. Tessadori, F., van Weerd, J.H., Burkhard, S.B., Verkerk, A.O., de Pater, E., Boukens, B.J., Vink, A., Christoffels, V.M., Bakkers, J., 2012. Identification and functional characterization of cardiac pacemaker cells in zebrafish. PLoS One 7 (10), e47644. Uyttebroek, L., Shepherd, I.T., Harrisson, F., Hubens, G., Blust, R., Timmermans, J.P., Van Nassauw, L., 2010. Neurochemical coding of enteric neurons in adult and embryonic zebrafish (Danio rerio). J. Comp. Neurol. 518 (21), 4419–4438. Vikenes, K., Farstad, M., Nordrehaug, J.E., 1999. Serotonin is associated with coronary artery disease and cardiac events. Circulation 100 (5), 483–489. Votavova, M., Boullin, D.J., Costa, E., 1971. Specificity of action of 6-hydroxydopamine in peripheral cat tissues: depletion of noradrenaline without depletion of 5-hydroxytryptamine. Life Sci. 10 (2), 87–91. Xu, J., Jian, B., Chu, R., Lu, Z., Li, Q., Dunlop, J., Rosenzweig-Lipson, S., McGonigle, P., Levy, R.J., Liang, B., 2002. Serotonin mechanisms in heart valve disease II: the 5-HT2 receptor and its signaling pathway in aortic valve interstitial cells. Am. J. Pathol. 161 (6), 2209–2218. Yabanoglu, S., Akkiki, M., Seguelas, M.H., Mialet-Perez, J., Parini, A., Pizzinat, N., 2009. Platelet derived serotonin drives the activation of rat cardiac fibroblasts by 5-HT2A receptors. J. Mol. Cell. Cardiol. 46 (4), 518–525. Yamano, M., Kamato, T., Nishida, A., Ito, H., Yuki, H., Tsutsumi, R., Honda, K., Miyata, K., 1994. Serotonin (5-HT) 3-receptor antagonism of 4,5,6,7-tetrahydrobenzimidazole derivatives against 5-HT-induced bradycardia in anesthetized rats. Jpn. J. Pharmacol. 65 (3), 241–248. Yavarone, M.S., Shuey, D.L., Tamir, H., Sadler, T.W., Lauder, J.M., 1993. Serotonin and cardiac morphogenesis in the mouse embryo. Teratology 47 (6), 573–584.
Frishman, W.H., Grewall, P., 2000. Serotonin and the heart. Ann. Med. 32 (3), 195–209. Gorman, J.M., Sloan, R.P., 2000. Heart rate variability in depressive and anxiety disorders. Am. Heart J. 140 (4), S77–S83. Henn, F.A., Hamberger, A., 1971. Glial cell function: uptake of transmitter substances. Proc. Natl. Acad. Sci. 68 (11), 2686–2690. Irisawa, H., 1978. Comparative physiology of the cardiac pacemaker mechanism. Physiol. Rev. 58 (2), 461–498. Jackson, R., Braubach, O.R., Bilkey, J., Zhang, J., Akimenko, M.A., Fine, A., Croll, R.P., Jonz, M.G., 2013. Expression of sall4 in taste buds of zebrafish. Dev. Neurobiol. 73 (7), 543–558. Jacobs, L., Comroe, J.H., 1971. Reflex apnea, bradycardia, and hypotension produced by serotonin and phenyldiguanidecting on the nodose ganglia of the cat. Circ. Res. 29 (2), 145–155. Jaffré, F., Bonnin, P., Callebert, J., Debbabi, H., Setola, V., Doly, S., Monassier, L., Mettauer, B., Blaxall, B.C., Launay, J.M., Maroteaux, L., 2009. Serotonin and angiotensin receptors in cardiac fibroblasts coregulate adrenergic-dependent cardiac hypertrophy. Circ. Res. 104 (1), 113–123. Jonz, M.G., Fearon, I.M., Nurse, C.A., 2004. Neuroepithelial oxygen chemoreceptors of the zebrafish gill. J. Physiol. 560 (3), 737–752. Levy, R.J., 2006. Serotonin transporter mechanisms and cardiac disease. Circulation 113 (1), 2–4. Li, N., Csepe, T.A., Hansen, B.J., Dobrzynski, H., Higgins, R.S., Kilic, A., Mohler, P.J., Janssen, P.M., Rosen, M.R., Biesiadecki, B.J., Fedorov, V.V., 2015. Molecular mapping of sinoatrial node HCN channel expression in the human heart. Circ. Arrhythm. Electrophysiol. 8 (5), 1219–1227. MacCanon, D.M., Horvath, S.M., 1954. Some effects of serotonin in pentobarbital anesthetized dogs. Am. J. Physiol.—Legacy Content 179 (1), 131–134. Madan, B.R., Khanna, N.K., Godhwani, J.L., Pendse, V.K., 1970. Changes in the 5-hydroxytryptamine (5-HT) content of the heart during ectopic ventricular arrhythmia and consequent to its reversion by quinidine. Indian J. Med. Res. 58 (1), 130. Mangoni, M.E., Nargeot, J., 2008. Genesis and regulation of the heart automaticity. Physiol. Rev. 88 (3), 919–982. Maximino, C., Puty, B., Benzecry, R., Araújo, J., Lima, M.G., Batista, E.D.J.O., de Matos Oliveira, K.R., Crespo-Lopez, M.E., Herculano, A.M., 2013. Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models. Neuropharmacology 71, 83–97. Nebigil, C.G., Maroteaux, L., 2001. A novel role for serotonin in heart. Trends Cardiovasc. Med. 11 (8), 329–335. Nebigil, C.G., Maroteaux, L., 2003. Functional consequence of serotonin/5-HT2B receptor signaling in heart role of mitochondria in transition between hypertrophy and heart failure? Circulation 108 (7), 902–908. Nemtsas, P., Wettwer, E., Christ, T., Weidinger, G., Ravens, U., 2010. Adult zebrafish heart as a model for human heart? An electrophysiological study. J. Mol. Cell. Cardiol. 48 (1), 161–171. Newton, C.M., Stoyek, M.R., Croll, R.P., Smith, F.M., 2014. Regional innervation of the heart in the goldfish, Carassius auratus: a confocal microscopy study. J. Comp. Neurol. 522 (2), 456–478. Olsson, C., 2009. Autonomic innervation of the fish gut. Acta Histochem. 111 (3), 185–195. Olsson, C., Holmberg, A., Holmgren, S., 2008. Development of enteric and vagal innervation of the zebrafish (Danio rerio) gut. J. Comp. Neurol. 508 (5), 756–770. Pacher, P., Ungvari, Z., Nanasi, P.P., Furst, S., Kecskemeti, V., 1999. Speculations on difference between tricyclic and selective serotonin reuptake inhibitor antidepressants on their cardiac effects. Is there any? Curr. Med. Chem. 6 (6), 469–480. Park, K.S., Kong, I.D., Park, K.C., Lee, J.W., 1999. Fluoxetine inhibits L-type Ca2 + and transient outward K+ currents in rat ventricular myocytes. Yonsei Med. J. 40 (2), 144–151. Pauza, D.H., Saburkina, I., Rysevaite, K., Inokaitis, H., Jokubauskas, M., Jalife, J., Pauziene, N., 2013. Neuroanatomy of the murine cardiac conduction system: a combined stereomicroscopic and fluorescence immunohistochemical study. Auton. Neurosci. 176 (1), 32–47. Pauza, D.H., Rysevaite, K., Inokaitis, H., Jokubauskas, M., Pauza, A.G., Brack, K.E., Pauziene, N., 2014. Innervation of sinoatrial nodal cardiomyocytes in mouse. A combined approach using immunofluorescent and electron microscopy. J. Mol. Cell. Cardiol. 75, 188–197. Prieto, M.J., Gutierrez, H.C., Arévalo, R.A., Chiaramoni, N.S., del Valle, Alonso S., 2012. Effect of risperidone and fluoxetine on the movement and neurochemical changes of zebrafish. Open J. Med. Chem. 2 (4), 129–138.
8