Psychopharmacology DOI 10.1007/s00213-014-3713-0
ORIGINAL INVESTIGATION
Differential effects of dopamine D1 and D2/3 receptor antagonism on motor responses Steven Tran & Magda Nowicki & Arrujyan Muraleetharan & Robert Gerlai
Received: 13 May 2014 / Accepted: 5 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Rationale The zebrafish dopaminergic system is thought to be evolutionarily conserved and may be amenable to pharmacological manipulation using drugs developed for mammalian receptors. However, only few studies have examined the role of specific receptor subtypes in behaviour of adult zebrafish. Objectives The objectives of this study are to determine the translational relevance of the zebrafish and examine the psychopharmacology of specific dopamine receptors in this species. Methods Using a behavioural pharmacological approach, we examine the effect of D1 and D2/3 receptor antagonisms on motor patterns of adult zebrafish during acute drug exposure and withdrawal. Results Acute exposure to SCH-23390 (D1 receptor antagonist) decreased total distance travelled in a dose-dependent manner. Exposure to amisulpride (D2/3 receptor antagonist) induced a biphasic dose-response in total distance travelled and in angular velocity. The results provide support for the existence of structurally and functionally conserved postsynaptic D1 and D2 receptors, as well as presynaptic D2 autoreceptors in the zebrafish brain. The behavioural effects of the employed antagonists did not persist following 30 min of withdrawal. Conclusion The results suggest that zebrafish, a cheaper and simpler model organism compared to the rat and the mouse, may be an efficient translationally relevant tool for the analysis of the psychopharmacology of receptors of the vertebrate dopaminergic system. S. Tran (*) : R. Gerlai Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road North, Rm 1022D, Mississauga, Ontario L5L 1C6, Canada e-mail: [email protected] M. Nowicki : A. Muraleetharan : R. Gerlai Department of Psychology, University of Toronto Mississauga, Mississauga, Canada
Keywords Dopamine antagonists . SCH-23390 . Amisulpride . Zebrafish . Behaviour
The zebrafish is an increasingly popular model organism in behavioural pharmacology. It is a small vertebrate with biological features and pharmacological targets homologous to those of mammals (Gerlai et al. 2000; Kily et al. 2008; Rico et al. 2011). Furthermore, the nucleotide sequence of zebrafish genes shares a high degree of similarity with those of humans, making the zebrafish a powerful animal model for translational research (Barbazuk et al. 2000; Klee et al. 2012). These similarities imply that pharmacological tools developed for mammals may be employed with zebrafish too. Many of these compounds are water soluble and may allow the investigator to employ a less invasive drug delivery method: immersion of the entire fish in the solution (Tran et al. 2014; Sivamani et al. 2013). Activation of dopamine receptors mediates a number of reward and motivational processes (Di Chiara and Bassareo 2007; Berridge and Robison 1998) and motor responses in mammals, e.g. rats (Meyer and Shults 1993; Klinker et al. 2013). In mammals, dopamine receptors are coupled to Gproteins and are classified as either D1-like or D2-like based on their downstream signalling. D1-like receptors, including D1 and D5, have excitatory effects on neurotransmission. Binding of dopamine to D1-like receptors leads to activation of stimulatory G-proteins coupled to adenylyl cyclase, which increases intracellular levels of cAMP. D2-like receptors (D2, D3 and D4) have inhibitory effects on neurotransmission, are coupled to inhibitory G-proteins, inhibit adenylyl cyclase and decrease intracellular levels of cAMP (Beaulieu and Gainetdinov 2011). Activation of D1-like and D2-like dopamine receptors affects synaptic transmission and is expected to influence a variety of motor and reward-related behavioural processes. For example, activation of D1 receptors initiates
Psychopharmacology
maternal behaviour in female ovariectomized rats through stimulation of adenylyl cyclase (Stolzenberg et al. 2010). In addition to the direct effects on synaptic transmission, changes in the levels of cAMP mediate the activity of protein kinases and transcription factors leading to enhancement/suppression of the expression of specific target genes (Jia et al. 2013). The dopaminergic system is conserved across a number of vertebrate species including zebrafish (Klee et al. 2012). This conservation may have practical relevance. Zebrafish receptors may be amenable to pharmacological manipulation with compounds developed for corresponding mammalian receptors. Dopamine D1, D2, D3 and D4 subtype receptors have all been identified in the zebrafish brain (Boehmler et al. 2004, 2007; Li et al. 2007). The D1 receptor is encoded by the drd1 gene which shares a high sequence homology with the D1 receptor genes found in other vertebrate species (Li et al. 2007). Full-length complementary (c)DNAs of three D2 receptor genes (drd2a, drd2b, drd2c), a single D3 receptor gene (drd3) and three D4 receptor genes (drd4a, drd4b, drd4c) have also been identified in zebrafish, and sequence comparisons have provided support that they are orthologs of the mammalian D2, D3 and D4 receptors, respectively (Boehmler et al. 2004, 2007). In certain mammalian species, the D2 receptor exists as genetic variants, D2S and D2L, generated by alternative splicing (Monsma et al. 1989; Giros et al. 1989). The D2 gene encodes five exons, and the fifth exon, which translates a polypeptide of 29 amino acids in length, is spliced out in the D2S isoform but not in the D2L isoform. The product of the fifth exon is thought to be important in localizing the two isoforms to their respective presynaptic or postsynaptic membranes in mammals (Boehmler et al. 2004). The D2S receptor is predominantly found on the presynaptic terminal and acts as an autoreceptor to mediate negative feedback. The D2L isoform is located on the postsynaptic terminal and facilitates inhibitory neurotransmission through inhibitory G-coupled proteins (De Mei et al. 2009). In the zebrafish, unlike in mammals, the fifth exon of all D2 receptor cDNAs is retained. The lack of alternative splicing in all D2 isoforms in zebrafish implies the absence of pre- versus postsynaptic differential localization. However, there have been few attempts to examine the spatial location of the D2 receptor isoforms, and the behavioural function of these receptors remains unknown, at least in the zebrafish. Here, we have taken a psychopharmacological approach to investigate the function of D1-like and D2-like dopamine receptors in zebrafish. Since the predicted amino acid sequence for the drd1 receptor in zebrafish is 71 % similar to the human D1 receptor (Li et al. 2007), and the drd2a, drd2b and drd2c receptors in zebrafish is 71, 66 and 71 %, respectively, similar to the human D2 dopamine receptor (Boehmler et al. 2007), drugs designed to target dopamine receptors in mammals may have similar binding affinities in zebrafish. We
employed two dopamine receptor antagonists, SCH-23390 and amisulpride, which possess different affinity for pre- and postsynaptic dopamine receptor subtypes. SCH-23390 is a D1-like receptor-selective antagonist which binds to postsynaptic dopamine receptors in mammals (Bourne 2001). Amisulpride is a D2/3-specific antagonist with preferential binding for receptors expressed in limbic structures in the mammalian brain (Perrault et al. 1997). At lower concentrations, amisulpride is selective for presynaptic mammalian dopamine autoreceptors and has been shown to inhibit D2 autoreceptor-mediated behaviours. At higher concentrations (five to ten times higher), it binds postsynaptic D2/3 receptors and has been shown to impair postsynaptic receptor-mediated behaviours in mice and rats (Perrault et al. 1997; Schoemaker et al. 1997). Comparison of the effects of these drugs as well as exploiting the differential binding affinity of amisulpride for different receptor subtypes at high and low concentrations may provide insights into the roles of pre- and postsynaptic dopamine receptors in zebrafish. In the current study, we examine the time-course of behavioural responses (swim path characteristics quantified using video-tracking) to acute exposure to a range of doses of SCH23390 and amisulpride (0, 0.1, 0.5 and 1.0 mg/L) in adult zebrafish. In addition, we also follow the time-course of behavioural changes induced by acute withdrawal from these two dopamine antagonists over a period of 30 min to determine how long the behavioural effects persist.
Methods Animal housing Adult AB zebrafish (progenitors obtained from the ZFIN Center (Eugene, Oregon, USA)) were bred at the University of Toronto Mississauga Vivarium (Mississauga, Ontario, Canada). The AB strain shows homozygosity at over 80 % of loci and is frequently used in behavioural neuroscience and high throughput mutagenesis studies (Guryev 2006). Fish were raised and kept in 37-L housing tanks as described elsewhere (Tran and Gerlai 2013a; Pannia et al. 2014). Experimental design and procedure The experiment was designed to characterize the behavioural dose-response to acute administration of two dopamine receptor antagonists, a D1 antagonist (R(+)-SCH-23390 hydrochloride) and a D2/3 antagonist (amisulpride). Zebrafish were taken from their housing tanks and randomly assigned to different concentration groups of SCH-23390 or amisulpride (0, 0.1, 0.5 1 mg/L). Individual zebrafish were exposed to the drug for 30 min in a 1.5-L tank containing 1 L of the appropriate drug concentration (n=16) in which the fish could be observed and
Psychopharmacology
its behavioural responses recorded. The concentration of SCH-23390 was based on a previous study (Scerbina et al. 2012). The concentration of amisulpride used in the current study corresponds to 100 times, 500 times and 1,000 times its
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Ki value for the D2 receptor and its differential binding affinity for pre- and postsynaptic receptors in mammals (Perrault et al. 1997; Schoemaker et al. 1997). Following the 30-min drug exposure period, zebrafish were placed into a 37-L tank
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1 minute intervals Fig. 1 The time-dependent changes in freezing behaviour are shown for 1-min intervals for DMSO exposure (drug exposure tank panel a; withdrawal tank panel b), SCH-23390 exposure (drug exposure tank panel c; withdrawal tank panel d) and amisulpride exposure (drug exposure tank panel e; withdrawal tank panel f). Mean±S.E.M. are shown. The average of the last 10 min in the drug exposure tank (full penetration of drug is expected) is used to compare different concentration groups using Tukey’s post hoc HSD tests, shown as bar graphs, insets of panels a, c
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1 minute intervals and e. The average of the last 10 min of drug withdrawal (minimal drug content in the brain) is used to compare different concentration groups using Tukey’s post hoc HSD tests, shown as bar graphs, insets of panels b, d and f. Significant (p0.05). Effects of D1 receptor antagonism on zebrafish motor patterns Zebrafish in the drug exposure and withdrawal tank exhibited a significant time-dependent decrease in freezing (Fig. 1), absolute angular velocity and relative angular velocity, as well as a time-dependent increase in total distance travelled and distance to bottom (Fig. 3) (see Table 1 for details of the results of statistical analyses). Analysis of average performance in the last 10 min of drug exposure determined a significant main effect of drug exposure on the total distance travelled, freezing and distance to bottom. Tukey’s post hoc multiple comparison test showed that zebrafish treated with the highest dose of the D1 receptor (D1R) antagonist moved significantly less (p= 0.046) and froze significantly more (p=0.028) compared to controls. All zebrafish treated with the D1R antagonist also spent more time near the top of the tank compared to controls (p
ORIGINAL INVESTIGATION
Differential effects of dopamine D1 and D2/3 receptor antagonism on motor responses Steven Tran & Magda Nowicki & Arrujyan Muraleetharan & Robert Gerlai
Received: 13 May 2014 / Accepted: 5 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Rationale The zebrafish dopaminergic system is thought to be evolutionarily conserved and may be amenable to pharmacological manipulation using drugs developed for mammalian receptors. However, only few studies have examined the role of specific receptor subtypes in behaviour of adult zebrafish. Objectives The objectives of this study are to determine the translational relevance of the zebrafish and examine the psychopharmacology of specific dopamine receptors in this species. Methods Using a behavioural pharmacological approach, we examine the effect of D1 and D2/3 receptor antagonisms on motor patterns of adult zebrafish during acute drug exposure and withdrawal. Results Acute exposure to SCH-23390 (D1 receptor antagonist) decreased total distance travelled in a dose-dependent manner. Exposure to amisulpride (D2/3 receptor antagonist) induced a biphasic dose-response in total distance travelled and in angular velocity. The results provide support for the existence of structurally and functionally conserved postsynaptic D1 and D2 receptors, as well as presynaptic D2 autoreceptors in the zebrafish brain. The behavioural effects of the employed antagonists did not persist following 30 min of withdrawal. Conclusion The results suggest that zebrafish, a cheaper and simpler model organism compared to the rat and the mouse, may be an efficient translationally relevant tool for the analysis of the psychopharmacology of receptors of the vertebrate dopaminergic system. S. Tran (*) : R. Gerlai Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road North, Rm 1022D, Mississauga, Ontario L5L 1C6, Canada e-mail: [email protected] M. Nowicki : A. Muraleetharan : R. Gerlai Department of Psychology, University of Toronto Mississauga, Mississauga, Canada
Keywords Dopamine antagonists . SCH-23390 . Amisulpride . Zebrafish . Behaviour
The zebrafish is an increasingly popular model organism in behavioural pharmacology. It is a small vertebrate with biological features and pharmacological targets homologous to those of mammals (Gerlai et al. 2000; Kily et al. 2008; Rico et al. 2011). Furthermore, the nucleotide sequence of zebrafish genes shares a high degree of similarity with those of humans, making the zebrafish a powerful animal model for translational research (Barbazuk et al. 2000; Klee et al. 2012). These similarities imply that pharmacological tools developed for mammals may be employed with zebrafish too. Many of these compounds are water soluble and may allow the investigator to employ a less invasive drug delivery method: immersion of the entire fish in the solution (Tran et al. 2014; Sivamani et al. 2013). Activation of dopamine receptors mediates a number of reward and motivational processes (Di Chiara and Bassareo 2007; Berridge and Robison 1998) and motor responses in mammals, e.g. rats (Meyer and Shults 1993; Klinker et al. 2013). In mammals, dopamine receptors are coupled to Gproteins and are classified as either D1-like or D2-like based on their downstream signalling. D1-like receptors, including D1 and D5, have excitatory effects on neurotransmission. Binding of dopamine to D1-like receptors leads to activation of stimulatory G-proteins coupled to adenylyl cyclase, which increases intracellular levels of cAMP. D2-like receptors (D2, D3 and D4) have inhibitory effects on neurotransmission, are coupled to inhibitory G-proteins, inhibit adenylyl cyclase and decrease intracellular levels of cAMP (Beaulieu and Gainetdinov 2011). Activation of D1-like and D2-like dopamine receptors affects synaptic transmission and is expected to influence a variety of motor and reward-related behavioural processes. For example, activation of D1 receptors initiates
Psychopharmacology
maternal behaviour in female ovariectomized rats through stimulation of adenylyl cyclase (Stolzenberg et al. 2010). In addition to the direct effects on synaptic transmission, changes in the levels of cAMP mediate the activity of protein kinases and transcription factors leading to enhancement/suppression of the expression of specific target genes (Jia et al. 2013). The dopaminergic system is conserved across a number of vertebrate species including zebrafish (Klee et al. 2012). This conservation may have practical relevance. Zebrafish receptors may be amenable to pharmacological manipulation with compounds developed for corresponding mammalian receptors. Dopamine D1, D2, D3 and D4 subtype receptors have all been identified in the zebrafish brain (Boehmler et al. 2004, 2007; Li et al. 2007). The D1 receptor is encoded by the drd1 gene which shares a high sequence homology with the D1 receptor genes found in other vertebrate species (Li et al. 2007). Full-length complementary (c)DNAs of three D2 receptor genes (drd2a, drd2b, drd2c), a single D3 receptor gene (drd3) and three D4 receptor genes (drd4a, drd4b, drd4c) have also been identified in zebrafish, and sequence comparisons have provided support that they are orthologs of the mammalian D2, D3 and D4 receptors, respectively (Boehmler et al. 2004, 2007). In certain mammalian species, the D2 receptor exists as genetic variants, D2S and D2L, generated by alternative splicing (Monsma et al. 1989; Giros et al. 1989). The D2 gene encodes five exons, and the fifth exon, which translates a polypeptide of 29 amino acids in length, is spliced out in the D2S isoform but not in the D2L isoform. The product of the fifth exon is thought to be important in localizing the two isoforms to their respective presynaptic or postsynaptic membranes in mammals (Boehmler et al. 2004). The D2S receptor is predominantly found on the presynaptic terminal and acts as an autoreceptor to mediate negative feedback. The D2L isoform is located on the postsynaptic terminal and facilitates inhibitory neurotransmission through inhibitory G-coupled proteins (De Mei et al. 2009). In the zebrafish, unlike in mammals, the fifth exon of all D2 receptor cDNAs is retained. The lack of alternative splicing in all D2 isoforms in zebrafish implies the absence of pre- versus postsynaptic differential localization. However, there have been few attempts to examine the spatial location of the D2 receptor isoforms, and the behavioural function of these receptors remains unknown, at least in the zebrafish. Here, we have taken a psychopharmacological approach to investigate the function of D1-like and D2-like dopamine receptors in zebrafish. Since the predicted amino acid sequence for the drd1 receptor in zebrafish is 71 % similar to the human D1 receptor (Li et al. 2007), and the drd2a, drd2b and drd2c receptors in zebrafish is 71, 66 and 71 %, respectively, similar to the human D2 dopamine receptor (Boehmler et al. 2007), drugs designed to target dopamine receptors in mammals may have similar binding affinities in zebrafish. We
employed two dopamine receptor antagonists, SCH-23390 and amisulpride, which possess different affinity for pre- and postsynaptic dopamine receptor subtypes. SCH-23390 is a D1-like receptor-selective antagonist which binds to postsynaptic dopamine receptors in mammals (Bourne 2001). Amisulpride is a D2/3-specific antagonist with preferential binding for receptors expressed in limbic structures in the mammalian brain (Perrault et al. 1997). At lower concentrations, amisulpride is selective for presynaptic mammalian dopamine autoreceptors and has been shown to inhibit D2 autoreceptor-mediated behaviours. At higher concentrations (five to ten times higher), it binds postsynaptic D2/3 receptors and has been shown to impair postsynaptic receptor-mediated behaviours in mice and rats (Perrault et al. 1997; Schoemaker et al. 1997). Comparison of the effects of these drugs as well as exploiting the differential binding affinity of amisulpride for different receptor subtypes at high and low concentrations may provide insights into the roles of pre- and postsynaptic dopamine receptors in zebrafish. In the current study, we examine the time-course of behavioural responses (swim path characteristics quantified using video-tracking) to acute exposure to a range of doses of SCH23390 and amisulpride (0, 0.1, 0.5 and 1.0 mg/L) in adult zebrafish. In addition, we also follow the time-course of behavioural changes induced by acute withdrawal from these two dopamine antagonists over a period of 30 min to determine how long the behavioural effects persist.
Methods Animal housing Adult AB zebrafish (progenitors obtained from the ZFIN Center (Eugene, Oregon, USA)) were bred at the University of Toronto Mississauga Vivarium (Mississauga, Ontario, Canada). The AB strain shows homozygosity at over 80 % of loci and is frequently used in behavioural neuroscience and high throughput mutagenesis studies (Guryev 2006). Fish were raised and kept in 37-L housing tanks as described elsewhere (Tran and Gerlai 2013a; Pannia et al. 2014). Experimental design and procedure The experiment was designed to characterize the behavioural dose-response to acute administration of two dopamine receptor antagonists, a D1 antagonist (R(+)-SCH-23390 hydrochloride) and a D2/3 antagonist (amisulpride). Zebrafish were taken from their housing tanks and randomly assigned to different concentration groups of SCH-23390 or amisulpride (0, 0.1, 0.5 1 mg/L). Individual zebrafish were exposed to the drug for 30 min in a 1.5-L tank containing 1 L of the appropriate drug concentration (n=16) in which the fish could be observed and
Psychopharmacology
its behavioural responses recorded. The concentration of SCH-23390 was based on a previous study (Scerbina et al. 2012). The concentration of amisulpride used in the current study corresponds to 100 times, 500 times and 1,000 times its
10 8 6 4 2 0 0
DMSO (% v/v)
10
A
Time spent freezing (seconds)
Time spent freezing (seconds)
12
Ki value for the D2 receptor and its differential binding affinity for pre- and postsynaptic receptors in mammals (Perrault et al. 1997; Schoemaker et al. 1997). Following the 30-min drug exposure period, zebrafish were placed into a 37-L tank
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1 minute intervals Fig. 1 The time-dependent changes in freezing behaviour are shown for 1-min intervals for DMSO exposure (drug exposure tank panel a; withdrawal tank panel b), SCH-23390 exposure (drug exposure tank panel c; withdrawal tank panel d) and amisulpride exposure (drug exposure tank panel e; withdrawal tank panel f). Mean±S.E.M. are shown. The average of the last 10 min in the drug exposure tank (full penetration of drug is expected) is used to compare different concentration groups using Tukey’s post hoc HSD tests, shown as bar graphs, insets of panels a, c
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1 minute intervals and e. The average of the last 10 min of drug withdrawal (minimal drug content in the brain) is used to compare different concentration groups using Tukey’s post hoc HSD tests, shown as bar graphs, insets of panels b, d and f. Significant (p0.05). Effects of D1 receptor antagonism on zebrafish motor patterns Zebrafish in the drug exposure and withdrawal tank exhibited a significant time-dependent decrease in freezing (Fig. 1), absolute angular velocity and relative angular velocity, as well as a time-dependent increase in total distance travelled and distance to bottom (Fig. 3) (see Table 1 for details of the results of statistical analyses). Analysis of average performance in the last 10 min of drug exposure determined a significant main effect of drug exposure on the total distance travelled, freezing and distance to bottom. Tukey’s post hoc multiple comparison test showed that zebrafish treated with the highest dose of the D1 receptor (D1R) antagonist moved significantly less (p= 0.046) and froze significantly more (p=0.028) compared to controls. All zebrafish treated with the D1R antagonist also spent more time near the top of the tank compared to controls (p
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