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Pharmacol Biochem Behav. Author manuscript; available in PMC 2016 November 01. Published in final edited form as: Pharmacol Biochem Behav. 2015 November ; 138: 32–39. doi:10.1016/j.pbb.2015.09.008.
Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio) Magda Nowicki1, Steven Tran2, Diptendu Chatterjee1, and Robert Gerlai1,2,* 1Department
of Psychology,University of Toronto Mississauga
2Department
of Cell and System Biology, University of Toronto
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Abstract
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Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine’s metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol’s locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.
Keywords alcohol; ethanol; dopamine; locomotor activity; tyrosine hydroxylase; zebrafish
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1. Introduction Alcoholism is a debilitating disorder associated with frequent or prolonged alcohol consumption (Hemmingsson & Lundberg, 2001). Understanding the neurobiological mechanisms of compulsive alcohol use may lead to the development of efficacious treatment for this disorder (Gilpin & Koob, 2008). Commonly classified as a sedative drug,
*
Corresponding author at: Department of Psychology, University of Toronto Mississauga, 3359 Mississauga Road North, Rm CCT4004, Mississauga, Ontario L5L 1C6, Canada, Tel: 905-569-4255 (office), Tel: 905-569-4257 (lab), Fax: 905-569-4326, [email protected].
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alcohol (ethanol) exerts biphasic effects across both dose and time. It has stimulant effects in humans and other animals following low to moderate doses, while high doses induce sedation (Addicott et al., 2007; Rodd et al., 2006; Tambour et al., 2006). The timedependent biphasic effect of alcohol has been associated with changes in blood alcohol concentration. In humans, a single acute dose of alcohol induces stimulation during the ascending trajectory of the blood alcohol curve (BAC) and sedation during the descending trajectory of the BAC (Holdstock & de Wit, 1998). Similar time-dependent biphasic effects of alcohol have been shown in other mammalian species (Read et al., 1960) and now in zebrafish too (Tran & Gerlai, 2013a).
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Sensitivity to the stimulant effects of alcohol is considered a risk factor for excessive alcohol consumption that may eventually lead to alcohol dependence (King et al., 2011, 2014). Therefore, the investigation of the stimulatory effects of alcohol is hoped to lead to the development of treatments or early interventions. Animal models have greatly improved our understanding of alcohol dependence because of the behavioural and pharmacological similarities of alcohol’s effects across humans and many other species (Brabant et al., 2014).
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The zebrafish (Danio rerio) is a small freshwater teleost that is rapidly emerging as an important animal model in many research areas including developmental biology, biomedical research and more recently, behavioural neuroscience (Zon & Peterson, 2005; Dawid, 2004; Gerlai, 2011). Zebrafish share many similarities with humans and other mammals on the genetic, molecular, and neuronal level (Barbazuk et al., 2000; Rico et al., 2011). The zebrafish genome has been sequenced, and zebrafish genes have been found to possess high nucleotide sequence similarity with human homologues (Barbazuk et al., 2000). In addition, conserved neurotransmitter systems in zebrafish permit the elucidation of molecular mechanisms underlying drug effects, neurological diseases or psychopathology (Rico et al., 2011). Besides the opportunity for translational research, there are practical advantages to studying this small vertebrate. Zebrafish breed frequently and produce large clutches of eggs (up to 300 eggs per female per spawning). Their small body size (4 cm long when full grown) allows their space efficient maintenance in the laboratory, and their high tolerance to a range of water conditions makes them a robust species even under artificial conditions (Gerlai, 2015).
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The zebrafish may be particularly suitable for the analysis of the mechanisms of in vivo actions of alcohol (Chatterjee et al., 2014; Puttonen et al., 2013; Tran & Gerlai, 2013a; Tran et al., 2015a) for many reasons. First, alcohol can be administered by immersing zebrafish into the alcohol solution, a non-invasive and practical alternative to injections or vapour inhalation employed in rodents (Tran et al., 2015a). Zebrafish also exhibit robust time- and dose-dependent behavioural responses to alcohol that are comparable to those described in humans and other mammals. For example, a 60-minute acute exposure to low or moderate concentrations of alcohol (up 1% v/v) induces a dose-dependent increase in locomotor activity in adult zebrafish, and reduces anxiety-like behavioural responses (Tran et al., 2015a). On the other hand, higher concentrations of alcohol induce sedative effects. For example, exposure to 3% alcohol immediately decreases swimming speed in zebrafish larvae (Puttonen et al., 2013). Adult zebrafish also exhibit temporal changes in motor activity in response to exposure to 1% alcohol. Motor activity (quantified by the total
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distance travelled) rises and peaks around 20-30 minutes after the start of alcohol treatment. Between 30-60 minutes, zebrafish motor activity remains elevated but it begins to gradually decline (Tran & Gerlai, 2013a). Like in mammals, motor activity also strongly correlates with brain alcohol concentration in zebrafish (Tran et al., 2015a).
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The neural underpinnings of acute alcohol exposure are complex and have not yet been entirely deciphered due to the involvement of numerous neurotransmitter systems and signal transduction mechanisms. It has been suggested that a common neurobiological mechanism, the dopaminergic system in the mesolimbic dopaminergic system, mediates the reinforcing and stimulant effects of addictive drugs, including alcohol (Brabant et al., 2014). Like other major drugs of abuse, alcohol has been shown to target the dopaminergic system in humans and other mammals (Boileau et al., 2003; Tang et al., 2003). However, the precise mechanism by which dopamine (DA) regulates acute alcohol responses and addiction remains elusive. Acute alcohol exposure has been shown to increase extracellular brain DA concentrations in rats (Di Chiara & Imperato, 1988) and levels of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in mice (Saeed & Wooles, 1984). The increase in DA levels has been proposed to underlie the stimulating properties of alcohol, as DA depletion and DA receptor antagonists block alcohol-induced locomotor activity (Bainton et al., 2000; Camarini et al., 2011). Furthermore, alcohol causes a dose-dependent accumulation of 3, 4-dihydroxyphenylalanine (DOPA) after inhibition of DOPA decarboxylase in the brains of mice (Saeed & Wooles, 1984).
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The rate limiting enzyme of DA synthesis is tyrosine hydroxylase (TH), which catalyzes the hydroxylation of tyrosine to form DOPA. Based on these findings, increased concentrations of DA may be a result of increased DA synthesis in response to alcohol exposure. This hypothesis has been supported by studies in mice that have suppressed the motor activating effects of alcohol through pharmacological inhibition of TH (Carlsson et al., 1972; Engel et al., 1974).
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The zebrafish may be appropriate for the analysis of the behavioural stimulatory effects of alcohol. Areas of the zebrafish telencephalon are thought to be homologous to regions of the mammalian basal ganglia, a crucial region involved in movement. Based on axonal projections, neurochemistry and gene expression, the dorsal nucleus of the ventral telencephalon is suggested to be the zebrafish homologue of the mammalian striatum. The area is rich in dopaminergic nerve terminals projecting from well-defined groups of dopaminergic nuclei in the telencephalon and diencephalon (Rink & Wullimann, 2004). Two putative TH genes have been identified in zebrafish: th1 and th2, however, th1 has been found to be most closely related to the mammalian TH (Candy & Collet, 2005). Similarly to mammals, activation of dopaminergic neurotransmission has been found to mediate alcohol’s stimulatory behavioural effects also in zebrafish. 60-minute exposure to alcohol (doses ranging from 0.25 to 1.00% v/v) has been shown to induce a dose-dependent increase in DA, DOPAC and TH activity in the zebrafish brain corresponding closely to the dosedependent increase found in motor activity (Tran et al., 2015a). TH activity increases in response to acute alcohol exposure in both mammals (Saeed & Wooles, 1984) and zebrafish (Chatterjee et al., 2014). However, the exact relationship
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between TH and the stimulant effects of alcohol is not well understood. An increase in DA synthesis in response to acute alcohol exposure may be a result of two distinct mechanisms: upregulation of gene expression of TH or activation of the enzyme. Acute alcohol increases expression of TH mRNA, for example, in the ventral tegmental area and substantia nigra of rats after 1 hour of alcohol administration (Oliva et al., 2007). A similar response to acute alcohol has been found in zebrafish larvae, in which a 10-minute long acute alcohol exposure increased swimming distance and levels th1 mRNA in the brain (Puttonen et al., 2013). On the other hand, cocaine was found to increase synaptic DA by binding to the DA transporter, and a positive feedback loop resulted in the phosphorylation of TH, and the thus activated TH further increased DA levels (Yao et al., 2010).
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DA is metabolized by two enzymes consecutively: monoamine oxidase A (MAO-A) or monoamine oxidase B (MAO-B), and aldehyde dehydrogenase 2 (ALDH2). First, DA is deaminated by MAO-A or MAO-B to 3, 4-dihydroxyphenylacetaldehyde (DOPAL), a transient and reactive intermediate, that is further metabolized to DOPAC by ALDH2 (Yao et al., 2010). Recently, a highly selective ALDH2 inhibitor was found to suppress cocaine self-administration and cocaine-seeking behaviour in rats by increasing DOPAL, enabling it to condense with DA to form tetrahydropapaveroline (THP). THP is an endogenous inhibitor of phosphorylated TH (p-TH) (approximately 75 times more selective for p-TH than unphosphorylated TH) and blocks the cocaine-induced increase in DA production (Yao et al., 2010). In addition to blocking cocaine addictive behaviour, the ALDH2 inhibitor has been shown to suppress alcohol intake and alcohol seeking in heavy drinking rats (Arolfo et al., 2009). It also prevents the alcohol-induced DA increase in the nucleus accumbens, without altering baseline DA levels (Arolfo et al., 2009) suggesting that the alcohol-induced DA increase may be mediated by phosphorylation of TH.
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Currently, it is unknown whether activation of TH by phosphorylation could underlie alcohol’s locomotor stimulant effects in addition to its addictive properties. To test this hypothesis, we investigated whether selective inhibition of p-TH by THP can block alcoholinduced hyperactivity in zebrafish and alcohol induced increase of DA levels in the brain of zebrafish. First, we characterized the dose-dependent effects of THP on zebrafish motor responses. Subsequently, we treated zebrafish with the highest dose of THP that did not alter baseline motor activity. The THP pre-treated zebrafish were then exposed to a dose of alcohol that would exert robust behavioural and neurochemical effects. We hypothesized that THP pre-treatment would attenuate the alcohol-induced increase of brain DA levels and locomotor activity. In addition, we also examined DOPAC levels in the brain following THP and alcohol treatment to investigate the effects of p-TH inhibition on downstream DA metabolism.
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2. Methods 2.1 Animal Housing 197 sexually mature, 10 month old (young adult), male and female zebrafish (Danio rerio) of the AB strain were used in this study. The AB strain was selected since it is most frequently studied and is genetically well-characterized, showing homozygosity at over 80% of its loci (Guryev et al., 2006). Furthermore, behavioural and neurochemical responses to Pharmacol Biochem Behav. Author manuscript; available in PMC 2016 November 01.
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alcohol have been thoroughly investigated for this strain (Chatterjee & Gerlai, 2009; Tran et al., 2015a; Tran & Gerlai, 2013a). Fish were bred and raised in the vivarium of the University of Toronto Mississauga (Mississauga, Ontario, Canada). Animals were housed in 37 L tanks with mechanical filters containing approximately 20 fish per tank. Zebrafish were kept on a 13 hour light-dark cycle with lights turning on at 08:00h and off at 21:00h. Water quality parameters were maintained at optimal conductivity levels (100-300 microsiemens), temperature (28°C-30°C), and pH (6.8-7.2). Upon hatching to 3 weeks post-fertilization, larvae were fed Larval AP 100. After 3 weeks post-fertilization, fish were fed twice a day, alternating between brine shrimp (Artemia salina) and a mixture of 2 parts flake food (Scientific Hatcheries Diet) and 1 part powdered spirulina. 2.2 Experiment 1
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Locomotor activity—The purpose of Experiment 1 was to characterize the dosedependent locomotor response of zebrafish to multiple concentrations of THP with the goal of determining the concentration that does not alter baseline motor responses. THP was delivered to zebrafish by immersing them in tank water containing the appropriate concentration of the compound. Immersion-based drug delivery was used because of its less invasive nature compared to other methods of administration (e.g. injection) and frequent use in adult zebrafish research (Nowicki et al., 2014; Tran et al., 2015b; Levin et al., 2007; Conners et al., 2014; Sackerman et al., 2010).
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THP (Santa Cruz Biotechnology, Inc., Dallas, TX, U.S.A.) was stored at 4°C until use. A stock solution of THP (7 mM) was prepared by diluting 128.8 mg of THP in 50 mL of distilled water. 1.5 L tanks were filled with 1 L of system water supplemented with 100 mg/L InstantOcean Sea Salt to adjust conductivity and salinity to appropriate levels as employed before (e.g. Tran et al., 2015a; 2015b). The THP stock solution was diluted in the tanks to achieve final bath concentrations of 50, 100 and 200 μM. No THP was added to the control tank, which only contained system water. The concentrations of THP were chosen to selectively inhibit the phosphorylated form of TH. The lowest concentration of THP was determined by multiplying the binding affinity of the inhibitor (Ki) by 1000 based on a previous study that determined 1/1000th of an external drug concentration in the water bath reaches the zebrafish brain (Sackerman et al., 2010). Higher doses were multiples of the lowest THP concentration by a factor of 2.
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Individual zebrafish were exposed to THP (n= 16 per group) in 1.5 L tanks for 90 minutes, a session length that corresponded to the total exposure session length employed in experiment 2. Water quality parameters (conductivity, temperature and pH) matched those of housing tanks. Video recordings from the front of the tanks were taken during drug exposure for subsequent behavioural quantification. All testing tanks were made of transparent plexi-glass with the lateral and back sides covered with white corrugated plastic to provide a uniform environment and obscure external cues during testing. Behavioural recordings during drug exposure were analyzed using automated video-tracking software, EthoVision XT 8.0. The dependent variable quantified was total distance travelled (cm), a measure of locomotor activity in zebrafish.
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2.3 Experiment 2
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Locomotor activity—Experiment 2 intended to determine whether pre-treatment with THP inhibits the ethanol-induced increases in locomotor activity and DA levels. Zebrafish used in Experiment 2 (n=114) were first repeatedly exposed to the dosing container in order to reduce anxiety induced by the novel handling procedure and environment. These predosing trials were conducted by placing individual fish in 1.5 L tanks containing system water for a 5 minute period. Fish were subsequently removed from the tank with a net and immediately transferred into another 1.5 L tank containing system water for 5 minutes. All zebrafish in Experiment 2 underwent three pre-dosing trials prior to testing, a procedure that was based upon prior results (Tran & Gerlai, 2013b). One pre-dosing session was performed per day, and the experiment was performed on the day following the last pre-dosing session.
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The highest THP concentration that did not alter baseline motor activity in Experiment 1 was determined to be 50 μM. In Experiment 2 we employed a 2 × 2 between subject factorial design with THP pre-treatment (0 or 50 μM) and ethanol exposure (0 or 1%) as the between subject factors. Furthermore, zebrafish that were exposed to THP first, were also exposed to THP concurrently during the ethanol exposure phase to ensure continued enzyme inhibition and to avoid potential confounding effects of THP withdrawal. The sample size (n) for each of the four drug conditions was 26-28. 1.5 L tanks were filled with 1 L of system water supplemented with 100 mg/L of Instant Ocean Sea Salt. Water quality parameters were ensured to match those of the housing tanks (temperature, pH and conductivity). A 5 mM stock solution of THP (73.6 mg in 40 ml of distilled water) was prepared and further diluted to 50 μM in the appropriate tanks using system water. Similarly, 100% anhydrous ethyl alcohol (Commercial Alcohols, Brampton, ON, CA) was diluted to achieve a final concentration of 1% in the required tanks.
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Zebrafish were individually pre-treated with 50 μM of THP or system water for 30 minutes in the 1.5 L tanks. System water pre-treated fish were immediately transferred to tanks containing system water or 1% ethanol for 60 minutes. THP pre-treated fish were transferred to tanks containing 50 μM THP or tanks containing 1% ethanol and 50 μM THP for 60 minutes. The length of exposure to THP and to ethanol was determined by pilot studies and was also based upon data published previously on zebrafish (e.g. Tran 3t ail, 2015a). Video recordings were taken from the front of the testing tanks during the 60 minute exposure. The he frontal view allows better video-detection as the fish appear brighter and more distinguishable from the background, and this method has been shown to allow psychopharmacological analysis and quantification of a number of natural behavioural responses in zebrafish (Nowicki et al., 2014). EthoVision XT 8.0 (Noldus Info Tech, Wageningen, The Netherlands) was used to analyze the behavioural recordings and quantify the total distance travelled (cm) fish swam. Analysis of the dopaminergic system—Whole brain DA and DOPAC levels were measured from the four treatment groups of zebrafish (n= 4-5 per group). Following the 60 minute exposure, zebrafish were sacrificed by decapitation and their heads were stored at −40°C until processing. Dopaminergic neurons of different brain regions are not expected to respond in opposite manner to the drug treatment employed here and due to this, and
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because the brain of the zebrafish is rather small, we chose to analyze whole brain extracts instead of attempting to sample separate brain areas. Whole brains were dissected and placed individually into 1.5 ml micro-centrifuge tubes on ice. Brains were sonicated in 10 μl of 0.025 M ascorbic acid in artificial cerebrospinal fluid and sonicates were used for a subsequent protein assay in order to standardize measures of DA and DOPAC. 1 μL of each sonicate and 1.5 ml of protein assay reagent were added to individual 5 ml test tubes. The reagent was prepared by a 1:4 dilution of Coomassie brilliant blue G-250 dye in distilled water. Dilutions of 2 μg/ml bovine serum albumin were used as protein standards. The test tubes were then incubated at room temperature for 20 minutes and 200 μl of the mixtures were transferred to individual wells of a 96 well, 330 μl microplate. The optical density at 595 nm was measured using a microplate reader (Synergy-HT Bio-Tek). Remaining sonicates were stored at −40°C until further use.
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DA and DOPAC levels were quantified by high-precision liquid chromatography (HPLC) using a method adopted for zebrafish (Chatterjee & Gerlai, 2009). First, sonicates were thawed and placed on ice. 1 μl of 0.5 N perchloric acid was added to each sonicate, the mixtures were vortexed and then centrifuged at 10 000 rpm for 20 minutes at 4 °C. The supernatants were collected and stored at −40°C until use. HPLC analysis was performed using a BAS 461 MICROBORE-HPLC system with a Uniget C18 reverse phase microbore column as the stationary phase. The mobile phase was a buffer (consisting of 14.5 mM sodium phosphate, 30 mM sodium citrate, 27 μM EDTA, 10 mM diethylamine hydrochloride, and 2.2 mM sodium octyl sulphate), acetonitrile and tetrahydrofuran at a ratio of 95:4:1 (pH 3.4). Supernatants were thawed and 5 μl of each was injected into the HPLC system for analysis. To serve as standards for quantification of peaks on the chromatograph, DA and DOPAC were obtained from Sigma Aldrich (Oakville, ON, CA).
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2.4 Statistical Analysis
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To compare the effects of the different THP doses on locomotor responses in Experiment 1, a two-way repeated measures ANOVA was conducted with THP concentration as the between-subject factor and time as the repeated measures factor. The dependent variable was the total distance travelled (cm) over 90 minutes of drug exposure. For Experiment 2, total distance travelled was analyzed with a three-way repeated measures ANOVA with THP treatment (0 or 50 μM THP) and alcohol exposure (0 or 1% alcohol) as the betweensubject factors, and time as the repeated measures factor. In case of significant interactions, a two-way univariate ANOVA (with THP and alcohol exposure as the between-subject factors) was conducted on total distance travelled in the last 10 minutes of exposure since Tukey’s Honestly Significant (HSD) post-hoc multiple comparison tests are not appropriate for repeated measures ANOVA. The last 10 minutes was the chosen time interval since behavioural effects are the most stable and robust due to intra-trial habituation during this period. Two-way univariate ANOVAs were also performed to analyze DA and DOPAC levels in whole zebrafish brains. Tukey’s HSD post-hoc tests were performed to compare locomotor activity, DA and DOPAC in all four groups. Significant differences were reported when the probability of null hypothesis (p) was less than 0.05.
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3. Results 3.1 Experiment 1 Locomotor activity—Zebrafish significantly changed their locomotor activity during the 90-minute testing period (F(89, 5340)= 6.61, p< 0.001, figure 1). There was also a significant main effect of THP, with THP exposure leading to reduced locomotor activity, F(3, 60)= 9.33, p< 0.001. Lastly, no significant THP × time interaction (F(267, 5340)= 0.79, p= 0.994) was found. As the inhibitory effect of THP was found to be independent of time, we compared zebrafish of the different treatment groups for the total distance travelled during the entire 90 minutes using Tukey’s HSD post-hoc multiple comparison test. This analysis showed that fish treated with 100 μM and 200 μM of THP swam significantly less than controls during the 90-minute period (p< 0.05), however, treatment with 50 μM of THP did not significantly alter locomotor activity compared to control fish (p= 0.310).
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3.2 Experiment 2
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Locomotor activity—Overall, zebrafish exhibited a time-dependent change in the total distance travelled, F(59, 6490)= 4.33, p< 0.001. The overall trajectory of this change appeared slightly different from what we saw during the first experiment, perhaps resulting from the different handling and procedures. The effect of THP was found significant (F(1, 110)= 5.94, p= 0.016) and independent of time (time × THP interaction, F(59, 6490)= 0.99, p= 0.503). In contrast, alcohol had a stimulatory effect on total distance travelled (F(1, 110)= 64.34, p< 0.001), with a significant time × alcohol interaction (F(59, 6490)= 6.17, p< 0.001), where alcohol-exposed zebrafish became increasingly hyperactive during the first 30 min of the recording/exposure session. Although no significant alcohol × THP interaction was found by repeated measures ANOVA (F(1, 110)= 2.48, p= 0.118), there was a significant THP × alcohol × time interaction (F(59, 6490)= 1.41, p= 0.021), with THP treatment attenuating the time-dependent alcohol-induced increase in locomotor activity (Figure 2). Also notably, fish that received no alcohol but received either freshwater or THP pre-treatment did not significantly differ from each other (THP treatment effect F(1, 56) = 0.546, p = 0.463; time × THP interaction F(59, 3304) = 1.212, p = 0.130).
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To further investigate the significant three-way interaction (THP × alcohol × time), a twoway univariate ANOVA was conducted on total distance travelled in the last 10 minutes of exposure (Figure 3). There was a significant inhibitory effect of THP (F(1, 110)= 6.52, p= 0.012) and stimulatory effect of alcohol treatment (F(1, 110)= 61.06, p< 0.001), with the THP × alcohol interaction approaching significance (F(1, 110)= 3.84, p= 0.053). Since ANOVA is known to be underpowered to detect interaction between main factors (Wahlsten, 1990) and since the interaction term was approaching significance, Tukey’s HSD test was conducted to compare all four groups of the 2 × 2 experimental design. The analysis revealed that fish exposed to alcohol without THP swam significantly further compared to controls, THP-treated fish, and fish treated concurrently with THP and alcohol respectively (p< 0.05), demonstrating that THP reduced alcohol-induced hyperactivity. Notably, THP exposure alone did not reduce baseline locomotor activity, as the total distance travelled did not differ significantly in THP-treated fish and controls (p= 0.974).
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Analysis of the dopaminergic neurotransmitter system—Exposure to alcohol significantly increased the levels of DA in the zebrafish brain, F(1, 15)= 53.08, p< 0.001. THP treatment, on the other hand, reduced DA levels (F(1, 15)= 29.70, p< 0.001) and there was a significant alcohol × THP interaction, F(1, 15)= 20.70, p< 0.001. Figure 4a shows that THP did not alter baseline DA in the brain: DA levels were not significantly different between THP-exposed fish and controls (p= 0.912). However, compared to controls, there was an increase in DA following alcohol treatment (p< 0.001). Furthermore, and importantly, we also found fish concurrently exposed to THP and alcohol to significantly (p < 0.001) differ from fish exposed to alcohol alone, but not from controls (p= 0.600), demonstrating a robust attenuation of alcohol-induced DA increase by THP.
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In contrast, there was no significant main effect of alcohol on DOPAC levels in the brain, F(1, 15)= 0.06, p= 0.811. Nevertheless, we found a significant main effect of THP treatment (F(1, 15)= 5.69, p= 0.031) as THP-treated fish had reduced DOPAC levels in the brain compared to fish unexposed to THP. Lastly, no significant alcohol × THP interaction was found (F(1, 15)= 0.83, p= 0.376). Although ANOVA found a significant THP induced reduction of DOPAC levels independently of alcohol treatment, Tukey HSD revealed no significant differences between the groups (p> 0.05). Results are shown in Figure 4b.
4. Discussion The current study shows that alcohol-induced hyperactivity is attenuated by the inhibition of p-TH. Although neuroanatomical locale specific changes could not be investigated due to technical limitations, our results suggests that alcohol’s actions are, at least partially, mediated by phosphorylation induced activation of tyrosine hydroxylase leading to increased DA synthesis in the zebrafish brain.
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For the first time, THP - a potent and selective inhibitor of p-TH (Yao et al., 2010) - was administered to zebrafish and found to alter behavioural responses. Zebrafish were exposed to multiple doses of THP to determine the concentration that does not reduce baseline locomotor activity and therefore selectively targets the phosphorylated form of TH over the unphosphorylated form. The two highest doses were found to reduce locomotor activity over a period of 90 minutes (Figure 1), suggesting that these concentrations may have inhibited unphosphorylated TH. It is unlikely that inhibition of movement occurred via oxidative stress in the brain, because concentrations of THP up to 15 μM have shown to be noncytotoxic (Kim et al., 2005), and the THP concentration reaching the brain was likely in the nanomolar range. The role of the dopaminergic system in movement is well established in humans and other mammals (Chinta & Andersen, 2005; Zhou & Palmiter, 1995), and these results support the importance of dopaminergic activity in zebrafish locomotion. Inhibition of basal, unphosphorylated tyrosine hydroxylase may have reduced DA synthesis and DA levels in the zebrafish brain. Less DA release and reduced binding of DA to receptors may have limited the activation of second messenger pathways which contribute to baseline motor activity. This hypothesis is supported by studies involving the inhibition of dopaminergic receptors. For example, antagonism of D1 receptors, the most abundant dopaminergic receptor subtype in the zebarfish brain, reduces baseline motor activity in zebrafish (Tran et al., 2015b).
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In Experiment 2, 60-minute exposure to 1% alcohol time-dependently increased motor activity over the first 30 minutes of exposure, and activity levels plateaued and remained high over the subsequent 30 minutes. This effect is consistent with previous findings (Tran & Gerlai, 2013a). Zebrafish were pre-treated with the THP dose that did not alter baseline locomotor activity in Experiment 1 (50 μM). This was done to ensure that any attenuation in alcohol-induced hyperactivity was not an additive effect of independent locomotor inhibition by THP exposure. In addition, during the 60-minute exposure, zebrafish that were pre-treated with THP were exposed to 1% alcohol concurrently with same dose of THP as the previous treatment. This functioned to eliminate any confounding effects of THP withdrawal and to ensure continued enzymatic inhibition. We found that inhibition of p-TH by THP blunted the time-dependent increase of locomotor activity induced by alcohol exposure, and this attenuation was maintained in the last 10 minutes of alcohol exposure (Figures 2, 3).
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We found that 60-minute exposure to 1% alcohol increased DA levels in the zebrafish brain, a result that corroborates the findings of previous studies using the same dose and duration of exposure (Chatterjee & Gerlai, 2009; Tran et al., 2015a). Importantly, THP treatment did not reduce baseline DA levels. Under basal conditions, DA-producing cells contain traces of p-TH (Pocotte et al., 1986). Thus, exposure to THP at a concentration that selectively targets p-TH does not inhibit basal levels of unphosphorylated TH and likely as a result, baseline DA is not affected. Following certain physiologically relevant stimuli, TH is phosphorylated and this results in increased enzymatic activity and, as a consequence, increased DA production. The current results demonstrate that alcohol-induced increase of dopamine levels is blocked by THP, suggesting that the alcohol induced increase of dopamine synthesis is due to phosphorylation induced activation of TH in the zebrafish brain.
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It has been previously shown that alcohol stimulates locomotor activity at doses that increase brain DOPAC levels in both zebrafish and rodents (Tran et al., 2015a; Imperato & Di Chiara, 1986). In the current study, alcohol was not found to increase DOPAC levels (Figure 4b). Compared to previous studies (Tran et al., 2015; Chatterjee & Gerlai, 2009), brain DOPAC levels were high in zebrafish from all groups, including controls, and it is possible that a ceiling effect prevented the detection of further increases in brain DOPAC levels following alcohol exposure. Overall, THP treatment was found to reduce DOPAC in the zebrafish brain, however, no significant differences were detected between groups. The reduction in DOPAC by THP treatment is unlikely to be a result of off-target enzymatic inhibition by THP. It has previously been shown that THP does not inhibit the activity of enzymes involved in DA metabolism, including MAO-A, MAO-B and ALDH2 (Yao et al., 2010). Nevertheless, it is possible that DA metabolizing enzymes are sensitive to DA levels, and may have adjusted their activity to reduce DA metabolism in response to THP. Figure 2 demonstrates that during the first 20 minutes of alcohol exposure, both control fish that were pre-treated with system water and THP-pre-treated fish exhibited a rapid rise in locomotor activity. After 20 minutes of alcohol exposure, locomotor activity of controls continued to increase and remained elevated up to 60 minutes of alcohol treatment. However, while the initial alcohol induced locomotor activation was observable in fish with or without THP-pretreatment, total distance travelled stopped rising after 20 min in the THP
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pre-treated fish (Figure 2). The findings demonstrate that inhibition of p-TH prevents further increases in motor activity and prevents the maintenance of heightened activity levels in zebrafish during alcohol exposure. This suggests that the initial alcohol-induced increase in motor activity is perhaps independent of TH phosphorylation, and increased synthesis of DA comes into play later on during alcohol exposure. It has been previously shown in zebrafish that during immersion in 1% alcohol, the brain alcohol concentration rapidly rises during the first 20-30 minutes of exposure, after which it stabilizes (Tran et al., 2015a). Rising alcohol concentrations have been suggested to cause a release of DA from synaptic vesicles into the synaptic cleft, which may underlie the rapid, and TH-phosphorylation independent, increase in locomotion during the first 20 minutes of ethanol exposure. However, following neurotransmitter release and breakdown of DA, activation of TH may need to occur to maintain heightened DA levels in the brain. This is supported by the finding that release of catecholamines is not followed by decreases in their levels within tissues due to an increase in the rate of catecholamine synthesis that is closely coupled to neurotransmitter release (Zigmond et al., 1989). Thus, it appears that alcohol’s locomotor stimulant effects are mediated, at least partially, by an increased production of DA but only after alcohol has reached stable concentrations in the brain. Although activity of TH can be modulated by other mechanisms (i.e. transcriptional and translational regulation) (Dunkley et al., 2004), the significant attenuation of the alcohol-induced DA increase and motor hyperactivity by THP suggests that TH phosphorylation may be one of the primary mechanisms responsible for the increase of DA levels following alcohol exposure. Moreover, it suggests that heightened DA levels are closely associated with alcohol’s motor activating effects.
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Many neurotransmitter systems have been implicated in alcohol’s mechanism of action, however, the dopaminergic system has been argued to be a primary system involved in both the stimulatory and rewarding properties of alcohol (Gilpin & Koob, 2008; Phillips & Shen, 1996). More importantly, sensitivity to the stimulant properties of alcohol has been identified as a risk factor for alcohol addiction (King et al., 2011, 2014). The current study was based on the hypothesis that alcohol may increase locomotor activity through mechanisms related to reinforcement by other drugs of abuse, such as cocaine. The ability to block the alcohol-induced DA increase and hyperactivity using THP closely parallels the prevention of cocaine-seeking behaviour by THP. Similar to alcohol, cocaine elevates DA levels in the brain and induces addictive behaviours (i.e. cocaine self-administration). In rats, THP inhibits the cocaine-stimulated DA production at concentrations targeting p-TH, without reducing basal DA production. The ability of THP to block the effects of two different psychoactive drugs in two entirely different species suggests that the stimulatory and rewarding properties of drugs of abuse may be partially mediated by a common evolutionarily conserved molecular pathway. The implications of such a finding are noteworthy for two reasons. One, common underpinnings of addiction can be exploited for the development of more effective treatments for many addictive disorders (Nestler, 2005), a goal in addiction research that is already beginning to be explored. For example, disulfiram, which is prescribed to treat alcoholism, has been shown to also reduce cocaine use (Suh et al., 2006). Two, conserved mechanisms and our ability to study them in a simple vertebrate such as zebrafish opens the possibility of the development of efficient and low cost animal models of complex human disorders including drug addiction.
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Acknowledgements RG supported by NSERC Discovery Grant (311637) and NIH/NIAAA (R01 AA14357-01A2), ST supported by NSERC Graduate Scholarship.
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Figure 1.
THP dose dependently reduces locomotor activity of zebrafish. Total distance experimental zebrafish swam (cm) is shown as a function of time (1-minute intervals) during a 90-minute acute exposure to different concentrations of THP (0, 50, 100, 200 μM external bath concentration). Means ± S.E.M. are shown. Note the lack of significant difference between control and 50μM THP exposed fish.
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Figure 2.
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Alcohol induced increase of total distance travelled (cm) is attenuated by THP pre-treatment during a 60-minute drug exposure/behavioural observation session. Means ± S.E.M. are shown. Note the robust hyperactivity of fish that received 1% alcohol alone (light grey squares) and the significant attenuation of this hyperactivity in fish that received alcohol and THP concurrently (black squares). Also note the lack of significant difference between control fish (0 alcohol and 0 THP, white squares) and fish that received 50 μM THP but no alcohol (dark grey squares).
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Figure 3.
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Alcohol elevates activity and THP pre-treatment attenuates the hyperactivity inducing effects of alcohol during the last ten minutes of the drug exposure/recording session. Mean ± S.E.M. are shown for the total distance travelled (cm) in the last 10 minutes of a 60-minute exposure session. The treatment conditions are indicated by the legends (THP concentration) and the X-axis labels (alcohol concentration) for the four treatment groups: (1) system water; (2) 1% ethanol; (3) 50 μM THP; and (4) 1% ethanol + 50 μM THP. Fish in treatment conditions (1) and (2) were pre-treated with system water for 30 minutes, and fish in conditions (3) and (4) were pre-treated with 50 μM THP for 30 minutes. Significant differences (p< 0.05) among groups are indicated by the letters above the bars. Bars that do not share the same letter designation are significantly different.
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The level of dopamine in the brain of zebrafish is increased by alcohol but pre-treatment with THP abolishes this alcohol effect. Mean ± S.E.M are shown for dopamine (panel A) and DOPAC (panel B) (expressed as ng neurochemical/mg brain protein) analyzed from whole brain tissue obtained from fish following a 60-minute exposure to four different treatments: (1) system water; (2) 1% ethanol; (3) 50 μM THP; and (4) 1% ethanol + 50 μM THP (THP concentrations are indicated by the legends and alcohol concentrations by the Xaxis labels). Fish in treatment conditions (1) and (2) were pre-treated with system water for 30 minutes, and fish in conditions (3) and (4) were pre-treated with 50 μM THP for 30 minutes. Significant differences (p< 0.05) among groups are indicated by the letters above the bars. Bars that do not share the same letter are significantly different.
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Pharmacol Biochem Behav. Author manuscript; available in PMC 2016 November 01. Published in final edited form as: Pharmacol Biochem Behav. 2015 November ; 138: 32–39. doi:10.1016/j.pbb.2015.09.008.
Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio) Magda Nowicki1, Steven Tran2, Diptendu Chatterjee1, and Robert Gerlai1,2,* 1Department
of Psychology,University of Toronto Mississauga
2Department
of Cell and System Biology, University of Toronto
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Abstract
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Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine’s metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol’s locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.
Keywords alcohol; ethanol; dopamine; locomotor activity; tyrosine hydroxylase; zebrafish
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1. Introduction Alcoholism is a debilitating disorder associated with frequent or prolonged alcohol consumption (Hemmingsson & Lundberg, 2001). Understanding the neurobiological mechanisms of compulsive alcohol use may lead to the development of efficacious treatment for this disorder (Gilpin & Koob, 2008). Commonly classified as a sedative drug,
*
Corresponding author at: Department of Psychology, University of Toronto Mississauga, 3359 Mississauga Road North, Rm CCT4004, Mississauga, Ontario L5L 1C6, Canada, Tel: 905-569-4255 (office), Tel: 905-569-4257 (lab), Fax: 905-569-4326, [email protected].
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alcohol (ethanol) exerts biphasic effects across both dose and time. It has stimulant effects in humans and other animals following low to moderate doses, while high doses induce sedation (Addicott et al., 2007; Rodd et al., 2006; Tambour et al., 2006). The timedependent biphasic effect of alcohol has been associated with changes in blood alcohol concentration. In humans, a single acute dose of alcohol induces stimulation during the ascending trajectory of the blood alcohol curve (BAC) and sedation during the descending trajectory of the BAC (Holdstock & de Wit, 1998). Similar time-dependent biphasic effects of alcohol have been shown in other mammalian species (Read et al., 1960) and now in zebrafish too (Tran & Gerlai, 2013a).
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Sensitivity to the stimulant effects of alcohol is considered a risk factor for excessive alcohol consumption that may eventually lead to alcohol dependence (King et al., 2011, 2014). Therefore, the investigation of the stimulatory effects of alcohol is hoped to lead to the development of treatments or early interventions. Animal models have greatly improved our understanding of alcohol dependence because of the behavioural and pharmacological similarities of alcohol’s effects across humans and many other species (Brabant et al., 2014).
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The zebrafish (Danio rerio) is a small freshwater teleost that is rapidly emerging as an important animal model in many research areas including developmental biology, biomedical research and more recently, behavioural neuroscience (Zon & Peterson, 2005; Dawid, 2004; Gerlai, 2011). Zebrafish share many similarities with humans and other mammals on the genetic, molecular, and neuronal level (Barbazuk et al., 2000; Rico et al., 2011). The zebrafish genome has been sequenced, and zebrafish genes have been found to possess high nucleotide sequence similarity with human homologues (Barbazuk et al., 2000). In addition, conserved neurotransmitter systems in zebrafish permit the elucidation of molecular mechanisms underlying drug effects, neurological diseases or psychopathology (Rico et al., 2011). Besides the opportunity for translational research, there are practical advantages to studying this small vertebrate. Zebrafish breed frequently and produce large clutches of eggs (up to 300 eggs per female per spawning). Their small body size (4 cm long when full grown) allows their space efficient maintenance in the laboratory, and their high tolerance to a range of water conditions makes them a robust species even under artificial conditions (Gerlai, 2015).
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The zebrafish may be particularly suitable for the analysis of the mechanisms of in vivo actions of alcohol (Chatterjee et al., 2014; Puttonen et al., 2013; Tran & Gerlai, 2013a; Tran et al., 2015a) for many reasons. First, alcohol can be administered by immersing zebrafish into the alcohol solution, a non-invasive and practical alternative to injections or vapour inhalation employed in rodents (Tran et al., 2015a). Zebrafish also exhibit robust time- and dose-dependent behavioural responses to alcohol that are comparable to those described in humans and other mammals. For example, a 60-minute acute exposure to low or moderate concentrations of alcohol (up 1% v/v) induces a dose-dependent increase in locomotor activity in adult zebrafish, and reduces anxiety-like behavioural responses (Tran et al., 2015a). On the other hand, higher concentrations of alcohol induce sedative effects. For example, exposure to 3% alcohol immediately decreases swimming speed in zebrafish larvae (Puttonen et al., 2013). Adult zebrafish also exhibit temporal changes in motor activity in response to exposure to 1% alcohol. Motor activity (quantified by the total
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distance travelled) rises and peaks around 20-30 minutes after the start of alcohol treatment. Between 30-60 minutes, zebrafish motor activity remains elevated but it begins to gradually decline (Tran & Gerlai, 2013a). Like in mammals, motor activity also strongly correlates with brain alcohol concentration in zebrafish (Tran et al., 2015a).
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The neural underpinnings of acute alcohol exposure are complex and have not yet been entirely deciphered due to the involvement of numerous neurotransmitter systems and signal transduction mechanisms. It has been suggested that a common neurobiological mechanism, the dopaminergic system in the mesolimbic dopaminergic system, mediates the reinforcing and stimulant effects of addictive drugs, including alcohol (Brabant et al., 2014). Like other major drugs of abuse, alcohol has been shown to target the dopaminergic system in humans and other mammals (Boileau et al., 2003; Tang et al., 2003). However, the precise mechanism by which dopamine (DA) regulates acute alcohol responses and addiction remains elusive. Acute alcohol exposure has been shown to increase extracellular brain DA concentrations in rats (Di Chiara & Imperato, 1988) and levels of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in mice (Saeed & Wooles, 1984). The increase in DA levels has been proposed to underlie the stimulating properties of alcohol, as DA depletion and DA receptor antagonists block alcohol-induced locomotor activity (Bainton et al., 2000; Camarini et al., 2011). Furthermore, alcohol causes a dose-dependent accumulation of 3, 4-dihydroxyphenylalanine (DOPA) after inhibition of DOPA decarboxylase in the brains of mice (Saeed & Wooles, 1984).
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The rate limiting enzyme of DA synthesis is tyrosine hydroxylase (TH), which catalyzes the hydroxylation of tyrosine to form DOPA. Based on these findings, increased concentrations of DA may be a result of increased DA synthesis in response to alcohol exposure. This hypothesis has been supported by studies in mice that have suppressed the motor activating effects of alcohol through pharmacological inhibition of TH (Carlsson et al., 1972; Engel et al., 1974).
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The zebrafish may be appropriate for the analysis of the behavioural stimulatory effects of alcohol. Areas of the zebrafish telencephalon are thought to be homologous to regions of the mammalian basal ganglia, a crucial region involved in movement. Based on axonal projections, neurochemistry and gene expression, the dorsal nucleus of the ventral telencephalon is suggested to be the zebrafish homologue of the mammalian striatum. The area is rich in dopaminergic nerve terminals projecting from well-defined groups of dopaminergic nuclei in the telencephalon and diencephalon (Rink & Wullimann, 2004). Two putative TH genes have been identified in zebrafish: th1 and th2, however, th1 has been found to be most closely related to the mammalian TH (Candy & Collet, 2005). Similarly to mammals, activation of dopaminergic neurotransmission has been found to mediate alcohol’s stimulatory behavioural effects also in zebrafish. 60-minute exposure to alcohol (doses ranging from 0.25 to 1.00% v/v) has been shown to induce a dose-dependent increase in DA, DOPAC and TH activity in the zebrafish brain corresponding closely to the dosedependent increase found in motor activity (Tran et al., 2015a). TH activity increases in response to acute alcohol exposure in both mammals (Saeed & Wooles, 1984) and zebrafish (Chatterjee et al., 2014). However, the exact relationship
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between TH and the stimulant effects of alcohol is not well understood. An increase in DA synthesis in response to acute alcohol exposure may be a result of two distinct mechanisms: upregulation of gene expression of TH or activation of the enzyme. Acute alcohol increases expression of TH mRNA, for example, in the ventral tegmental area and substantia nigra of rats after 1 hour of alcohol administration (Oliva et al., 2007). A similar response to acute alcohol has been found in zebrafish larvae, in which a 10-minute long acute alcohol exposure increased swimming distance and levels th1 mRNA in the brain (Puttonen et al., 2013). On the other hand, cocaine was found to increase synaptic DA by binding to the DA transporter, and a positive feedback loop resulted in the phosphorylation of TH, and the thus activated TH further increased DA levels (Yao et al., 2010).
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DA is metabolized by two enzymes consecutively: monoamine oxidase A (MAO-A) or monoamine oxidase B (MAO-B), and aldehyde dehydrogenase 2 (ALDH2). First, DA is deaminated by MAO-A or MAO-B to 3, 4-dihydroxyphenylacetaldehyde (DOPAL), a transient and reactive intermediate, that is further metabolized to DOPAC by ALDH2 (Yao et al., 2010). Recently, a highly selective ALDH2 inhibitor was found to suppress cocaine self-administration and cocaine-seeking behaviour in rats by increasing DOPAL, enabling it to condense with DA to form tetrahydropapaveroline (THP). THP is an endogenous inhibitor of phosphorylated TH (p-TH) (approximately 75 times more selective for p-TH than unphosphorylated TH) and blocks the cocaine-induced increase in DA production (Yao et al., 2010). In addition to blocking cocaine addictive behaviour, the ALDH2 inhibitor has been shown to suppress alcohol intake and alcohol seeking in heavy drinking rats (Arolfo et al., 2009). It also prevents the alcohol-induced DA increase in the nucleus accumbens, without altering baseline DA levels (Arolfo et al., 2009) suggesting that the alcohol-induced DA increase may be mediated by phosphorylation of TH.
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Currently, it is unknown whether activation of TH by phosphorylation could underlie alcohol’s locomotor stimulant effects in addition to its addictive properties. To test this hypothesis, we investigated whether selective inhibition of p-TH by THP can block alcoholinduced hyperactivity in zebrafish and alcohol induced increase of DA levels in the brain of zebrafish. First, we characterized the dose-dependent effects of THP on zebrafish motor responses. Subsequently, we treated zebrafish with the highest dose of THP that did not alter baseline motor activity. The THP pre-treated zebrafish were then exposed to a dose of alcohol that would exert robust behavioural and neurochemical effects. We hypothesized that THP pre-treatment would attenuate the alcohol-induced increase of brain DA levels and locomotor activity. In addition, we also examined DOPAC levels in the brain following THP and alcohol treatment to investigate the effects of p-TH inhibition on downstream DA metabolism.
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2. Methods 2.1 Animal Housing 197 sexually mature, 10 month old (young adult), male and female zebrafish (Danio rerio) of the AB strain were used in this study. The AB strain was selected since it is most frequently studied and is genetically well-characterized, showing homozygosity at over 80% of its loci (Guryev et al., 2006). Furthermore, behavioural and neurochemical responses to Pharmacol Biochem Behav. Author manuscript; available in PMC 2016 November 01.
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alcohol have been thoroughly investigated for this strain (Chatterjee & Gerlai, 2009; Tran et al., 2015a; Tran & Gerlai, 2013a). Fish were bred and raised in the vivarium of the University of Toronto Mississauga (Mississauga, Ontario, Canada). Animals were housed in 37 L tanks with mechanical filters containing approximately 20 fish per tank. Zebrafish were kept on a 13 hour light-dark cycle with lights turning on at 08:00h and off at 21:00h. Water quality parameters were maintained at optimal conductivity levels (100-300 microsiemens), temperature (28°C-30°C), and pH (6.8-7.2). Upon hatching to 3 weeks post-fertilization, larvae were fed Larval AP 100. After 3 weeks post-fertilization, fish were fed twice a day, alternating between brine shrimp (Artemia salina) and a mixture of 2 parts flake food (Scientific Hatcheries Diet) and 1 part powdered spirulina. 2.2 Experiment 1
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Locomotor activity—The purpose of Experiment 1 was to characterize the dosedependent locomotor response of zebrafish to multiple concentrations of THP with the goal of determining the concentration that does not alter baseline motor responses. THP was delivered to zebrafish by immersing them in tank water containing the appropriate concentration of the compound. Immersion-based drug delivery was used because of its less invasive nature compared to other methods of administration (e.g. injection) and frequent use in adult zebrafish research (Nowicki et al., 2014; Tran et al., 2015b; Levin et al., 2007; Conners et al., 2014; Sackerman et al., 2010).
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THP (Santa Cruz Biotechnology, Inc., Dallas, TX, U.S.A.) was stored at 4°C until use. A stock solution of THP (7 mM) was prepared by diluting 128.8 mg of THP in 50 mL of distilled water. 1.5 L tanks were filled with 1 L of system water supplemented with 100 mg/L InstantOcean Sea Salt to adjust conductivity and salinity to appropriate levels as employed before (e.g. Tran et al., 2015a; 2015b). The THP stock solution was diluted in the tanks to achieve final bath concentrations of 50, 100 and 200 μM. No THP was added to the control tank, which only contained system water. The concentrations of THP were chosen to selectively inhibit the phosphorylated form of TH. The lowest concentration of THP was determined by multiplying the binding affinity of the inhibitor (Ki) by 1000 based on a previous study that determined 1/1000th of an external drug concentration in the water bath reaches the zebrafish brain (Sackerman et al., 2010). Higher doses were multiples of the lowest THP concentration by a factor of 2.
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Individual zebrafish were exposed to THP (n= 16 per group) in 1.5 L tanks for 90 minutes, a session length that corresponded to the total exposure session length employed in experiment 2. Water quality parameters (conductivity, temperature and pH) matched those of housing tanks. Video recordings from the front of the tanks were taken during drug exposure for subsequent behavioural quantification. All testing tanks were made of transparent plexi-glass with the lateral and back sides covered with white corrugated plastic to provide a uniform environment and obscure external cues during testing. Behavioural recordings during drug exposure were analyzed using automated video-tracking software, EthoVision XT 8.0. The dependent variable quantified was total distance travelled (cm), a measure of locomotor activity in zebrafish.
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2.3 Experiment 2
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Locomotor activity—Experiment 2 intended to determine whether pre-treatment with THP inhibits the ethanol-induced increases in locomotor activity and DA levels. Zebrafish used in Experiment 2 (n=114) were first repeatedly exposed to the dosing container in order to reduce anxiety induced by the novel handling procedure and environment. These predosing trials were conducted by placing individual fish in 1.5 L tanks containing system water for a 5 minute period. Fish were subsequently removed from the tank with a net and immediately transferred into another 1.5 L tank containing system water for 5 minutes. All zebrafish in Experiment 2 underwent three pre-dosing trials prior to testing, a procedure that was based upon prior results (Tran & Gerlai, 2013b). One pre-dosing session was performed per day, and the experiment was performed on the day following the last pre-dosing session.
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The highest THP concentration that did not alter baseline motor activity in Experiment 1 was determined to be 50 μM. In Experiment 2 we employed a 2 × 2 between subject factorial design with THP pre-treatment (0 or 50 μM) and ethanol exposure (0 or 1%) as the between subject factors. Furthermore, zebrafish that were exposed to THP first, were also exposed to THP concurrently during the ethanol exposure phase to ensure continued enzyme inhibition and to avoid potential confounding effects of THP withdrawal. The sample size (n) for each of the four drug conditions was 26-28. 1.5 L tanks were filled with 1 L of system water supplemented with 100 mg/L of Instant Ocean Sea Salt. Water quality parameters were ensured to match those of the housing tanks (temperature, pH and conductivity). A 5 mM stock solution of THP (73.6 mg in 40 ml of distilled water) was prepared and further diluted to 50 μM in the appropriate tanks using system water. Similarly, 100% anhydrous ethyl alcohol (Commercial Alcohols, Brampton, ON, CA) was diluted to achieve a final concentration of 1% in the required tanks.
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Zebrafish were individually pre-treated with 50 μM of THP or system water for 30 minutes in the 1.5 L tanks. System water pre-treated fish were immediately transferred to tanks containing system water or 1% ethanol for 60 minutes. THP pre-treated fish were transferred to tanks containing 50 μM THP or tanks containing 1% ethanol and 50 μM THP for 60 minutes. The length of exposure to THP and to ethanol was determined by pilot studies and was also based upon data published previously on zebrafish (e.g. Tran 3t ail, 2015a). Video recordings were taken from the front of the testing tanks during the 60 minute exposure. The he frontal view allows better video-detection as the fish appear brighter and more distinguishable from the background, and this method has been shown to allow psychopharmacological analysis and quantification of a number of natural behavioural responses in zebrafish (Nowicki et al., 2014). EthoVision XT 8.0 (Noldus Info Tech, Wageningen, The Netherlands) was used to analyze the behavioural recordings and quantify the total distance travelled (cm) fish swam. Analysis of the dopaminergic system—Whole brain DA and DOPAC levels were measured from the four treatment groups of zebrafish (n= 4-5 per group). Following the 60 minute exposure, zebrafish were sacrificed by decapitation and their heads were stored at −40°C until processing. Dopaminergic neurons of different brain regions are not expected to respond in opposite manner to the drug treatment employed here and due to this, and
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because the brain of the zebrafish is rather small, we chose to analyze whole brain extracts instead of attempting to sample separate brain areas. Whole brains were dissected and placed individually into 1.5 ml micro-centrifuge tubes on ice. Brains were sonicated in 10 μl of 0.025 M ascorbic acid in artificial cerebrospinal fluid and sonicates were used for a subsequent protein assay in order to standardize measures of DA and DOPAC. 1 μL of each sonicate and 1.5 ml of protein assay reagent were added to individual 5 ml test tubes. The reagent was prepared by a 1:4 dilution of Coomassie brilliant blue G-250 dye in distilled water. Dilutions of 2 μg/ml bovine serum albumin were used as protein standards. The test tubes were then incubated at room temperature for 20 minutes and 200 μl of the mixtures were transferred to individual wells of a 96 well, 330 μl microplate. The optical density at 595 nm was measured using a microplate reader (Synergy-HT Bio-Tek). Remaining sonicates were stored at −40°C until further use.
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DA and DOPAC levels were quantified by high-precision liquid chromatography (HPLC) using a method adopted for zebrafish (Chatterjee & Gerlai, 2009). First, sonicates were thawed and placed on ice. 1 μl of 0.5 N perchloric acid was added to each sonicate, the mixtures were vortexed and then centrifuged at 10 000 rpm for 20 minutes at 4 °C. The supernatants were collected and stored at −40°C until use. HPLC analysis was performed using a BAS 461 MICROBORE-HPLC system with a Uniget C18 reverse phase microbore column as the stationary phase. The mobile phase was a buffer (consisting of 14.5 mM sodium phosphate, 30 mM sodium citrate, 27 μM EDTA, 10 mM diethylamine hydrochloride, and 2.2 mM sodium octyl sulphate), acetonitrile and tetrahydrofuran at a ratio of 95:4:1 (pH 3.4). Supernatants were thawed and 5 μl of each was injected into the HPLC system for analysis. To serve as standards for quantification of peaks on the chromatograph, DA and DOPAC were obtained from Sigma Aldrich (Oakville, ON, CA).
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2.4 Statistical Analysis
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To compare the effects of the different THP doses on locomotor responses in Experiment 1, a two-way repeated measures ANOVA was conducted with THP concentration as the between-subject factor and time as the repeated measures factor. The dependent variable was the total distance travelled (cm) over 90 minutes of drug exposure. For Experiment 2, total distance travelled was analyzed with a three-way repeated measures ANOVA with THP treatment (0 or 50 μM THP) and alcohol exposure (0 or 1% alcohol) as the betweensubject factors, and time as the repeated measures factor. In case of significant interactions, a two-way univariate ANOVA (with THP and alcohol exposure as the between-subject factors) was conducted on total distance travelled in the last 10 minutes of exposure since Tukey’s Honestly Significant (HSD) post-hoc multiple comparison tests are not appropriate for repeated measures ANOVA. The last 10 minutes was the chosen time interval since behavioural effects are the most stable and robust due to intra-trial habituation during this period. Two-way univariate ANOVAs were also performed to analyze DA and DOPAC levels in whole zebrafish brains. Tukey’s HSD post-hoc tests were performed to compare locomotor activity, DA and DOPAC in all four groups. Significant differences were reported when the probability of null hypothesis (p) was less than 0.05.
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3. Results 3.1 Experiment 1 Locomotor activity—Zebrafish significantly changed their locomotor activity during the 90-minute testing period (F(89, 5340)= 6.61, p< 0.001, figure 1). There was also a significant main effect of THP, with THP exposure leading to reduced locomotor activity, F(3, 60)= 9.33, p< 0.001. Lastly, no significant THP × time interaction (F(267, 5340)= 0.79, p= 0.994) was found. As the inhibitory effect of THP was found to be independent of time, we compared zebrafish of the different treatment groups for the total distance travelled during the entire 90 minutes using Tukey’s HSD post-hoc multiple comparison test. This analysis showed that fish treated with 100 μM and 200 μM of THP swam significantly less than controls during the 90-minute period (p< 0.05), however, treatment with 50 μM of THP did not significantly alter locomotor activity compared to control fish (p= 0.310).
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3.2 Experiment 2
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Locomotor activity—Overall, zebrafish exhibited a time-dependent change in the total distance travelled, F(59, 6490)= 4.33, p< 0.001. The overall trajectory of this change appeared slightly different from what we saw during the first experiment, perhaps resulting from the different handling and procedures. The effect of THP was found significant (F(1, 110)= 5.94, p= 0.016) and independent of time (time × THP interaction, F(59, 6490)= 0.99, p= 0.503). In contrast, alcohol had a stimulatory effect on total distance travelled (F(1, 110)= 64.34, p< 0.001), with a significant time × alcohol interaction (F(59, 6490)= 6.17, p< 0.001), where alcohol-exposed zebrafish became increasingly hyperactive during the first 30 min of the recording/exposure session. Although no significant alcohol × THP interaction was found by repeated measures ANOVA (F(1, 110)= 2.48, p= 0.118), there was a significant THP × alcohol × time interaction (F(59, 6490)= 1.41, p= 0.021), with THP treatment attenuating the time-dependent alcohol-induced increase in locomotor activity (Figure 2). Also notably, fish that received no alcohol but received either freshwater or THP pre-treatment did not significantly differ from each other (THP treatment effect F(1, 56) = 0.546, p = 0.463; time × THP interaction F(59, 3304) = 1.212, p = 0.130).
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To further investigate the significant three-way interaction (THP × alcohol × time), a twoway univariate ANOVA was conducted on total distance travelled in the last 10 minutes of exposure (Figure 3). There was a significant inhibitory effect of THP (F(1, 110)= 6.52, p= 0.012) and stimulatory effect of alcohol treatment (F(1, 110)= 61.06, p< 0.001), with the THP × alcohol interaction approaching significance (F(1, 110)= 3.84, p= 0.053). Since ANOVA is known to be underpowered to detect interaction between main factors (Wahlsten, 1990) and since the interaction term was approaching significance, Tukey’s HSD test was conducted to compare all four groups of the 2 × 2 experimental design. The analysis revealed that fish exposed to alcohol without THP swam significantly further compared to controls, THP-treated fish, and fish treated concurrently with THP and alcohol respectively (p< 0.05), demonstrating that THP reduced alcohol-induced hyperactivity. Notably, THP exposure alone did not reduce baseline locomotor activity, as the total distance travelled did not differ significantly in THP-treated fish and controls (p= 0.974).
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Analysis of the dopaminergic neurotransmitter system—Exposure to alcohol significantly increased the levels of DA in the zebrafish brain, F(1, 15)= 53.08, p< 0.001. THP treatment, on the other hand, reduced DA levels (F(1, 15)= 29.70, p< 0.001) and there was a significant alcohol × THP interaction, F(1, 15)= 20.70, p< 0.001. Figure 4a shows that THP did not alter baseline DA in the brain: DA levels were not significantly different between THP-exposed fish and controls (p= 0.912). However, compared to controls, there was an increase in DA following alcohol treatment (p< 0.001). Furthermore, and importantly, we also found fish concurrently exposed to THP and alcohol to significantly (p < 0.001) differ from fish exposed to alcohol alone, but not from controls (p= 0.600), demonstrating a robust attenuation of alcohol-induced DA increase by THP.
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In contrast, there was no significant main effect of alcohol on DOPAC levels in the brain, F(1, 15)= 0.06, p= 0.811. Nevertheless, we found a significant main effect of THP treatment (F(1, 15)= 5.69, p= 0.031) as THP-treated fish had reduced DOPAC levels in the brain compared to fish unexposed to THP. Lastly, no significant alcohol × THP interaction was found (F(1, 15)= 0.83, p= 0.376). Although ANOVA found a significant THP induced reduction of DOPAC levels independently of alcohol treatment, Tukey HSD revealed no significant differences between the groups (p> 0.05). Results are shown in Figure 4b.
4. Discussion The current study shows that alcohol-induced hyperactivity is attenuated by the inhibition of p-TH. Although neuroanatomical locale specific changes could not be investigated due to technical limitations, our results suggests that alcohol’s actions are, at least partially, mediated by phosphorylation induced activation of tyrosine hydroxylase leading to increased DA synthesis in the zebrafish brain.
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For the first time, THP - a potent and selective inhibitor of p-TH (Yao et al., 2010) - was administered to zebrafish and found to alter behavioural responses. Zebrafish were exposed to multiple doses of THP to determine the concentration that does not reduce baseline locomotor activity and therefore selectively targets the phosphorylated form of TH over the unphosphorylated form. The two highest doses were found to reduce locomotor activity over a period of 90 minutes (Figure 1), suggesting that these concentrations may have inhibited unphosphorylated TH. It is unlikely that inhibition of movement occurred via oxidative stress in the brain, because concentrations of THP up to 15 μM have shown to be noncytotoxic (Kim et al., 2005), and the THP concentration reaching the brain was likely in the nanomolar range. The role of the dopaminergic system in movement is well established in humans and other mammals (Chinta & Andersen, 2005; Zhou & Palmiter, 1995), and these results support the importance of dopaminergic activity in zebrafish locomotion. Inhibition of basal, unphosphorylated tyrosine hydroxylase may have reduced DA synthesis and DA levels in the zebrafish brain. Less DA release and reduced binding of DA to receptors may have limited the activation of second messenger pathways which contribute to baseline motor activity. This hypothesis is supported by studies involving the inhibition of dopaminergic receptors. For example, antagonism of D1 receptors, the most abundant dopaminergic receptor subtype in the zebarfish brain, reduces baseline motor activity in zebrafish (Tran et al., 2015b).
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In Experiment 2, 60-minute exposure to 1% alcohol time-dependently increased motor activity over the first 30 minutes of exposure, and activity levels plateaued and remained high over the subsequent 30 minutes. This effect is consistent with previous findings (Tran & Gerlai, 2013a). Zebrafish were pre-treated with the THP dose that did not alter baseline locomotor activity in Experiment 1 (50 μM). This was done to ensure that any attenuation in alcohol-induced hyperactivity was not an additive effect of independent locomotor inhibition by THP exposure. In addition, during the 60-minute exposure, zebrafish that were pre-treated with THP were exposed to 1% alcohol concurrently with same dose of THP as the previous treatment. This functioned to eliminate any confounding effects of THP withdrawal and to ensure continued enzymatic inhibition. We found that inhibition of p-TH by THP blunted the time-dependent increase of locomotor activity induced by alcohol exposure, and this attenuation was maintained in the last 10 minutes of alcohol exposure (Figures 2, 3).
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We found that 60-minute exposure to 1% alcohol increased DA levels in the zebrafish brain, a result that corroborates the findings of previous studies using the same dose and duration of exposure (Chatterjee & Gerlai, 2009; Tran et al., 2015a). Importantly, THP treatment did not reduce baseline DA levels. Under basal conditions, DA-producing cells contain traces of p-TH (Pocotte et al., 1986). Thus, exposure to THP at a concentration that selectively targets p-TH does not inhibit basal levels of unphosphorylated TH and likely as a result, baseline DA is not affected. Following certain physiologically relevant stimuli, TH is phosphorylated and this results in increased enzymatic activity and, as a consequence, increased DA production. The current results demonstrate that alcohol-induced increase of dopamine levels is blocked by THP, suggesting that the alcohol induced increase of dopamine synthesis is due to phosphorylation induced activation of TH in the zebrafish brain.
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It has been previously shown that alcohol stimulates locomotor activity at doses that increase brain DOPAC levels in both zebrafish and rodents (Tran et al., 2015a; Imperato & Di Chiara, 1986). In the current study, alcohol was not found to increase DOPAC levels (Figure 4b). Compared to previous studies (Tran et al., 2015; Chatterjee & Gerlai, 2009), brain DOPAC levels were high in zebrafish from all groups, including controls, and it is possible that a ceiling effect prevented the detection of further increases in brain DOPAC levels following alcohol exposure. Overall, THP treatment was found to reduce DOPAC in the zebrafish brain, however, no significant differences were detected between groups. The reduction in DOPAC by THP treatment is unlikely to be a result of off-target enzymatic inhibition by THP. It has previously been shown that THP does not inhibit the activity of enzymes involved in DA metabolism, including MAO-A, MAO-B and ALDH2 (Yao et al., 2010). Nevertheless, it is possible that DA metabolizing enzymes are sensitive to DA levels, and may have adjusted their activity to reduce DA metabolism in response to THP. Figure 2 demonstrates that during the first 20 minutes of alcohol exposure, both control fish that were pre-treated with system water and THP-pre-treated fish exhibited a rapid rise in locomotor activity. After 20 minutes of alcohol exposure, locomotor activity of controls continued to increase and remained elevated up to 60 minutes of alcohol treatment. However, while the initial alcohol induced locomotor activation was observable in fish with or without THP-pretreatment, total distance travelled stopped rising after 20 min in the THP
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pre-treated fish (Figure 2). The findings demonstrate that inhibition of p-TH prevents further increases in motor activity and prevents the maintenance of heightened activity levels in zebrafish during alcohol exposure. This suggests that the initial alcohol-induced increase in motor activity is perhaps independent of TH phosphorylation, and increased synthesis of DA comes into play later on during alcohol exposure. It has been previously shown in zebrafish that during immersion in 1% alcohol, the brain alcohol concentration rapidly rises during the first 20-30 minutes of exposure, after which it stabilizes (Tran et al., 2015a). Rising alcohol concentrations have been suggested to cause a release of DA from synaptic vesicles into the synaptic cleft, which may underlie the rapid, and TH-phosphorylation independent, increase in locomotion during the first 20 minutes of ethanol exposure. However, following neurotransmitter release and breakdown of DA, activation of TH may need to occur to maintain heightened DA levels in the brain. This is supported by the finding that release of catecholamines is not followed by decreases in their levels within tissues due to an increase in the rate of catecholamine synthesis that is closely coupled to neurotransmitter release (Zigmond et al., 1989). Thus, it appears that alcohol’s locomotor stimulant effects are mediated, at least partially, by an increased production of DA but only after alcohol has reached stable concentrations in the brain. Although activity of TH can be modulated by other mechanisms (i.e. transcriptional and translational regulation) (Dunkley et al., 2004), the significant attenuation of the alcohol-induced DA increase and motor hyperactivity by THP suggests that TH phosphorylation may be one of the primary mechanisms responsible for the increase of DA levels following alcohol exposure. Moreover, it suggests that heightened DA levels are closely associated with alcohol’s motor activating effects.
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Many neurotransmitter systems have been implicated in alcohol’s mechanism of action, however, the dopaminergic system has been argued to be a primary system involved in both the stimulatory and rewarding properties of alcohol (Gilpin & Koob, 2008; Phillips & Shen, 1996). More importantly, sensitivity to the stimulant properties of alcohol has been identified as a risk factor for alcohol addiction (King et al., 2011, 2014). The current study was based on the hypothesis that alcohol may increase locomotor activity through mechanisms related to reinforcement by other drugs of abuse, such as cocaine. The ability to block the alcohol-induced DA increase and hyperactivity using THP closely parallels the prevention of cocaine-seeking behaviour by THP. Similar to alcohol, cocaine elevates DA levels in the brain and induces addictive behaviours (i.e. cocaine self-administration). In rats, THP inhibits the cocaine-stimulated DA production at concentrations targeting p-TH, without reducing basal DA production. The ability of THP to block the effects of two different psychoactive drugs in two entirely different species suggests that the stimulatory and rewarding properties of drugs of abuse may be partially mediated by a common evolutionarily conserved molecular pathway. The implications of such a finding are noteworthy for two reasons. One, common underpinnings of addiction can be exploited for the development of more effective treatments for many addictive disorders (Nestler, 2005), a goal in addiction research that is already beginning to be explored. For example, disulfiram, which is prescribed to treat alcoholism, has been shown to also reduce cocaine use (Suh et al., 2006). Two, conserved mechanisms and our ability to study them in a simple vertebrate such as zebrafish opens the possibility of the development of efficient and low cost animal models of complex human disorders including drug addiction.
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Acknowledgements RG supported by NSERC Discovery Grant (311637) and NIH/NIAAA (R01 AA14357-01A2), ST supported by NSERC Graduate Scholarship.
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Figure 1.
THP dose dependently reduces locomotor activity of zebrafish. Total distance experimental zebrafish swam (cm) is shown as a function of time (1-minute intervals) during a 90-minute acute exposure to different concentrations of THP (0, 50, 100, 200 μM external bath concentration). Means ± S.E.M. are shown. Note the lack of significant difference between control and 50μM THP exposed fish.
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Figure 2.
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Alcohol induced increase of total distance travelled (cm) is attenuated by THP pre-treatment during a 60-minute drug exposure/behavioural observation session. Means ± S.E.M. are shown. Note the robust hyperactivity of fish that received 1% alcohol alone (light grey squares) and the significant attenuation of this hyperactivity in fish that received alcohol and THP concurrently (black squares). Also note the lack of significant difference between control fish (0 alcohol and 0 THP, white squares) and fish that received 50 μM THP but no alcohol (dark grey squares).
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Figure 3.
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Alcohol elevates activity and THP pre-treatment attenuates the hyperactivity inducing effects of alcohol during the last ten minutes of the drug exposure/recording session. Mean ± S.E.M. are shown for the total distance travelled (cm) in the last 10 minutes of a 60-minute exposure session. The treatment conditions are indicated by the legends (THP concentration) and the X-axis labels (alcohol concentration) for the four treatment groups: (1) system water; (2) 1% ethanol; (3) 50 μM THP; and (4) 1% ethanol + 50 μM THP. Fish in treatment conditions (1) and (2) were pre-treated with system water for 30 minutes, and fish in conditions (3) and (4) were pre-treated with 50 μM THP for 30 minutes. Significant differences (p< 0.05) among groups are indicated by the letters above the bars. Bars that do not share the same letter designation are significantly different.
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The level of dopamine in the brain of zebrafish is increased by alcohol but pre-treatment with THP abolishes this alcohol effect. Mean ± S.E.M are shown for dopamine (panel A) and DOPAC (panel B) (expressed as ng neurochemical/mg brain protein) analyzed from whole brain tissue obtained from fish following a 60-minute exposure to four different treatments: (1) system water; (2) 1% ethanol; (3) 50 μM THP; and (4) 1% ethanol + 50 μM THP (THP concentrations are indicated by the legends and alcohol concentrations by the Xaxis labels). Fish in treatment conditions (1) and (2) were pre-treated with system water for 30 minutes, and fish in conditions (3) and (4) were pre-treated with 50 μM THP for 30 minutes. Significant differences (p< 0.05) among groups are indicated by the letters above the bars. Bars that do not share the same letter are significantly different.
Author Manuscript Author Manuscript Pharmacol Biochem Behav. Author manuscript; available in PMC 2016 November 01.
