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. Author manuscript; available in PMC: 2018 Jun 1.
Published in final edited form as: Pharmacol Biochem Behav. 2017 Apr 10;157:1–8. doi: 10.1016/j.pbb.2017.01.005

Dopaminergic control of anxiety in young and aged zebrafish

Victoria Kacprzak 1, Neil A Patel 1, Elizabeth Riley 1, Lili Yu 1, Jing-Ruey J Yeh 2, Irina V Zhdanova 1,✉
PMCID: PMC5505502  NIHMSID: NIHMS871380  PMID: 28408289

Abstract

Changes in the expression of the dopamine transporter (DAT), or the sensitivity of dopamine receptors, are associated with aging and substance abuse and may underlie some of the symptoms common to both conditions. In this study, we explored the role of the dopaminergic system in the anxiogenic effects of aging and acute cocaine exposure by comparing the behavioral phenotypes of wildtype (WT) and DAT knockout zebrafish (DAT-KO) of different ages. To determine the involvement of specific dopamine receptors in anxiety states, antagonists to D1 (SCH23390) and D2/D3 (sulpiride) were employed. We established that DAT-KO results in a chronic anxiety-like state, seen as an increase in bottom-dwelling and thigmotaxis. Similar effects were produced by aging and acute cocaine administration, both leading to reduction in DAT mRNA abundance (qPCR). Inhibition of D1 activity counteracted the anxiety-like effects associated with DAT deficit, independent of its origin. Inhibition of D2/D3 receptors reduced anxiety in young DAT-KO, enhanced the anxiogenic effects of cocaine in WT, but did not affect aged WT or DAT-KO fish. These findings provide new evidence that the dopaminergic system plays a critical role in anxiety-like states, and suggest that adult zebrafish provide a sensitive diurnal vertebrate model for elucidating the molecular mechanisms of anxiety and a platform for anxiolytic drug screens.

Keywords: zebrafish, aging, anxiety, dopamine, dopamine receptors, dopamine transporter, cocaine, sulpiride, SCH23390

Introduction

Dopamine (DA) is a critical neurotransmitter, involved in a broad range of physiological functions, including cognitive, visual and motor (Dunnett, 2005). Properties of the dopaminergic system, including DA synthesis and release from neurons, and the presence of both presynaptic and postsynaptic DA receptors, are highly conserved in vertebrates. DA levels in the synaptic cleft, and therefore its effects on receptors, depend on the rate of DA re-uptake by nerve terminals. The re-uptake rate is controlled by a presynaptic membrane-bound protein, dopamine transporter (DAT), which is critical for maintaining DA homeostasis and controlling the duration of DA signals (Kahlig and Galli, 2003).

As a result, genetic or pharmacologically-induced changes in DAT expression and function modulate the entire DA pathway, significantly affecting health and disease states. DAT polymorphisms are associated with extreme behavioral traits and disorders, including angry-impulsive personality and borderline personality disorder (Joyce et al., 2009, n.d.), binge-eating (Shinohara et al., 2004), disruptive behavior disorder (Lee et al., 2007), attention-deficit and hyperactivity disorder (Franke et al., 2008) and alcohol dependence (Ueno et al., 1999).

A variety of addictive drugs, from opiates to psychostimulants, inhibit DAT expression or function and thus extend the duration of DA effects on its receptors, both postsynaptic (e.g., D1) and autoregulatory presynaptic (e.g., D2) (Jia et al., 2005; Liang et al., 2014; Wilson et al., 1996; Yuan et al., 2015). This, in turn, affects drug-seeking behaviors and associated cognitive and emotional states (Wang and Deutch, 2007). Notably, DAT is a direct target of cocaine and both the reinforcing and drug-withdrawal effects of cocaine administration are linked to its ability to inhibit DAT function or modify DAT expression levels (Thomsen et al., 2009).

There is evidence that DAT expression in humans declines in normal aging. Imaging studies have revealed gradual and region-specific reductions in DAT density with age (Shingai et al., 2014; Volkow et al., 1998, 1996), supporting post-mortem findings of significant loss of nigrostriatal DA neurons even in the absence of neurodegenerative disease (Fearnley and Lees, 1991). Additionally, several major age-dependent disorders are known to be associated with DAT polymorphisms, such as Alzheimer’s disease (Lin et al., 2012) and Parkinson’s disease (le Couteur et al., 1997; Ritz et al., 2009). DAT polymorphisms have also been linked to reduced life-expectancy in humans (Hadi et al., 2015). However, despite a known correlation between decline in DAT or dopamine receptor markers and cognitive performance in the elderly (Bäckman, Nyberg, & Farde, 2006; Li, Arime, Hall, Uhl, & Sora, 2010), the role of DAT deficiency in emotional and cognitive changes during normal aging remains to be fully elucidated.

To understand the DAT-dependent mechanisms involved in symptoms that are common to both aging and drug addiction, e.g., anxiety, and to develop ways to reverse these symptoms, the use of potent and preferably high throughput animal models is required. The recent and remarkable discovery that birds and reptiles lack the DAT transporter (Lovell et al., 2015) reduces the choice of vertebrate models to mammals and fish. Among those, the diurnal vertebrate zebrafish model offers multiple advantages, including its well-developed DA system (Panula et al., 2010), gradual aging (Kishi et al., 2009) and sensitivity to multiple drugs of abuse and their withdrawal (Bencan et al., 2009; López Patiño et al., 2008a; Riley et al., 2015; Rinkwitz et al., 2011).

In this study, we addressed the role of the dopaminergic system in the anxiogenic effects of both aging and cocaine by comparing the behavioral phenotypes of wildtype (WT) and DAT knockout zebrafish (DAT-KO). We determined that DAT knockout results in an anxiety-like state, which is remarkably similar to that of aged zebrafish. It is also similar to the behavioral response of zebrafish to transient inhibition of DAT by acute cocaine exposure. Moreover, we found that such anxiety-like behavior in both DAT-KO and WT fish is under differential control of dopamine receptors. Together, this implies a major role of the dopaminergic system in anxiety and suggests that the zebrafish is a sensitive model for defining the mechanisms involved and designing effective treatments against anxiety associated with drug addiction or aging.

Materials and Methods

Animals

Juvenile (3–4 months of age), adult (4–18 months), and aged (over 18 months) male zebrafish (Danio rerio) of both DAT knockout strain (DAT-KO, Foley et al., 2009) and genetic background wild-type control (WT) were born and raised in the lab. Animals were housed in 14L:10D light:dark cycle, in 3L tanks, 6–10 fish per tank, in a 26.5 °C, pH 7.0–7.4 controlled multi-tank recirculating water system (Aquaneering, San Diego, CA, USA). Animals were fed twice a day with live brine shrimp (BrineShrimpDirect, Ogden, Utah. USA) and flake food (TetraMin, Tetra, Blacksburg, VA, USA). Two weeks prior to the initiation of experimental procedures, fish were moved to individual 1L housing tanks for adaptation, and separated from each other by opaque partitions. They remained in the 1L tanks throughout the experimental period. All experiments were approved by Boston University Institutional Animal Care and Use Committee and were performed in accordance with the guidelines described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Dopamine transporter mutation

The dopamine transporter (DAT, also known as slc6a3) mutant was generated using engineered zinc finger nucleases (Foley et al., 2009). This mutant line has a 4-basepair insertion in exon 12 (Fig. S1).

Behavioral assays

To document locomotor activity patterns, 1L tanks containing individual fish were placed on an experimental rack, with the lateral wall of each tank facing the camera, to document the speed and depth at which fish were swimming. Activity in the entire tank (100 mm water column) and within 3 equally-spaced areas of the tank (the top, middle and bottom) was documented for up to 24-h periods using VideoTrack image analysis software (ViewPoint, Canada). Fish were not able to see each other during recording. Fluorescent lighting at 100 lx was provided during the day only, with infrared illumination during the night. All recording was accomplished with cameras equipped with infrared pass filters, to ensure uniform images throughout the day and night. Consistent with our earlier studies (López Patiño et al., 2008a), high-speed swimming activity was defined as above 15.0 cm/s, low-speed as 0.1–15.0 cm/s, and inactivity as below 0.1 cm/s. To document thigmotaxis, a tendency to stay close to the walls, fish behavior in a 9 L tank (27.3 × 19.7 cm floor area) was video-recorded from above and the percent time spent within 5 cm of the walls versus the remaining central part of the tank was documented. To determine changes in locomotor activity across the lifespan, the swimming patterns of juvenile, adult and aged fish were recorded in parallel. Behavioral studies involved 6–22 fish per group.

Treatments

Stock solution of cocaine hydrochloride (NIDA) was prepared in water and, following baseline recording, added directly to the tanks containing fish, with final cocaine concentration in the tank ranging from 1 to 33 μM. Control groups received water. Behavioral assessment was conducted for 1 h for each dose (n=6/group). The dose range was based on our earlier dose-dependency studies, in which cocaine-induced stereotypy or thigmotaxis was documented (López Patiño et al., 2008a).

For dopamine receptor antagonists, sulpiride (Sigma-Aldrich, D2/D3 receptor antagonist, dissolved in dimethyl sulfoxide) and SCH23390 (Tocris Bioscience, D1 receptor antagonist, dissolved in water) were used. Vehicle groups received either dimethyl sulfoxide or water. Following a baseline behavioral assessment (1 h), the appropriate receptor antagonist was added to tank water to achieve a final concentration of 10 μM (Darland et al., 2012). Behavioral assessment then continued for another hour.

Each DA receptor antagonist (10 μM) was also tested in combination with low (5 μM) or high (33 μM) cocaine dose (n=6/group). Experiments had three 1 h phases. Following a baseline behavioral assessment (1 h), fish were treated with cocaine (1 h) and then appropriate receptor antagonist was added to tank water (1 h). Behavioral assessment continued throughout the experiment.

Diazepam (DzP, Abbott Laboratories, Chicago, IL, USA) was dissolved in 10% ethanol to produce a 17.5 mM working solution and administered directly into the fish tank, with final concentration of 5 μM. The benzodiazepine receptor inverse agonist, N-methyl-β-carboline-3-carboxamide (FG-7142, Tocris Cookson Inc., Ellisville, MO, USA) was dissolved in 10% ethanol to produce a 3.5 mM working solution, with final concentration in the tank of 0.5 μM. Both concentrations were based on earlier dose-dependence studies (López Patiño et al., 2008a). The control animals were exposed to a 0.003% final concentration of ethanol in tank water. Behavioral recording was conducted for 1h, in parallel.

Real-Time quantitative RT-PCR (qPCR)

Fish (n=6/group) were dissected on dry ice and total RNA was extracted from individual brains using RNAeasy kit for high lipid content tissue (Qiagen, Chatsworth, CA, USA), according to the manufacturer’s protocol. The quantity and quality of RNA was determined spectrophotometrically, at 260 nm and 260/280 nm respectively. The same amount of RNA from each sample was converted into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instruction. qPCR was performed using a TaqMan® Universal PCR Master Mix and ABI Prism 7300 Real Time PCR System (ABI, Foster City, CA, USA). The following TaqMan® primers and probes {5′ FAM, 3′ TAMRA} for dopamine transporter (zDAT) were used (ABI): Forward, 5′-GTCTGGAAGATCTGCCCCATATT-3′, Reverse, 5′-CACATACAGCGAGATCAGGATCAC-3′; {AAGCCCACGCCTTTG}. Gene expression was normalized using β-actin expression level for each individual fish sample. Relative mRNA expression level was calculated using the standard comparative delta-Ct method. The data are presented as fold change, relative to mean control group result, shown as “1” on Y-axis.

Statistical Analysis

The statistical significance of the effects of pharmacological treatments was determined by a linear mixed model analysis with repeated measures using SPSS software (SPSS Statistics, NY, USA), with a significance level of p < 0.05. For t-tests, the significance level was also p < 0.05. Data are presented as mean ± SEM. Linear regressions were used to assess correlations, with R2 values reported in the results or figures.

Results

Anxiety-like behavioral phenotype in DAT-KO zebrafish

Adult DAT-KO zebrafish had a 21.4 ± 0.52 fold decrease in DAT mRNA abundance compared to controls and manifested a striking behavioral phenotype. They swam predominantly at the bottom of the tank and close to the tank walls, thus displaying bottom-dwelling and thigmotaxis (Fig. 1A). Behavioral analysis over a 24-h period showed that the DAT-KO bottom dwelling phenotype was present throughout the day and night (Fig. 1B, double-plotted). This was in contrast to controls, which displayed bottom-dwelling mainly during nighttime sleep period (Fig. 1B). During the daytime, the percent time spent in the bottom third of the tank was 76.8 ± 4.27% for DAT-KO fish vs. 36.4 ± 3.59% for control fish (t-test, n = 12, p < 0.001). DAT-KO fish spent 10.1 ± 1.92% of their time in the central region of the tank, compared with 21.42 ± 3.22% in controls (t-test, n = 44, p = 0.03).

Fig. 1.

Fig. 1

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Swimming behavior of DAT-KO vs. wildtype zebrafish. (A) The bottom dwelling in DAT-KO fish is in contrast to regular swimming pattern in genetic control wild-type (WT) zebrafish; (B) Double-plot of daily variation in swimming depth in DAT-KO vs. WT, as per % time spent in the bottom third of the tank. ZT0 (Zeitgeber time 0) = lights on time; (C) % time spent in the middle third of the tank, * p < 0.05; (D) % time spent in the top third of the tank,* p < 0.05; (E) % time spent in the bottom third of the tank for WT (white) and DAT-KO (black) animals after control, FG7142 and diazepam treatments, * p < 0.05 between control and FG7142 treatment for WT animals, # p < 0.05 between control and diazepam treatment for DAT-KO animals.

Bottom-dwelling and thigmotaxis were earlier described as associated with anxiety-like states in zebrafish (Egan et al., 2009; López Patiño et al., 2008a). The translational value of such a model of anxiety in fish was further tested through exposure to the known anxiogenic benzodiazepine receptor inverse agonist FG-7142, and the anxiolytic benzodiazepine receptor agonist diazepam (Fig. 1E). FG-7142 increased bottom dwelling in WT animals (t-test, n = 6, p = 0.009), with no significant changes observed in DAT-KO (t-test, n = 6, p = 0.073), presumably due to a “floor effect”. In contrast, diazepam significantly reduced bottom-dwelling in DAT-KO animals (t-test, n = 6, p = 0.002), while reduction in this behavior in WT fish did not reach statistical significance (t-test, n=6, p=0.095). Together, these results further confirmed that bottom-dwelling reflects an anxiety-like state in zebrafish.

Anxiety-like behavioral phenotype in aged zebrafish

We documented typical locomotor activity patterns across the lifespan, from juvenile to aged WT and DAT-KO fish. In WT, there was a strong association (R2 = 0.97) between age and bottom-dwelling (Fig. 2A), with the oldest WT fish spending 5.3 times more time at the bottom of the tank than the youngest fish tested. This pattern, which suggested increased prevalence of an anxiety-like state in aged animals, was also associated with a modest increase in time spent inactive (Fig. 2D) and a reduction in both average high and low swimming speed (Fig. 2E, F).

Fig. 2.

Fig. 2

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Age-dependent changes in locomotor activity of DAT-KO and wildtype zebrafish.

Percent time spent in the one third of the tank: (A) bottom; (B) middle; (C) top. (D) % time spent inactive (<0.1 cm/s); (E) Average speed at High range (>15.0 cm/s); (F) Average speed at Low range (0.1–15.0 cm/s). Mean (SEM); n=4/group.

There was also a strong association (R2 = 0.76) between age and bottom-dwelling in DAT-KO zebrafish, although even juvenile DAT-KO fish already spent the majority of time at the tank bottom (Fig. 2A). Due to this “bottom effect”, our oldest mutant fish spent only 1.3 times more time at the bottom of the tank than the youngest DAT-KO fish. Unlike WT fish, with increasing age, DAT-KO fish tended to stay less time inactive and have faster average high and low swimming speeds (Fig. 2D–F).

We also found that aged WT fish had 2.1 ± 0.12 fold lower DAT mRNA abundance than young adult WT fish (t test, n = 4, p < 0.01). In DAT-KO fish, no further change in DAT mRNA was observed with aging.

Acute cocaine exposure induces anxiety-like behavior in zebrafish

Exposing WT zebrafish to cocaine resulted in dramatic changes in their behavior (Fig. 3A). A dose-dependent increase in bottom-dwelling reached significance at a concentration of 4 μM, with cocaine-treated WT animals spending around 60% of the time in the bottom third of the tank, compared to around 25% at baseline (Fig. 3A). Further increase in dose led to even more pronounced bottom-dwelling in WT, approaching the behavior characteristic of DAT-KO fish. Following administration of lower doses (Fig. 3C), bottom-dwelling was associated with a significant decline in time spent at the top of the tank, and at higher dose (16 μM), led to additional decline in the time spent in the middle of the tank (Fig. 3B). Bottom-dwelling corresponded to significant reduction in DAT mRNA abundance in the brains of WT fish exposed to cocaine (3.1 ± 0.23 fold below controls for 33 μM dose, t test, n = 12, p < 0.05), though it still remained significantly higher than in DAT-KO fish (p < 0.0001). Cocaine treatment in WT fish resulted in a significant decline in distance traveled at high speed (over 15 cm/s; mixed model, p < 0.01) but not low speed.

Fig. 3.

Fig. 3

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Dose-dependence of acute cocaine effects on swimming behavior of adult DAT-KO and wildtype zebrafish.

Percent time spent in the one third of the tank: (A) bottom; (B) middle; (C) top; Mean (SEM); n= 12/group; * p<0.01 between WT Control & WT Cocaine; # p<0.01 between DAT-KO Control & DAT-KO Cocaine.

In DAT-KO fish, cocaine also increased bottom-dwelling, though starting at a higher dose (8 μM) than in WT (Fig. 3A). This was at the expense of swimming in both the middle and top of the tank (Fig. 3B,C). No significant change in the overall distance traveled at high or low speed was found in DAT-KO exposed to cocaine. Similarly, cocaine did not affect DAT mRNA abundance in DAT-KO mutants.

Dopamine receptor antagonists can rescue DAT-KO behavioral phenotype but the effect is receptor-specific and age-dependent

Administration of SCH23390, a D1 receptor antagonist, led to significant (mixed model, p < 0.0001) reduction in bottom-dwelling in both WT and DAT-KO fish (Fig. 4A). Treatment with sulpiride, D2/D3 receptor antagonist, resulted in a modest but significant decrease in bottom-dwelling in DAT-KO fish (mixed model, p < 0.0001), though left WT behavior unchanged (Fig. 4B).

Fig. 4.

Fig. 4

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Effect of specific dopamine receptor antagonists on swimming behavior in adult DAT-KO and wildtype zebrafish.

Percent time spent at the bottom third of the tank for DAT-KO and WT zebrafish following treatment: (A) SCH23390 or vehicle; (B) Sulpiride or vehicle. Mean (SEM); n=12/group; * p<0.05 vs. corresponding control.

When aged fish were treated with DA receptor antagonists, the bottom-dwelling was significantly reduced following administration of SCH23390 in both aged WT (14.3 ± 0.52%, mixed model, p < 0.05) and aged DAT-KO (28.6 ± 0.43%, mixed model, p < 0.01) fish. In contrast, no significant effect of sulpiride was documented in either aged DAT-KO or aged WT fish.

Dopamine receptor antagonists interfere with cocaine-induced anxiety-like behavior in a receptor-specific manner

Administration of SCH23390 significantly reduced anxiety-like behavior induced in WT animals by low (5 μM) or high (33 μM) doses of cocaine (Fig. 5A,B). In striking contrast, administration of sulpiride augmented the characteristic anxiety-like response to low and high doses of cocaine in WT animals, significantly increasing the amount of time the fish spent at the bottom of the tank (Fig. 5C,D). In DAT-KO fish receiving both cocaine and SCH23390, the D1 antagonist reduced bottom-dwelling, independent of the cocaine dose used (Fig. 5A,B). Sulpiride failed to modify the behavior of cocaine-treated DAT-KO fish (Fig. 5,D).

Fig. 5.

Fig. 5

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Effects of dopamine receptor antagonist on cocaine-induced changes in swimming behavior of adult DAT-KO and wildtype zebrafish.

Percent time spent at the bottom third of the tank: (A) Baseline vs. Low-dose cocaine (5 μM) vs. 5 μM cocaine + 10 μM SCH23390 (D1 antagonist); (B) Baseline vs. High-dose cocaine (33 μM) vs. 33 μM cocaine + 10 μM SCH23390 (C) Baseline vs. Low-dose cocaine (5 μM) vs. 5 μM cocaine + 10 μM Sulpiride (D2/D3 antagonist); (D) Baseline vs. High-dose cocaine (33 μM) vs. 33 μM cocaine + 10 μM Sulpiride. Mean (SEM); n=12 per group; * p<0.01 antagonist + cocaine vs. cocaine alone, # p < 0.01 cocaine vs. baseline.

Discussion

In this study, we demonstrate that deficiency in DAT due to several independent factors, such as aging, acute cocaine administration or genetic DAT knockout, leads to similar anxiety-like behavior in zebrafish. The degree of DAT deficit correlates with the anxiety level, which is highest in DAT-KO fish, suggesting that anxiety results from acute or chronic increase in dopamine levels in certain brain structures. These findings provide the first characterization of DAT-KO zebrafish and are consistent with low DAT availability in patients with generalized anxiety disorder (Lee et al., 2015). They also highlight the overall critical role of dopaminergic transmission in the development of anxiety.

We document here that the anxiety-like state in zebrafish is mediated via specific DA receptors. The D1 and D2/D3 receptors contribute differently to the effect, with the former being post-synaptic receptors and the latter being present on both pre- and post-synaptic membranes (for a review, see Ford, 2014). Treatment with a D1 receptor antagonist counteracts anxiety in aged wild-type fish, and even reduces anxiety-like behaviors in young WT animals. The most dramatic effects of D1 antagonism can be observed in DAT-KO animals, where it leads to temporary rescue of the DAT-KO phenotype in both young and aged mutants. Furthermore, anxiety induced by acute cocaine administration in wild-type animals or maintained in cocaine-treated DAT-KO fish is also attenuated by D1 receptor antagonist. This provides strong evidence that, in zebrafish, enhanced post-synaptic DA transmission via D1 receptors is at the core of the anxiety-like state.

Counteracting the effects of dopamine on D2/D3 receptors produces relatively modest effects on anxiety in zebrafish, but shows remarkable dependence on the background characteristics of the animal. In DAT-KO fish, inhibition of D2/D3 receptor function results in a significant decrease in anxiety, though substantially less robust than after treatment with D1 antagonist. Although after cocaine exposure DAT-KO fish continue to display their typical anxiety-like behavior, such treatment with a psychostimulant prevents the D2/D3 antagonist from attenuating the phenotype. In striking contrast, cocaine-induced anxiety in young wild-type animals is further augmented by the inhibition of the D2/D3 receptors. This has also recently been shown by McGinnis et al. (Mcginnis et al., 2016), who hypothesize that the enhancement of the effect of cocaine by D3 inhibition is due to increased potency at DAT. There is also prior work showing that D2 deficiency leads to increased sensitivity to cocaine. Finally, in aged animals, whether wild-type or DAT-KO, administration of D2/D3 receptor antagonist produces no modification of anxiety-like state. Overall, aged animals did not respond to sulpiride under any of the conditions we tested, suggesting that expression of D2 and D3 receptors, in addition to expression of DAT, changes with normal aging.

The molecular mechanisms of anxiety are complex and involve several principal neurotransmitters and neuromodulators, and their specific receptors (Nikolaus et al., 2010). Our data suggest that zebrafish can provide a promising model for defining functional significance of DA receptors in health and disease, or developing treatments targeting specific receptor subtypes. This is further supported by a well-characterized dopaminergic system in zebrafish, with dopaminergic neurons located primarily in the telencephalon and midbrain (Parker et al., 2013; Rink and Wullimann, 2002). Zebrafish possess orthologs to all human dopamine system proteins (Boehmier et al., 2004; Li et al., 2007), explaining high sensitivity of this species to dopaminergic drugs. For example, a response to cocaine, a powerful DAT inhibitor, is seen during early zebrafish development (Riley et al., 2015; Shang and Zhdanova, 2007) and later in life (Darland & Dowling, 2001; Darland et al., 2012).

Anxiety disorders significantly affect quality of life, as well as the course of other somatic and psychiatric disorders (for review, Martin, 2003). They are also strongly associated with substance abuse and drug addiction, especially during withdrawal, and are known to potentiate relapse (Sinha, 2007). Earlier, we reported that adult zebrafish manifest anxiety-like behaviors following cocaine administration and during its withdrawal, with symptoms similar to those produced in this species by established anxiogenic drugs and involving dopaminergic system (López Patiño et al., 2008a, 2008b). Similarly, anxiety-like states were documented in zebrafish exposed to other drugs of abuse, alcohol and nicotine (Bencan et al., 2009; Blaser et al., 2010; Blaser and Rosemberg, 2012; Egan et al., 2009; Gerlai et al., 2000; Speedie and Gerlai, 2008; Stewart et al., 2011). The results of this study, demonstrating a link between the cocaine-induced anxiety and DA system in zebrafish, further emphasize the value of this in vivo model with high throughput capability and well-studied molecular biology in addressing the complex molecular mechanisms of anxiety of drug addiction.

Our study addresses the role of aging in anxiety and the extent to which this might involve dopaminergic system. Although in humans anxiety disorders are prevalent at all ages, in elderly the anxiety can be especially debilitating and associated with low quality of life (Meltzer et al., 2016). Importantly, anxiety is typically a chronic condition and is shown to accelerate the aging process, negatively affecting neuroplasticity, immune responses and other body systems and functions (for review, Tetzner & Schuth, 2016). Development of tolerance or addiction to existing anxiolytic medications complicates their long-term use (Lugoboni and Quaglio, 2014) and reports of damage to somatic health and brain functions raise major concerns (Baldwin et al., 2013; Ward et al., 2015). It is thus critical to better understand the mechanisms involved in anxiety of aging in order to develop safe methods targeting the primary pathways affected.

The results of our investigation demonstrate that, in zebrafish, aging is associated with DAT deficit and an anxiety-like state, and with distinctly different responses to the modulation of D1 vs. D2/D3 receptors. The anxiety-like aging phenotype in zebrafish develops over the lifespan, providing an opportunity to explore the dynamics of both pathogenetic and compensatory mechanisms involved. Notably, the zebrafish is quite unique among high-throughput vertebrate models in displaying gradual aging, reaching sexual maturation by 4 months of age, followed by up to 6-year life span in controlled laboratory conditions (Kishi et al., 2009)

Together, these findings provide new evidence that the dopaminergic system plays a critical role in anxiety-like states and suggest that zebrafish is an outstanding model for basic research on the molecular mechanisms of anxiety of different origin, and a promising platform for anxiolytic drug screens.

Highlights.

  • Dopamine transporter knockout leads to anxiety-like phenotype in zebrafish

  • Similar anxiety-like state found after cocaine exposure and in normal aging

  • Inhibition of D1 receptor, but not D2/3 receptor, rescued anxiety-like phenotype

  • Provides new evidence for role of dopaminergic system in anxiety

Acknowledgments

Funding source: This research was supported by grants from NIDA (DA1541801) and the Chaikin-Wile Foundation.

Footnotes

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