Characterization of D3 Autoreceptor Function in Whole Zebrafish Brain with Fast-Scan Cyclic Voltammetry

Abstract

Zebrafish (Danio rerio) is an ideal model organism for investigating nervous system function, both in health and disease. Nevertheless, functional characteristics of dopamine (DA) release and uptake regulation are still not well-understood in zebrafish. In this study, we assessed D3 autoreceptor function in the telencephalon of whole zebrafish brains ex vivo by measuring electrically stimulated DA release ([DA]_max) and uptake at carbon fiber microelectrodes with fast-scan cyclic voltammetry. Treatment with pramipexole and 7-OH-DPAT, selective D3 autoreceptor agonists, sharply decreased [DA]_max. Conversely, SB277011A, a selective D3 antagonist, nearly doubled [DA]_max and decreased k, the first order rate constant for dopamine uptake, to about 20% of its original value. Treatment with desipramine, a selective norepinephrine transporter blocker, failed to increase current, suggesting that our electrochemical signal arises solely from the release of DA. Furthermore, blockage of DA uptake with nomifensine reversed 7-OH-DPAT induced decreases in [DA]_max. Collectively, our data show that, as in mammals, D3 autoreceptors regulate DA release, likely by inhibiting uptake. The results of this study are useful in the further development of zebrafish as a model organism for DA-related neurological disorders such as Parkinson’s disease, schizophrenia, and drug addiction.

Graphical Abstract

Introduction

Dopamine (DA) levels in extracellular environment are highly regulated in many parts of the brain. Proteins that play important roles in this regulation include membrane-bound DA transporter (DAT) protein molecules, which rapidly take up DA from the extracellular space, and DA receptors, which play a crucial role in regulating dopaminergic signals transduction.^1,2 These receptors are members of the 7-transmembrane G-protein coupled receptors (GPCRs) family and consist of five types categorized according to their structure and function. D1 and D5 receptors belong to the D1-like DA receptor class and D2, D3, and D4 receptors belong to D2-like DA receptor class.³

Receptors in the D1-like subfamily are excitatory in that they stimulate DA signaling by coupling with Gα_s, which activates adenyl cyclase (AC) and increases cyclic adenosine monophosphate (cAMP)-dependent protein kinase levels.⁴ Conversely, D2 like receptors are inhibitory in that they inactivate AC, inhibit the formation of cAMP, and suppress the function of calcium channels by coupling to Gα_i/o proteins.⁵ D2, D3, and D4 receptors that are located on presynaptic terminals can act as autoreceptors, with their activation regulating the DA system by a feedback inhibitory mechanism.⁶

Stimulated D3 receptors decrease DA signaling by regulating DA release and synthesis by a feedback inhibition mechanism.^7,8 According to radiological binding studies, D3 receptors, which are present in brain limbic areas, such as the olfactory tubercle and nucleus accumbens in the striatum, have a high affinity for DA (K_i = 30 nM) compared to the other four types of DA receptors.^9,10,11 Therefore, minor changes in D3 receptor expression and function can dramatically affect synaptic dopaminergic signaling in these brain regions.

The DAT, which is part of the SLC6 gene family¹², takes up extracellular DA from the synaptic cleft with the support of the Na⁺ and Cl⁻ electrochemical gradients. The primary means of the termination of DA signal transduction in the synapse is through the DAT; consequently, many addictive and psychostimulant drugs are designed to interfere with DAT function. Previous research has shown that DATs are regulated by different presynaptic proteins, including the D3 and D2 autoreceptors in the human brain.^13,14 It has been reported that DA transport in the mouse striatum increased upon treatment with DA D2/D3 agonists and decreased with D2/D3 antagonist treatment.¹⁵ Moreover, several studies conducted with D2-DAT and D3-DAT co-transfected cells have found that treatment with DA receptor agonists elevates DA uptake and DAT recruitment to the plasma membrane.^16,17,18

The application of electrochemical, electrophysiological, and sampling methods to various model organisms, including fruit flies¹⁹, rodents²⁰, and non-human primates²¹, have yielded important information regarding the regulation of extracellular DA levels by DA receptors and transporters. Zebrafish (Danio rerio) represent a useful alternative to these model organisms since they are easy to genetically manipulate, are complex, contain about 10 million cells²², and are small enough to survive for many hours in a perfusion chamber ex vivo.²¹ Additionally, zebrafish are cheaper to use and can offer improved throughput over mammalian organisms.^23,24

Experimental evidence has confirmed that zebrafish have evolutionarily conserved dopaminergic pathways, homologous to human dopaminergic pathways.²⁵ Furthermore, zebrafish and human genes have a sequence homology of about 70%, with genes that encode D1-D5 receptors having a similar degree of homology (59–72%).^{26,27, 28} Zebrafish and human D1 and D3 receptors also share the same amino acid sequences in their binding pockets.²⁸ As a result, many of the drugs designed to act on the human DA receptors have similar effects on zebrafish as well.

Given the emerging role of D3 receptors as pharmacological targets, it is important to understand how they function in the zebrafish brain to develop this organism as a useful model to study neurological function. Recently, our group and others have measured the release and uptake of dopamine and other neurochemicals in living zebrafish brains with fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes^{29, 30}. In this report, we have pharmacologically characterized the function of D3 autoreceptors with FSCV in this preparation. Our results emphasize the importance of DAT function in DA regulation in zebrafish.

Materials and methods

Animals.

All animals were handled with the University of Kansas Institutional Animal Care and Use Committee approved procedures. Wild-type zebrafish were bred and housed in a recirculating tank system in which reverse osmosis water was filtered and treated before being introduced into the system. The pH, temperature, and conductivity were automatically controlled at 7.2, 28°C, and ~800 μS/m, respectively. Rapid chilling followed by decapitation was used to euthanize the zebrafish. A dissection stereoscope (Leica M125 Microsystem, Bannockburn, IL) was used to isolate the whole brain. The isolated brain was placed in the recording chamber and oxygenated (95% O₂ and 5% CO₂) artificial cerebrospinal fluid (aCSF) kept at a temperature of 28°C was continuously perfused through the recording chamber to keep the brain viable. The brain was retained in the perfusion chamber by positioning a metal harp (slice anchor) with Lycra^® threads on top.

Chemicals.

DA hydrochloride (Lot# BCBG8676V), pramipexole dihydrochloride (Batch# 0000069160), (±)-7-Hydroxy-2-(di-n-propylamino) tetralin hydrobromide (7-OH-DPAT, Lot# 037M4153V), desipramine hydrochloride (Lot# 107K0730) and nomifensine maleate (Lot# 087K4064) were purchased from Sigma-Aldrich (St. Louis, MO). SB277011A dihydrochloride (Batch# 3B/219051) was purchased from Tocris Bioscience (Bristol, UK). Purified (18.2 MΩ) water was used to prepare all aqueous solutions. The artificial cerebrospinal fluid (aCSF) modified for zebrafish contained 131 mM NaCl, 2 mM KCl, 1.25 mM KH₂PO₄, 20 mM NaHCO₃, 2 mM MgSO₄, 10 mM glucose, 2.5 mM CaCl₂·H₂O, and 10 mM HEPES (pH 7.4). Perchloric acid (0.2 M) was used to prepare the DA stock solutions and aCSF without glucose was used to dilute the stock solutions for electrode calibrations. Stock solutions of 50 μM 7-OH-DPAT, 50 μM pramipexole, 10 μM nomifensine, and 10 μM SB277011A, were freshly prepared before each experiment. 7-OH-DPAT and SB277011A stock solutions were prepared in DMSO and nomifensine and pramipexole stock solutions were prepared in aCSF.

Fast scan cyclic voltammetry.

A previously described procedure was used to fabricate the cylindrical carbon fiber microelectrodes. Briefly, a single carbon fiber (7 μm diameter, Goodfellow Cambridge LTD, Huntingdon, UK) was inserted into a glass capillary tube (1.2 mm D.D. and 0.68 mm LD, 4 in long; A-M Systems Inc, Carlsberg, WA) using a vacuum pump. A PE-22 heated coil puller (Narishige Int. USA, East Meadow NY) was used to pull the carbon fiber loaded glass capillary. The exposed carbon fiber, which served as the active sensing region, was trimmed to a length of 100 μm from the pulled glass tip. Carbon fibers were sealed by dipping the electrode tip into epoxy resin (EPON resin 815C and EPIKURE 3234 curing agent, Miller-Stephenson, Danbury, CT) for 45 s. Dipped electrodes were gently rinsed with toluene to remove excess resin and cured at 100 °C for 1 h. Just before use, each electrode was dipped in isopropanol for 10 min. A 0.5 M potassium acetate solution was used to back fill the electrodes to facilitate electrical connection and the exposed electroactive carbon surface was electrochemically pretreated by applying the triangular waveform −0.4 to +1.3 V to −0.4 V. The waveform frequency was kept at 60 Hz for 15 min and then changed to 10 Hz for 10 min. The reference electrode was an Ag/AgCl electrode made by a chlorided Ag wire. Cyclic voltammograms were collected by applying the waveform every 100 ms at a scan rate of 400 V/s.

The electrochemical workstation used for data collection and analysis consisted of a modified Dagan Chem-Clamp potentiostat (Dagan, Minneapolis, MN) allowing decreased gain settings and a breakout box. Tar Heel CV software (provided by R.M. Wightman, University of North Carolina, Chapel Hill, NC) was used with a personal computer and two National Instruments computer interface cards (PCI 6052 and PCI 6711, National Instruments, Austin, TX). To stimulate DA release in the whole brain, multiple biphasic stimulus pulses (25 pulses, 50 Hz, 2 ms, 350 μA) were applied locally to the dorsal nucleus of the ventral telencephalon (Vd)³¹ with two tungsten electrodes positioned 200 μm apart. Brains were given a 10 min recovery time between electrical stimulations.

Pharmacological measurements.

The zebrafish brains were equilibrated for 1 hour in the perfusion chamber before starting the DA release measurements by FSCV. Before adding any drug solution, pre-drug measurements were collected until a stable baseline DA release was observed (≥30 min). For the agonist experiments, after obtaining pre-drug measurements, a single dose of pramipexole (1 μM) and 7-OH-DPAT (2 μM) dissolved in aCSF was perfused through the zebrafish whole brain and collected post drug measurements for 30 minutes at 10-minute intervals. Drug washout measurements were obtained after perfusion of aCSF without the drug for another 30 minutes. For the cumulative dosing experiments, a series of pramipexole (0.005 to 3.0 μM) and 7-OH-DPAT (0.02 to 3.0 μM) solutions in aCSF was perfused through the zebrafish whole brains. The effect of each drug concentration was monitored by measuring DA release for 30 minutes. To check the effect of NET inhibition on D3 receptor function, a single dose of 7-OH-DPAT (1 μM) was perfused through the brains for 30 minutes and next, a drug mixture containing 7-OH-DPAT (1 μM) and desipramine (1 μM) was perfused for another 30 minutes. In the antagonist experiments, a single dose of L-741,626 (2.5 nM) and SB277011A (1 μM) prepared in aCSF was perfused through brains, and DA measurements were collected for 30 minutes. To evaluate the effect of DAT inhibition on D3 agonism, first, nomifensine (1 μM) was perfused through the brain for 30 minutes and after that, a series of drug solutions with a constant nomifensine (1 μM) concentration and increasing concentrations of 7-OH-DPAT (1.0 to 5.0 μM) was perfused through brains. The effect of each drug solution was monitored for 30 minutes by measuring DA release. The peak oxidation current of DA was converted to concentration using pre-and post-calibration of electrodes against DA standard solutions.

Data analysis and statistics.

Graph Pad Prism 6 (Graph Pad Software, Inc, La Jolla, CA) was used for statistical analyses. All the reported data are represented as a mean value plus or minus the standard error of the mean (SEM). For all experiments listed, the N value refers to the number of used zebrafish brains. Modeling to determine DA reuptake rate constants were performed using GraphPad. Briefly, the files were loaded into Tar Heel software, and the peak current (i_max) was determined. The portion of the plot in which current decreases from ~90% to ~40% of i_max was modeled to determine the kinetic parameters. If the current did not return to 40% of i_max, then the curve was modeled for 4 s from the 90% point. The x and y values of the 90% value and the x value were used as constraints in the nonlinear regression plateau followed by one phase decay model. Data files were excluded from modeling if they did not have a peak current over 0.8 nA and if there were less than 3 points between the 90% and 40% values. Once the modeling was completed, the 1^st order rate constant (k), and the half-life (t_1/2), were obtained.

Results

D3 agonists inhibit evoked DA release in the zebrafish brain.

We first determined the effect of pramipexole, a DA autoreceptor agonist with selectivity for D3 receptors³², on electrically evoked DA release in the Vd of zebrafish whole brains. Our representative raw data show that DA release decreased after 30 minutes of treatment with 1 μM pramipexole (Fig. 1A). We chose this concentration because it provided a measurable effect, based on the drug titration curve shown in Fig. 1B. The voltammetrically determined half-maximal effective concentration (EC50) of pramipexole was determined from this plot to be 1.02 ± 0.05 μM. The change in DA release over time with the addition of the drug is shown in Fig. 1C. The addition of pramipexole (1 μM) decreased DA release within 10 min while treatment for 30 minutes caused a significant decrease in release compared to pre-drug values (P < 0.005, t-test) (Figs. 1C, D). Next, we examined the effects of treatment with 7-OH-DPAT, a selective D3 autoreceptor agonist³³, on DA release (Fig. 2A). The EC50 value was determined from this curve to be 412 ± 87 nM. Like the results obtained with pramipexole, DA release decreased within the first 10 minutes (Fig. 2C) and continued to decrease over the course of 30 minutes (Fig. 2D). The release did not significantly increase after washout in the case of both drugs.

Fig 1. Pramipexole decreases evoked DA release in the zebrafish brain.

. (A) Stimulated release plots representing DA release before adding the drug (Pre drug), 30 min after 1 μM pramipexole administration (Post drug), and after the washing out of pramipexole with aCSF (Washout). CVs (insets) confirm the presence of dopamine. (B) Pramipexole dose-response curve. (C) Change in stimulated dopamine release over time. (D) Pooled measurements of dopamine release before (Pre) and after (Post) addition of pramipexole and after washout (Wash) (***p<0.00, **p<0.01 versus Pre, t-test, n=4 zebrafish whole brains).

Figure 2. Selective agonism of D3 receptors decreases evoked DA release in zebrafish brains.

(A) Representative stimulated dopamine release plots, before adding the drug (pre-drug), 30 min after 2 μM 7-OH-DPAT administration (post-drug), and after washing out the drug with aCSF (Washout). CVs (insets) confirm the presence of dopamine. (B) 7-OH-DPAT concentration-response curve. (C) Change in DA concentration over time. (D) Pooled measurements of dopamine release before (Pre) and after (Post) addition of 7-OH-DPAT and after washout (Wash) (***p<0.001 versus Pre, t-test, n=4 zebrafish whole brains).

Norepinephrine uptake inhibition does not affect DA release in the presence of a D3 agonist.

DA and NE are catecholamine neurotransmitters that have similar electrochemical properties and, therefore, their cyclic voltammograms are nearly identical.³⁴ Our group has previously compared the effects of DAT and NE transporter (NET) inhibition in the zebrafish telencephalon and the results have confirmed that the predominant neurotransmitter in the zebrafish telencephalon region is DA, not NE.³⁰ Nevertheless, Chu et al.(2004) report that 7-OH-DPAT inhibited basal and K⁺-evoked NE release in PC12 cells³⁵. Therefore, we wanted to determine if NE is present in the peak suppressed by 7-OH-DPAT in the zebrafish telencephalon region. To compare the relative signal contribution of NE to the DA signal, and possible inhibition of NE by 7-OH-DPAT, desipramine, a selective NET inhibitor, was applied after the DA signal was diminished with 7-OH-DPAT. Our results in Fig. 3 indicate that NET inhibition did not affect the magnitude of the DA signal suppressed by 7-OH-DPAT. This finding suggests that the D3 agonist, 7-OH-DPAT, is acting on DA in our measurements, but not on NE.

Figure 3. Desipramine administration has a negligible effect on D3 receptor agonism by 7-OH-DPAT.

(A) Representative stimulated release plots and CVs (insets) of DA release for pre-drug, 1 μM 7-OH-DPAT (7-OH), and 1 μM 7-OH-DPAT with 1 μM Desipramine (7-OH+Des). (B) Pooled measurements of dopamine release. (***p<0.001 versus pre-drug value, t-test, n=4 zebrafish whole brains).

Autoreceptor antagonists increase DA release and decrease reuptake rate.

We next examined the effect of D3 autoreceptor antagonism on DA release and uptake in whole zebrafish brains. After obtaining a consistent pre-drug DA signal, a selective D3 receptor antagonist^36,37 SB277011A (1 μM) was applied to zebrafish brains. Perfusion of SB277011A increased evoked DA release (Fig. 4A, B). We then modeled the stimulated release plots to determine the first-order rate constant (k) and then pooled these values. Perfusion with SB277011A significantly decreased k (p<0.005, t-test).

Figure 4. Selective antagonism of D3 receptors increases stimulated dopamine release and decreases the uptake rate.

(A) Representative stimulated release plots before (Pre-drug) and after adding the D3 antagonist SB277011A (1 μM). The blue line indicates reuptake modeling. (B) Pooled measurements (C) 1st order rate constant (k) values were obtained by modeling the I vs T curves of the pre SB277011A perfusion and post SB277011A measurements. (*p<0.05, t-test, n=4 zebrafish whole brains)

D3 receptor agonism decreases DA release in presence of D2 receptor antagonist.

The D3 agonist, 7-OH-DPAT, selectively binds to D3 receptors with high affinity compared to D2 receptors.³⁸ D3 receptors also bind with high affinity to DA (420-fold higher than D2 receptors); however, D2 receptors are more abundant than D3 receptors in the striatum.³⁹ Therefore, we sought to confirm that the observed DA signal suppression by 7-OH-DPAT occurs primarily by acting on D3 receptors compared to D2 receptors. First, brains were perfused with L-741,626 (~40 times greater affinity for D2 than D3 receptors) for 30 minutes. As shown in Fig. 5 and SF2, this perfusion resulted in about a 100% increase in stimulated DA release due to the blockage of the D2 autoreceptor. Next, a mixture of L-741,626 and 7-OH-DPAT was perfused for another 30 minutes. This perfusion mixture decreased DA release to about 45% of the pre-drug signal. The observed decrease in DA release after adding the D3 agonist (while D2 receptors are blocked with the D2 antagonist) suggests that D3 agonism counteracts D2 antagonism.

Figure 5. D3 agonism increases DA release in presence of a D2 antagonist.

(A) Representative stimulated release plots before (Pre-drug), after adding the D2 antagonist L-741,626 0.5 μM), and L-741,626 (0.5 μM) with 7-OH-DPAT (1 μM) (B) Pooled data representing pre-drug and post-drug dopamine measurements. Pre = pre-drug, L741 = L-741,626, 7-OH = 7-OH-DPAT (* represent comparisons with pre-drug, ^# represent comparisons with L-741,626, **p<0.01, ^###p<0.001 t-test, n=4 zebrafish whole brains).

DAT inhibition increases DA overflow even during D3 agonism.

The DAT is a critical regulator of extracellular DA levels. Apart from controlling DA synthesis and release, feedback inhibitory DA autoreceptors are also known to influence DAT function.^14,12 Here, we examined the effect of DAT inhibition on D3 function. Zebrafish whole brains were perfused with nomifensine, a well-known DA reuptake inhibitor,⁴⁰ and then with increasing concentrations of the D3 agonist, 7-OH-DPAT, with a constant concentration of nomifensine. Perfusion with aCSF containing 1 μM nomifensine resulted in an immediate increase in electrically evoked DA overflow due to the inhibition of DA reuptake (p<0.01, t-test) while increasing concentrations of 7-OH-DPAT failed to decrease release (Fig. 6). As depicted in Fig. 6C, nomifensine application significantly decreased the DA reuptake rate compared to the pre-drug value. However, the application of the increasing concentrations of 7-OH-DPAT, while keeping the nomifensine concentration constant, increased the DA reuptake rate constant compared to nomifensine. The half-life values of DA reuptake demonstrated an inverse trend when compared to the k values—nomifensine increased half-life and treatment with 7-OH-DPAT decreased it. These effects on uptake suggest that the potency of nomifensine was affected by D3 agonism.

Figure 6. Treatment of zebrafish brains with nomifensine masks the effects of D3 agonism.

(A) Representative stimulated release plots of DA release for pre-drug and post-drug with a constant nomifensine concentration and increasing 7-OH-DPAT concentrations: (a) pre-drug, (b) 1 μM Nomifensine, (c) 1 μM Nomifensine +1 μM 7-OH-DPAT, (d) 1 μM Nomifensine + 3 μM 7-OH-DPAT, (e) 1 μM Nomifensine + 5 μM 7-OH-DPAT). (B) Pooled measurements show that nomifensine affects the 7-OH-DPAT action (One-way ANOVA and Dunnett’s multiple comparisons test, *p<0.05, **p<0.01, ****p<0.0001 versus Pre, n=4 zebrafish whole brains). (C) Dopamine reuptake rate constants (k) obtained by nonlinear regression modeling of I vs T plots. (One-way ANOVA and Dunnett’s multiple comparisons test, n=4, # represent comparisons with 1 μM Nomifensine, * represent comparisons with pre-drug, ****p<0.0001, ^####p<0.0001, ^##p<0.01) (D) Half-life values obtained by dopamine reuptake modeling (One-way ANOVA and Dunnett’s multiple comparisons test, ^# represent comparisons with 1 μM Nomifensine, * represent comparisons with pre-drug ****p<0.0001, **p<0.01, ^####p<0.0001, n=4 zebrafish whole brains) [pre = pre-drug, Nom = nomifensine, 7-OH = 7-OH-DPAT).

In a follow-up experiment, we activated the D3 receptors with 7-OH-DPAT first, and after diminution of the DA signal, a mixture of 7-OH-DPAT and nomifensine was perfused. A significant increase in the DA concentration was observed upon perfusion of the 7-OH-DPAT and nomifensine mixture, indicating that nomifensine reverses the effects of 7-OH-DPAT (Fig. 7). Collectively, the findings in Figs. 6 and 7 suggest that DAT inhibition can both prevent and reverse the effects of D3 agonism on DA release.

Figure 7. Effect of DAT inhibition on evoked DA release of the brains treated with a D3 agonist.

(A) Representative current plots and CVs (insets) of DA release before adding drug (Pre-drug), after treating with 1 μM 7-OH-DPAT (7-OH), and after treating with 1 μM 7-OH-DPAT + 1 μM Nomifensine (7-OH + Nom). (B) Pooled measurements (* represent comparisons with pre-drug, ^# represent comparisons with 7-OH-DPAT *p<0.05, ^#p<0.05 t-test, n=4 zebrafish whole brains).

Discussion

This study revealed that DA release is tightly regulated by D2 and D3 autoreceptors in zebrafish, like that in mammals. Moreover, select agonists and antagonists of these receptors that have been used in mammalian systems affect DA release similarly in zebrafish. Our results also indicate that the primary catecholamine neurotransmitter released in the selected region of the zebrafish telencephalon is DA and not NE. Interestingly, it appeared that interactions between D3 autoreceptor function and uptake occurred: antagonism of D3 autoreceptors increased DA overflow and appeared to decrease uptake rate, while inhibition of DAT seemed to reverse the inhibitory effects of D3 agonism.

Pramipexole is a potent non-ergot D3 autoreceptor agonist⁴¹ that is currently approved as a palliative treatment for Parkinson’s disease and restless leg syndrome^42,43 and 7-OH-DPAT is a highly selective D3 receptor agonist.^38,44 Our findings that pramipexole and 7-OH-DPAT significantly decreased electrically stimulated DA release in a concentration-dependent manner (Figs. 1 and 2) demonstrate that D3 receptor agonism strongly inhibits DA release in zebrafish and, therefore, likely acts as an important regulator in vivo. Doses and affinities for these and the other drugs used in this study have not been measured in zebrafish before. However, although the affinity for D3 receptors may be less in zebrafish and any possible off-target effects should be investigated at a later point, our results are consistent with previous findings in human and mammalian research models.^45,46 In particular, PD patients treated with pramipexole have shown a reduction of phasic DA release in the nucleus accumbens (NAcc).^{47,48, 49} Furthermore, studies using rats and mice have also reported that D3 antagonism with pramipexole^{50, 51,52} and 7-OH-DPAT suppresses DA release.^{53, 54} The effects of these drugs persisted after a 30 min washout period. Nevertheless, the tissue likely remained viable, given that: 1) DA release measurements obtained in the absence of drug remained relatively constant over 120 minutes (SF. 1), 2) nomifensine reversed the inhibitory effects of 7-OH-DPAT (Fig. 6), and 3) previous results in which the effects of specific antipsychotics and D3 inhibitors have also shown persistent effects even after a washout period⁵⁵. We speculate that this effect is a characteristic of the zebrafish whole brain preparation. More studies are needed to determine the underlying causes of this phenomenon.

Apart from acting as a D3 agonist, 7-OH DPAT is also known to inhibit NE release. A study conducted using rat adrenal pheochromocytoma (PC12) cells has found that activation of D3 receptors by 7-OH-DPAT leads to inhibition of norepinephrine via inhibition of forskolin-stimulated cAMP accumulation and carbachol-induced calcium transients.³⁵ DA and NE have similar electrochemical properties and are difficult to distinguish by FSCV.⁵⁶ Therefore, we applied desipramine after the DA signal was diminished by 7-OH-DPAT to confirm that the effect we see in our FSCV data is entirely due to the suppression of DA release. Desipramine is a selective inhibitor of the norepinephrine transporter (NET).⁵⁷ Our results demonstrated that perfusion with desipramine did not affect DA release, depleted by 7-OH-DPAT (Fig. 3). Thus, NE was not present in appreciable amounts at the site of our recordings.

As shown in Fig. 4, antagonism of D3 receptors with SB277011A elevated DA overflow in the zebrafish brain. Several studies have used this drug to characterize D3 receptor function in rats. Acute administration of SB277011A in male Sprague–Dawley rats resulted in increased extracellular levels of DA, norephreneprine, and acetylcholine.⁵⁸ In another study, administration to rats dose-dependently reversed decreases of DA efflux caused by the non-selective DA receptor agonist quinelorane in the nucleus accumbens, while there was no effect in the striatum, accordant with the D3 receptor localization in the rat brain.³⁷ Anatomical data suggest that the Vd, the brain region from where we obtained measurements in zebrafish, is analogous to the nucleus accumbens in rodents.³¹ Therefore, our current findings on the effects of D3 preferring antagonists, as well as data showing that D2 receptors modulate DA³⁰ release (SF. 2), might be beneficial for modeling substance abuse disorders in zebrafish.

The abundance of D3 receptors in the brain is low compared with D2 receptors (2 orders of magnitude).¹⁰ The agonist, 7-OH-DPAT, is selective for D3 receptors, with 100-, 1000-, and 10,000- fold lower affinities for D2, D4, and D1 receptors respectively.¹⁰ In order to evaluate the selectivity of 7-OH-DPAT toward D3 receptors in the zebrafish brain, we blocked the D2 receptors with the selective antagonist L-741,626. We observed that D2 receptor blockade by L-741,626 increased DA release while subsequent cumulative dosing with 7-OH-DPAT in the presence of L-741,646 caused dopamine release to decrease. Thus, these data suggest that D3 agonism prevails over D2 antagonism.

Our findings also suggest that interactions between the D3 receptor and DAT may occur. Antagonism of DA receptors with SB277011A revealed an increase in DA overflow and a decrease in the first-order rate constant (k) that we obtained from curve modeling, indicating a decrease in uptake. This decrease in the uptake rate might indicate that antagonism of D3 receptors affects the DAT function. D3R antagonism has been known to reduce addiction-relevant behaviors in various animal models. Inhibition of cocaine and nicotine self administration as well as depletion of cravings for the drugs after withdrawal have been observed with SB277011A^{59, 60}. Selective blockade of D3 receptors with antagonists has attenuated the self-administration of opioids under several schedules of reinforcement along with opioid-seeking behaviors⁶¹. Furthermore, attenuation of reduced cue-, stress- and drug-influenced recurrence in cocaine self-administration models, as well as diminished cocaine governed place preference, have been shown by SB-277011-A.⁶² These behavioral responses relate to the modulation of DA dynamics by the effects of D3 antagonism on both DA overflow and clearance.

The modulation of DAT activity allows DA neurons to adjust clearance rates according to short-term or long-term physiological requirements.⁶³ Moreover, interactions between DAT and DA receptors are well known. Several proteins, including plasma membrane receptors and kinases, interact with DAT, thereby modulating its catalytic activity and trafficking.^64,13,65 A few studies have confirmed a physical interaction among intracellular loops of the D2 receptors and the DAT N-terminus.^66,67,68 Also, D3 receptor activation has previously been found to decrease DA uptake in the mammalian striatum.⁶⁹ However, the duration of agonist exposure may affect DAT function differentially,⁷⁰ with acute activation increasing DAT expression in the plasma membrane, thereby increasing reuptake.⁷¹ The pre-synaptic localization of D3 receptors might also facilitate these interactions between DAT and other DA receptors.⁷²

Treatment of brains with nomifensine significantly increased stimulated DA overflow.³⁰ When we treated brains with 7-OH-DPAT in the presence of nomifensine, [DA]_max was unaffected (Fig. 6). DA reuptake rate constants obtained by nonlinear regression modeling of stimulated release plots indicated an increase in k values after perfusion 7-OH-DPAT. It was previously reported that cocaine’s potential to impede DA uptake was enhanced by D3 receptor inhibition with SB277011A. However, this study found that cocaine-induced DA uptake was not affected by the application of the D3 agonist, (+)-PD 128907.⁷³ Another study has reported a significant (+)-PD 128907 induced elevation of the initial clearance rate of DA in the in tissue suspensions from nucleus accumbens of rats.⁷⁴ The results from our study indicate that D3 receptor activation with 7-OH-DPAT enhances DA uptake rate and, therefore, decreases nomifensine-induced DA efflux in the brain. Also, the addition of nomifensine overcame the inhibitory effects of D3 agonism with 7-OH-DPAT (Fig. 7). When taken with the results obtained while antagonizing the D3 receptors with SB277011A, these findings support a model in which blockade of D3 autoreceptors decreases DA uptake, and activation of the D3 receptors facilitates DA uptake, possibly by interactions of the receptors with DAT.⁷⁵ In summary, the results of the current study contribute to the growing body of evidence indicating that DAT function is affected by the D2/D3 receptor function.

Conclusion

In the present study, we used FSCV at carbon-fiber microelectrodes to electrochemically characterize D3 autoreceptor function in ex vivo zebrafish whole brains. We have shown that the D3 receptor activation by selective D3 agonists, pramipexole, and 7-OH-DPAT inhibited evoked DA release while D3 selective antagonist SB277011A increased the DA release and decreased the rate of DA reuptake. Additionally, inhibition of DAT by nomifensine masked the inhibitory effects of D3 autoreceptors exerted by 7-OH-DPAT. Together, these findings provide new evidence that the zebrafish D3 receptors have similar pharmacological effects from the administration of both clinical and experimental drugs targeting human D3 receptors compared to mammalian systems. D3 receptor activation may also affect the regulation of neuronal development and neuroprotection.⁷⁶ Given their strong affinity for DA, these receptors could be a useful target when developing therapies for dopaminergic disorders, such as Parkinson’s disease.^8,77 Therefore, this study enhances recent efforts to establish zebrafish as a useful model for studying human neurological disorders. To our knowledge, this is the first work published in the peer-reviewed literature that systematically examines the function of D3 receptors in zebrafish. In the future, it will be important to develop zebrafish as a model organism for neurological disorders related to D3 receptor signaling, such as Parkinson’s disease, schizophrenia, and substance abuse disorders.

Abbreviations:

aCSF

Artificial cerebrospinal fluid

DA

Dopamine

DAT

Dopamine transporter

FSCV

Fast scan cyclic voltammetry

GPCRs

G-protein coupled receptors

NE

Norepinephrine

Vd

ventral telencephalon