Neurochemistry International xxx (2013) xxx–xxx

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Neurochemistry International

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b-Phenylethylamine requires the dopamine transporter to increaseextracellular dopamine in Caenorhabditis elegans dopaminergicneurons

0197-0186/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.neuint.2013.10.010

Abbreviations: DA, dopamine; DAT, DA transporter; bPEA, b-Phenylethylamine;VMAT, vesicular monoamine transporter.⇑ Corresponding author. Address: Department of Basic Science, University of

North Dakota, 504 Hamline St., Grand Forks, ND 58203, United States. Tel.: +1 701777 2293.

E-mail address: lucia.carvelli@med.und.edu (L. Carvelli).1 Present address: Department of Biomedical Sciences, University of Creighton,

2500 California Plaza, Omaha, NE 68178, United States.

Please cite this article in press as: Hossain, M., et al. b-Phenylethylamine requires the dopamine transporter to increase extracellular dopamine inrhabditis elegans dopaminergic neurons. Neurochem. Int. (2013), http://dx.doi.org/10.1016/j.neuint.2013.10.010

Murad Hossain, Rochelle N. Wickramasekara 1, Lucia Carvelli ⇑Department of Basic Science, University of North Dakota, School of Medicine and Health Sciences, Grand Forks, ND 58202-9037, United States

a r t i c l e i n f o

Article history:Available online xxxx

Keywords:Dopamine transporterb-PhenylethylamineDopamine releaseElectrophysiologyCaenorhabditis elegans

a b s t r a c t

b-Phenylethylamine (bPEA) is an endogenous amine that has been shown to increase the synaptic levelsof dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT)plays a role in the effects generated by bPEA, however the mechanism through which bPEA affects DAThas not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1cells and neuronal cultures to investigate whether the bPEA-induced increase of extracellular DA requiredDAT-1. Our data show that bPEA increases extracellular dopamine both in DAT-1 transfected cells andcultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blockedthe bPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibitedthe increase of extracellular DA generated by bPEA. These results suggest that bPEA requires DAT-1 andother, not yet identified proteins, to increase extracellular DA when tested in a native system. Further-more, our results suggest that bPEA-induced increase of extracellular DA does not require functionalmonoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter,cat-1, did not compromise the ability of bPEA to increase extracellular DA. Finally, our electrophysiologydata show that bPEA caused fast-rising and self-inactivating amperometric currents in a subset ofwild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, thesedata demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegansneurons, bPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellularconcentration of DA.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

b-Phenylethylamine (bPEA) is an endogenous trace aminepresent in the central nervous system, however, its role in themammalian physiology is still unknown. Previous studiesdemonstrated that bPEA is synthesized in neurons that also containtyrosine hydroxylase and coexists with dopamine (DA) in thenigrostriatal brain regions (Juorio et al., 1991). In striatal tissue,bPEA synthesis occurs with a rate similar to that of DA, but sinceit is more efficiently metabolized by monoamine oxidase enzymes,striatal bPEA concentrations are about three orders of magnitude

lower than DA levels (Paterson et al., 1990). These data suggestthat it is crucial the DA neurons keep low concentrations of bPEAto guarantee a proper physiological activity. In fact, changes in uri-nary bPEA levels have been documented in various human disor-ders including schizophrenia, attention deficit hyperactivedisorder (ADHD) and depression (Baker et al., 1991; O’Reilly andDavis, 1994; Sandler et al., 1980). A direct interaction betweendopaminergic neurons and bPEA has also been demonstrated inin vivo and in vitro experiments showing that bPEA induces DA re-lease (Bailey et al., 1987; Ishida et al., 2005; Kuroki et al., 1990;Nakamura et al., 1998; Sotnikova et al., 2004; Yamada et al.,1998), and inhibits DA uptake (Liang and Rutledge, 1982; Raiteriet al., 1976). Moreover, in vivo studies showed that physiologicalbPEA concentrations directly and transiently inhibit the firing rateof the DA neurons through the activation of the DA D2 autorecep-tors (Ishida et al., 2005; Mercuri et al., 1997; Rodriguez andBarroso, 1995). Interestingly, the firing inhibition caused by bPEAas well as bPEA-induced behaviors (Barroso and Rodriguez, 1996)were not affected by pretreatment with the vesicular monoamine

Caeno-

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transporter (VMAT) blocker reserpine. These data suggested thatbPEA stimulates the release of DA from a non-vesicular cytoplas-mic pool.

In this study, we investigated the effect of bPEA on extracellularlevels of DA in Caenorhabditis elegans cultured neurons. We foundthat in isolated DA neurons, bPEA requires DAT to induce transientDA efflux. Furthermore, our data suggest that bPEA-induced DAefflux utilizes cytosolic DA since genetic ablation of VMAT didnot affect the increase of extracellular DA induced by bPEA.

transfected cells

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Fig. 1. bPEA increases the amount of extracellular DA in DAT-1 transfected cells.When DAT-1 expressing LLC-pk1 cells were treated for 1 min with 100 lM bPEA,we measured a significant increase of extracellular [3H]DA with respect to control-treated samples (⁄p = 0.0001). Cells treated with bPEA/RTI-55 showed a significantreduction in extracellular [3H]DA with respect to bPEA-treated cells (�p = 0.05, one-way ANOVA). RTI55 applied alone did not have any effect.

2. Materials and methods

2.1. C. elegans husbandry and transgenic animals

Wild-type animals (N2) and knockout animals for DAT-1(dat-1(ok157)III) and the C. elegans VMAT homologue CAT-1(cat-1(ok411) were obtained from the Caenorhabditis Genetic Cen-ter (University of Minnesota, Minneapolis, MN, USA). All C. elegansstrains were grown on bacteria lawns of NA22 and maintained at22–24 �C using standard methods (Brenner, 1974). For amperome-try recordings, we used the BY250 strain (gift from Dr. Blakely,Vanderbilt University) expressing cytosolic GFP under the controlof the dat-1.

2.2. Extracellular [3H]DA in DAT-1 transfected cells and C. elegansembryonic cultures

C. elegans embryonic cultures were prepared as previously de-scribed (Carvelli et al., 2004; Strange and Morrison, 2006). Embry-onic cells were seeded on 22 � 22 mm glass cover slips coated withpeanut lectin (Sigma) and maintained with L15 media (GIBCO) en-riched with 5% inactivated FBS and 100 units/ml of penicillin andstreptomycin. Cells were assayed 2 days after been seeded. LLC-pk1 (pig epithelial kidney) cells were grown in DMEM medium en-riched with 5% FBS, 2 mM L-glutamine, 100 units/ml penicillin and100 mg/ml streptomycin. 100,000 cells were seeded into 12 wellplates and after 18–24 h were transfected with 0.5 lg DAT-1 inthe pEGFP vector using X-tremeGENE HP DNA transfection reagent(Roche). Control cells were transfected with pEGFP vector alone.24–30 h after transfection, cells were washed 2 times with KRHbuffer (120 mM NaCl or MNDG+/4.7 mM KCl/1.2 mM KH2PO4/10 mM Hepes/2.2 CaCl2/10 mM glucose) containing 100 lM ofascorbic acid and tropolone (Sigma) and incubated for 30 min atroom temperature with 20 nM [3H]DA (PerkinElmer). Cells werethen washed 3 times with ascorbic acid/tropolone containingKRH buffer and treated with drugs (bPEA and/or RTI-55) or KRH(control) for 1 min. Supernatant was collected from each welland counted for radioactivity. We used the same protocol to mea-sure extracellular concentration of [3H]DA in cultured C. elegansneurons except 5 nM of [3H]DA was used and the buffer contained145 mM NaCl or NMDG+, 5 mM KCl, 1 mM CaCl2, 5 mM MgCl2,10 mM Hepes, and 20 mM D-glucose (pH 7.2 and 350 osmolarity).

2.3. Amperometric recordings

4–5 days after C. elegans embryonic cells were seeded in peanutlectin coated glass dishes (MatTech Corporation, Cincinnati, OH,USA), cells were washed twice with bath solution containing145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 5 mM MgCl2, 10 mM HEPES,and 20 mM D-glucose (pH 7.2; 325 osmolarity adjusted withsucrose). DA fluxes were induced by applying 50 lM bPEA and re-corded using a carbon fiber electrodes connected to an Axopatch200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Electrodeswere placed in proximity to an isolated dopaminergic neuronexpressing cytosolic GFP. Amperometric currents were digitized

Please cite this article in press as: Hossain, M., et al. b-Phenylethylamine requirhabditis elegans dopaminergic neurons. Neurochem. Int. (2013), http://dx.doi.

with an Axon Digidata 1440A, collected with pClamp 10.2(Molecular Devices), filtered with a low-pass Bessel filter set at5 Hz, and analyzed using pClampfit 10.2 (Molecular Devices) andOrigin 8.5 software (OriginLab Corp, Northampton, MA, USA).

3. Results and discussion

3.1. b-Phenylethylamine increases extracellular levels of DA in DAT-1transfected cells and C. elegans cultured embryonic cells

Previous studies showed that bPEA induces DA release and/orinhibits DA uptake. We investigated whether bPEA increased extra-cellular DA in cultures of LLC-pk1 cells expressing C. elegans DAT(DAT-1). After preloading with 20 nM [3H]DA, cells were treatedwith 100 lM bPEA for 1 min. As shown in Fig. 1, bPEA caused a sta-tistically significant increase (120 ± 20%) of extracellular [3H]DAwith respect to control treated samples (one-way ANOVA test;⁄p = 0.0001), whereas no change in extracellular [3H]DA was mea-sured in non-transfected cells (data not shown). Previous reportsdemonstrated that DA uptake through DAT-1 is specifically antag-onized by monoamine transporters inhibitors such as mazindol,nisoxetine and cocaine (Jayanthi et al., 1998). Here we testedwhether RTI-55, a cocaine homologue, was also capable of blockingthe bPEA-induced increase of extracellular [3H]DA in DAT-1 trans-fected cells. We found that 100 lM RTI-55 completely blocked theeffect of bPEA on the extracellular levels of [3H]DA (Fig. 1, gray bar).In fact, bPEA/RTI-55 treatment caused a decrease of extracellular[3H]DA comparable to control-treated samples, 109 ± 12% and100 ± 2%, respectively. The extracellular levels of [3H]DA followingbPEA/RTI-55 treatment were statistically different than those mea-sured after bPEA treatment (one-way ANOVA test, p = 0.05). To testwhether RTI-55 itself had an effect on extracellular [3H]DA, wetreated DAT-1 transfected cells with RTI-55 alone (stripe bar,Fig. 1) and found that 100 lM RTI-55 caused no effect on extracel-lular [3H]DA with respect to controls, 108 ± 12% and 100%, respec-tively. Taken together, these data demonstrate that bPEA causes anincrease of extracellular DA in DAT-1 transfected cells which isselectively blocked by the DAT-1 inhibitor RTI-55.

Previously, it was shown that cultured C. elegans embryoniccells are instrumental in studying the activity of proteins in theirnative cells. C. elegans embryonic cells, which undergo terminaldifferentiation within a few days after being seeded, have beenused to characterize protein function and regulation and to deter-mine specific gene expression patterns (Strange and Morrison,2006; Carvelli et al., 2008). Particularly, we used this preparationto investigate the ability of DAT-1 to accumulate [3H]DA in thedopaminergic neurons and showed that like the mammalianhomologue, C. elegans DAT-1 efficiently accumulated DA, whereas

res the dopamine transporter to increase extracellular dopamine in Caeno-org/10.1016/j.neuint.2013.10.010

M. Hossain et al. / Neurochemistry International xxx (2013) xxx–xxx 3

genetic and pharmacological ablation of DAT-1 prevented [3H]DAuptake (Carvelli et al., 2004). Importantly, our previous studiesdemonstrated that functional characterization of DAT-1 in differ-entiated neurons recapitulates the pharmacological properties ob-served in mammalian DAT expressed in heterologous systems. Inthis study, we used differentiated cultures of C. elegans embryoniccells to investigate the effects of bPEA on dopaminergic neurons.Three days after preparation, we preloaded C. elegans embryoniccells with 5 nM [3H]DA and then applied 100 lM bPEA for 1 min.Similarly to what we saw in DAT-1 transfected cells, bPEA signifi-cantly increased the amount of extracellular [3H]DA (one-way AN-OVA test, ⁄p = 0.0001). We measured a 157 ± 12% increase inextracellular [3H]DA following bPEA treatment with respect to con-trol treated samples (Fig. 2). When bPEA was applied together with100 lM RTI-55 we measured an increase of 85 ± 34% in extracellu-lar [3H]DA with respect to control-treated samples (Fig. 2, graybar). Interestingly, this value was statistically different to both con-trol- and bPEA-treated sample (⁄p = 0.0001 vs controls andp = 0.001 vs bPEA-treated samples). This result showed that in pri-mary cultures RTI-55 partly blocked the bPEA-induced increase inextracellular DA, and RTI-55 applied alone did not change theextracellular amount of [3H]DA with respect to controls (Fig. 2,stripe bar), 126 ± 11% and 100%, respectively.

In conclusion, these results demonstrate that bPEA caused anincrease of extracellular DA in DAT-1 transfected cells and differen-tiated C. elegans neurons. Moreover, the ability of bPEA to increaseextracellular DA was similar in DAT-1 transfected cells (120%) andneuronal cultures (157%), suggesting that DAT-1 accounts for theincrease of extracellular DA measured after bPEA treatment.Interestingly though, in neuronal cultures the DAT-1 blocker RTI-55 only partly inhibited the bPEA-induced increase of extracellularDA. This result suggests that in native neurons DAT-1 and other un-known proteins are required by bPEA to elevate extracellular DA.Perhaps the DA D2 receptors as well as low affinity neurotransmit-ter transporters might be involved (Ishida et al., 2005; Mercuri et al.,1997; Rodriguez and Barroso, 1995; Horton et al., 2013), however,we can not exclude that a dose-related dependency of bPEA and/or RTI-55 in transfected cells versus neuronal culture underliesthe different effect of RTI-55 seen in these two preparations.

3.2. b-Phenylethylamine-induced increase of extracellular dopaminedoes not involve vesicle-dependent dopamine release

Neurotransmitters like DA and serotonin are released in thesynaptic cleft through two different mechanisms: a vesicle-depen-dent mechanism or a Ca2+-independent and DAT mediated mecha-nism. In C. elegans, all major proteins involved in the dopaminergicsystem have been identified (Chase and Koelle, 2007), including

wild-type neurons

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Fig. 2. bPEA increases the amount of extracellular DA in C. elegans differentiatedneuronal cultures. 100 lM bPEA applied for 1 min in C. elegans cells caused asignificant increase of extracellular [3H]DA with respect to control-treated cells(⁄p = 0.0001, one-way ANOVA). RTI-55 partly inhibited the bPEA-induced effect(⁄p = 0.0001 vs. controls and p = 0.001 vs. bPEA-treated samples, one-way ANOVA),but caused no effect when applied alone.

Please cite this article in press as: Hossain, M., et al. b-Phenylethylamine requirhabditis elegans dopaminergic neurons. Neurochem. Int. (2013), http://dx.doi.

the vesicular monoamine transporter (VMAT) homologue CAT-1.Moreover, animals made knockout for cat-1 have been previouslycreated and characterized (Duerr et al., 1999). We investigatedwhether the effects of bPEA seen in Fig. 2 required intact DA vesi-cles. Thus, we measured the increase of extracellular [3H]DA in-duced by bPEA in cultures of differentiated cells obtained fromcat-1 knockout animals. We found that bPEA significantly(⁄p = 0.01; t-test) augmented the extracellular amount of [3H]DAof about 110 ± 32% with respect to control treated cells (Fig. 3A).The comparison of bPEA effects between cat-1 knockout and wildtype neurons did not show a statistically significant difference,110 ± 32 and 137 ± 12, respectively (Fig. 3B). These data demon-strated that genetic ablation of cat-1 did not compromise the abil-ity of bPEA to increase extracellular DA in native neuronal cultures,suggesting that CAT-1 (VMAT) does not play a significant role inthe bPEA-induced increase of extracellular DA. Therefore, thesedata suggest that the elevated levels of extracellular DA measuredduring bPEA treatment are mediated by DAT-1.

3.3. bPEA generates amperometric currents in a subset of C. elegans DAneurons

Previously, we demonstrated that amphetamine promoted DArelease through DAT-1 (efflux) in C. elegans dopaminergic neurons(Carvelli et al., 2010). Since our data (Fig. 3) demonstrated thatbPEA-induced increase of [3H]DA does not occur through vesiclerelease, we investigated whether bPEA induces DA efflux bymeasuring amperometric currents. Microamperometry is a tech-nique which allows detecting DA by measuring the electrons re-leased during its oxidizing reaction. When DA touches a carbonfiber electrode held at a voltage of +700 mV, each DA molecule re-leases two electrons that are detected by the same carbon fiberelectrode. Thus, DA efflux stimulated by bPEA can be detected bya carbon fiber electrode as a measure of DA oxidation (Carvelli,2010, Khoshbouei et al., 2003; Schroeder et al., 1994). When C. ele-gans DA neurons engineered to express cytosolic GFP (Fig. 4A) wereperfused with 50 lM bPEA, we observed fast-rising and outwardamperometric currents (0.26 ± 0.02 pA) in 44% of DA neuronstested (Fig. 4B and E). This result demonstrated that bPEAtransiently increases the extracellular levels of DA in proximityof a single dopaminergic neuron. However, 33 dopaminergic neu-rons of 59 total neurons tested exhibited no effect following bPEAapplication (Fig. 4C). Interestingly, the amperometric currents,which reached their maximal levels within 11 ± 0.5 s after bPEAperfusion, spontaneously returned to basal levels after 24 ± 3 seven though bPEA was still present in the recording chamber(Fig. 4B and F). To test whether DAT-1 was required in bPEA-in-duced currents, we performed recordings in DA neurons lackingexpression of DAT-1. bPEA did not cause significant amperometriccurrents (0.016 ± 0.004 pA) in any of the dat-1 knockout neurons(N = 20) we tested (Fig. 3D and E). This result demonstrated thatthe increase of extracellular DA measured during bPEA treatmentrequired a functional DAT-1 and suggested that bPEA needs DAT-1 to increase extracellular DA.

Taken together, these data demonstrate that in cultures of C.elegans DA neurons bPEA generates amperometric currents. More-over, they show that about 50% of dopaminergic neurons did notrespond to bPEA treatment suggesting, as previously shown formammalian DA neurons, that physiological differences might existamong different subtypes of C. elegans DA neurons (Grenhoff et al.,1988; Murata et al., 2009; Li et al., 2012; Weihe et al., 2006).Importantly, our data support that the amperometric currentsinduced by bPEA are mediated by DAT-1. In fact, in DA neuronslacking expression of dat-1, bPEA did not generate amperometriccurrents.

res the dopamine transporter to increase extracellular dopamine in Caeno-org/10.1016/j.neuint.2013.10.010

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Fig. 3. VMAT is not required by bPEA to increase the extracellular levels of DA. (A) In cat-1 (VMAT) knockout neurons, 100 lM bPEA caused a significant increase ofextracellular [3H]DA with respect to control-treated cells (⁄p = 0.01, t-test). (B) The increase of extracellular DA measured in cat-1 knockout neurons was comparable to thatmeasured in wild-type neurons.

Fig. 4. bPEA generates amperometric currents in a subset of dopaminergic neurons. (A) C. elegans dopaminergic neuron visualized with cytosolic GFP. (B) Representativerecording of fast-rising and self-inactivated amperometric currents generated by 50 lM bPEA. (C) Representative recording from a subset of dopaminergic neurons that didnot respond to bPEA. (D) Representative recording from a DAT-1 knockout neuron treated with 50 lM bPEA. (E) Average of amperometric currents measured in wild-type(N = 26) and dat-1 knockout (N = 20) neurons (⁄p = 0.0001, t-test). (F) Average of time required to activate and inactivate the bPEA-induced amperometric currents.

4 M. Hossain et al. / Neurochemistry International xxx (2013) xxx–xxx

4. Conclusion

In the brain, the amount of extracellular DA is controlled by atight balance between DA release and uptake events. In vitro andin vivo studies have showed that bPEA enhances the extracellularlevels of DA by inducing DA release and/or preventing DA uptakethrough mammalian DAT. Here we found that in C. elegans neuro-nal cultures, the bPEA-induced increase of extracellular DA waspartly inhibited by the DAT blocker RTI-55, whereas RTI-55 didnot have any effect when applied alone (Fig. 2). Moreover, our datademonstrate that bPEA-induced release of DA requires DAT. Thisconclusion was supported by two major observations: (I) the in-

Please cite this article in press as: Hossain, M., et al. b-Phenylethylamine requirhabditis elegans dopaminergic neurons. Neurochem. Int. (2013), http://dx.doi.

crease of extracellular DA induced by bPEA in cat-1 (VMAT) knock-out neurons was comparable to that measured in wild-typeneurons (Fig. 3B), and (II) transient amperometric DA currents gen-erated by bPEA were lost in neurons lacking expression of DAT-1(Fig. 4). Interestingly, our data show that in DAT-1 transfectedcells, RTI-55 completely blocked bPEA effect on extracellular levelsof [3H]DA whereas, in neuronal culture this effect was only partial.These results could simply reflect a different dose-related depen-dency of bPEA and/or RTI-55 in transfected cells versus neuronalculture or suggest that bPEA involved proteins other than DAT-1,e.g., low affinity neurotransmitter transporter and/or DA D2 recep-tors (Horton et al., 2013; Duan and Wang, 2010; Ishida et al., 2005;

res the dopamine transporter to increase extracellular dopamine in Caeno-org/10.1016/j.neuint.2013.10.010

M. Hossain et al. / Neurochemistry International xxx (2013) xxx–xxx 5

Mercuri et al., 1997; Rodriguez and Barroso, 1995). Finally, ourdata demonstrate that C. elegans represents a powerful geneticmodel to perform in vitro studies in differentiated dopaminergicneurons.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgment

The authors thank the support from NIH Grant R21 DA024797and the NIH funded COBRE P20 GM103329.

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