drosophila melanogaster as a genetic model system to study...

Neurochemistry International xxx (2014) xxx–xxx

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Neurochemistry International

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Review

Drosophila melanogaster as a genetic model system to studyneurotransmitter transporters

http://dx.doi.org/10.1016/j.neuint.2014.03.0150197-0186/� 2014 Published by Elsevier Ltd.

Abbreviations: 5HT, 5-hydroxytryptamine (serotonin); ACh, acetylcholine; ASD, autism spectrum disorder; ChT, choline transporter; ChAT, choline acetyl transfedopamine; Ddc, dopa decarboxylase; EAAT, excitatory amino acid transporter; GABA, gamma amino butyric acid; GAT, GABA transporter; KC, Kenyon cell; LDCV, larcore vesicle; Mas, Manduca sexta (tobacco hornworm); MAO, Monoamine Oxidase; MB, mushroom body; MDMA, 3,4-methylenedioxy-N-methylamphetamine (Ecstoctopamine; NMJ, neuromuscular junction; SV, synaptic vesicle; TNT, tetanus toxin; T. ni or Trn, Trichoplusia ni (cabbage looper caterpillar/moth); TbH, tyrhydroxylase; Tdc, tyrosine decarboxylase; TH, tyrosine hydroxylase; VGAT, vesicular GABA transporter; VGLUT, vesicular glutamate transporter; VMAT, vesicular motransporter; UAS, (yeast) upstream activating sequence.⇑ Corresponding author. Address: Department of Psychiatry and Biobehavioral Sciences and Semel Institute for Neuroscience and Human Behavior, Hatos C

Neuropharmacology, Gonda (Goldschmied) Neuroscience and Genetics Research Center, Room 3357C, 695 Charles Young Drive South, David Geffen School of MediAngeles, CA 90095-1761, United States. Tel.: +1 310 206 8508.

E-mail address: [email protected] (D.E. Krantz).

Please cite this article in press as: Martin, C.A., Krantz, D.E. Drosophila melanogaster as a genetic model system to study neurotransmitter transpNeurochem. Int. (2014), http://dx.doi.org/10.1016/j.neuint.2014.03.015

Ciara A. Martin a, David E. Krantz b,⇑a UCLA Interdepartmental Program in Molecular Toxicology, United Statesb Department of Psychiatry and Biobehavioral Sciences and Semel Institute for Neuroscience and Human Behavior, Hatos Center for Neuropharmacology, David Geffen Schoolof Medicine University of California, Los Angeles, CA 90095, United States

a r t i c l e i n f o

Article history:Available online xxxx

Keywords:DrosophilaNeurotransmitter transporterVesicular transporterVMATVGATVGLUTVAChTEAATDATSERTGATInebriatedSLC6SLC1SLC18SLC17DopamineSerotoninGABAOctopamineGlutamatePortabellaAcetylcholineChT1VNUT

a b s t r a c t

The model genetic organism Drosophila melanogaster, commonly known as the fruit fly, uses many of thesame neurotransmitters as mammals and very similar mechanisms of neurotransmitter storage, releaseand recycling. This system offers a variety of powerful molecular-genetic methods for the study of trans-porters, many of which would be difficult in mammalian models. We review here progress made usingDrosophila to understand the function and regulation of neurotransmitter transporters and discuss futuredirections for its use.

� 2014 Published by Elsevier Ltd.

1. Introduction transporters. These include a powerful molecular-genetic toolset,

Several features of the model genetic system Drosophila melano-gaster make it attractive for the study of neurotransmitter

a short lifespan, low cost and the availability of an essentiallylimitless supply of ‘‘test subjects’’. Here we provide an overviewof current studies on neurotransmitter transporters in Drosophila

rase; DA,ge denseasy); OA,amine bnoamine

enter forcine, Los

orters.

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2 C.A. Martin, D.E. Krantz / Neurochemistry International xxx (2014) xxx–xxx

and discuss potential uses for this system in the future. By includingadditional background, we hope to provide a general introductionto the field for both Drosophilists and non-Drosophilists alike.Conversely, for ‘‘fly people’’ with other primary interests, we alsoprovide an overview of neurotransmitter transporters in generaland some of the outstanding questions that remain unanswered.

2. Neurotransmitter transporters

Neurotransmitter transporters are responsible for the move-ment across biological membranes of classical neurotransmitters– the biogenic amines and acetylcholine – and the amino acid neu-rotransmitters – GABA, glutamate and glycine. Plasma membraneand vesicular neurotransmitter transporters represent two distinctactivities (Fig. 1). Plasma membrane neurotransmitter transportersare responsible for the termination of synaptic transmission andrecycling neurotransmitters after they are released (Blakely andEdwards, 2012) (Fig. 1). Vesicular neurotransmitter transporterslocalize to the membranes of secretory vesicles and are responsiblefor transport and storage of neurotransmitters into the vesiclelumen (Blakely and Edwards, 2012) (Fig. 1).

Vesicular transporters are required for the storage of neuro-transmitters in synaptic vesicles (SVs) as well as large dense corevesicles (LDCVs), which also store and release peptide neurotrans-mitters (Fei et al., 2008). However, peptides are not transportedinto LDCVs via vesicular transporters, but rather packaged intothe lumen of the vesicle as it is being formed (Dikeakos andReudelhuber, 2007). Peptides also do not undergo transport atthe plasma membrane. Similarly, ‘‘novel’’ neurotransmitters suchas nitrous oxide do not require specific transport proteins sincethey are synthesized on demand and pass relatively freely throughlipid membrane barriers (Boehning and Snyder, 2003). It remainsunclear whether lipid-based signaling molecules such as ananda-mide require specific transporters for movement across biologicalmembranes (Fowler, 2013).

All known plasma membrane and vesicular transporters aremembers of the Solute Carrier (SLC) family of proteins and shareseveral characteristic features (Blakely and Edwards, 2012). Allcontain multiple transmembrane domains and are responsible formoving relatively small (�75–200 Da) hydrophilic moleculesacross lipid membranes. As ‘‘active’’ transporters, all use energyto drive the movement of neurotransmitter against a concentrationgradient, and as ‘‘secondary’’ active transporters, the energy is sup-plied by an ion gradient rather than by direct hydrolysis of ATP. Bycontrast, the activities of ‘‘facilitated’’ transporters allow solutemovement down a concentration gradient and do not require

HNT

Glia

PresynapticSynapseNeuron

NaNT

Na NT

PlasmaMembrane

SynapticVesicle

PostsynapticNeuron

Fig. 1. Vesicular and plasma membrane neurotransmitter transporters. Vesicularand plasma membrane neurotransmitter transporters differ in several respects,including their localization and bioenergetics. In general, vesicular transporterslocalize to secretory vesicles, including synaptic vesicles in the presynaptic neuronsthat synthesize and release neurotransmitter. Depending on their subtype (see text)plasma membrane transporters may localize to either presynaptic neurons orsurrounding glia. The movement of neurotransmitter via vesicular and plasmamembrane transporters are coupled to proton and sodium gradients respectively.Vesicular transporters move protons (H) and neurotransmitter (NT) in oppositedirections (antiport), using the high concentration of lumenal protons to drivetransport. At the plasma membrane, sodium (Na) and neurotransmitters move inthe same direction (symport).

Please cite this article in press as: Martin, C.A., Krantz, D.E. Drosophila melanoNeurochem. Int. (2014), http://dx.doi.org/10.1016/j.neuint.2014.03.015

additional energy (e.g. (Richter and Hargreaves, 2013)), whileprimary active transporters directly hydrolyze ATP (e.g.(Dermauw and Van Leeuwen, 2014).

At the plasma membrane, neurotransmitter transporters exploitthe steep sodium gradient experienced by most eukaryotic cells.The sodium gradient drives the movement of the transporter andis coupled to neurotransmitter flux in a stoichiometrically definedmanner, and in some cases with additional ions facilitating trans-port (Grewer et al., 2014; Kanai et al., 2014; Pramod et al., 2013;Rudnick et al., 2014). Since both sodium and the neurotransmittermove in the same direction at the plasma membrane, this is by def-inition a ‘‘symport’’ mechanism. Vesicular transporters use a dis-tinct but related ‘‘antiport’’ mechanism in which a protongradient is used to drive the movement of neurotransmitter inthe opposite direction and into the lumen of the secretory vesicle(Parsons, 2000) (Fig. 1).

The manner in which the transporters couple the movement ofions and neurotransmitter is not yet fully understood, but bio-chemical experiments and more recent crystallographic and mod-eling studies have begun to unravel the underlying mechanisms(Penmatsa and Gouaux, 2014; Zhao et al., 2011). Plasma membranetransporters may also function in an uncoupled or loosely coupledmode characterized by relatively large ionic currents (Fairmanet al., 1995; Galli et al., 1997; Schicker et al., 2013). The potentialfunction of these currents in synaptic physiology is not yet clear.Efflux represents an additional functional mode in which neuro-transmitter moves out of rather than into the cell (Fleckensteinet al., 2007; Sulzer, 2011). The mechanism by which this occursis a very active area of investigation, and includes work in Drosoph-ila described in more detail below (Pizzo et al., 2014, 2013).

Vesicular transporters generally localize to secretory vesicles inthe presynaptic neurons that synthesize and release neurotrans-mitter (Blakely and Edwards, 2012). Plasma membrane transport-ers for biogenic amines also localize to presynaptic neurons(Blakely and Edwards, 2012). By contrast plasma membrane gluta-mate and GABA transporters may be expressed in either neurons orglia (Fig. 1) depending on their subtype (Zhou and Danbolt, 2013).We discuss the localization of specific Drosophila transportersbelow.

A circumscribed set of phylogenetic families encodes theplasma membrane and vesicular transporters (see Tables 1 and2). Glutamate or excitatory amino acid transporters (EAATs) aremembers of SLC1; this family also contains neutral amino acidtransporters (Grewer et al., 2014; Kanai et al., 2014; Sheldon andRobinson, 2007; Yang et al., 2009). All other known plasma mem-brane transporters, including those for GABA and the biogenicamines, are members of SLC6 (Pramod et al., 2013; Rudnicket al., 2014). SLC6 also includes a number of amino acid transport-ers, some of which are specific for insects, as well as a few remain-ing ‘‘orphans’’, whose substrate is not known (Boudko, 2012;Boudko et al., 2005; Caveney et al., 2006). Acetylcholine is hydro-lyzed before reuptake and there is no plasma membrane acetylcho-line transporter. Rather, following ACh hydrolysis, choline istransported into the presynaptic neuron by a high affinity cholinetransporter, a member of SLC5 (Ferguson et al., 2004; Ribeiro et al.,2006).

Vesicular transporter subfamilies include SCL32, of which VGATis the lone member (Schioth et al., 2014). SLC18 includes vesiculartransporters for the biogenic amines and acetylcholine (Lawal andKrantz, 2013) and SLC17 includes the vesicular glutamate trans-porters as well as other transporters with divergent functions suchas organic anion uptake at the plasma membrane (Reimer, 2013).Another member of SLC17 was recently identified in mammals asa vesicular ATP or nucleotide transporter (VNUT) (Sawada et al.,2008); a possible VNUT ortholog is present in the fly genome buthas not yet been characterized (see Table 1).

gaster as a genetic model system to study neurotransmitter transporters.


Table 1Mammalian and Drosophila vesicular transporters. In both mammals and flies, known vesicular transporters are members of the SLC17, 18 or 32 subfamilies. See text for details.

SLC17 SLC18 SLC32

Mammals Drosophila Mammals Drosophila Mammals Drosophila

VGLUT1 dVGLUT VMAT1 dVMAT VGAT dVGATVGLUT2 VMAT2VGLUT3 VAChT dVAChTVNUT (SLC17A9) CG15438 prt

Table 2Mammalian and Drosophila plasma membrane transporters. In both mammals andflies, known plasma membrane neurotransmitter transporters are members of theSLC1 and 6 subfamilies. As described in the text, dEAAT2 may not function as aglutamate transporter. The choline transporter is a member of SLC5, and although it isnot a neurotransmitter transporter per se, it is responsible for reuptake of choline intothe presynaptic neuron after acetylcholine is metabolized in the synapse. Thepredicted fly gene CG11880 appears to be most similar to mammalian cholinetransporters, but has not been characterized. CG5549 is listed as a putative glycinetransporter based on its similarity to mammalian glycine (and proline) transporters,but its function is not known; it also remains unclear whether glycine functions as aneurotransmitter in the fly. The gene CG7075, recently named NeurotransmitterTransporter-Like (Ntl) is also a putative glycine transporter but found only in malegermline cells (Chatterjee et al., 2011). SLC6 also includes amino acid transportersrequired for nutrient uptake and several additional members whose functions are notknown (not shown) as well as inebriated, which may transport a metabolite ofhistamine (see text).

SLC1 SLC6 (SLC5)

Mammals Drosophila Mammals Drosophila Mammals Drosophila

EAAT1 dEAAT1dEAAT2

DAT dDAT ChT1 CG11880?

EAAT2 SERT dSERTEAAT3 GAT1 dGATEAAT4 GAT2EAAT5 GAT3

GlyT1 (CG7075)CG5549?

GlyT2NET

(Inebriated)

C.A. Martin, D.E. Krantz / Neurochemistry International xxx (2014) xxx–xxx 3

3. Current research topics

Current research on neurotransmitter transporters includesinvestigations into the precise mechanisms by which they movesolutes across membranes. Although some of the binding sitesfor ions and the neuorotransmitters have been mapped, muchremains unclear. At present, we primarily have a static structuralview of the binding sites, with only modeling able to capture themoment-to-moment movement of substrates (Penmatsa andGouaux, 2014; Zhao et al., 2011). A surprisingly large number ofcommonly used drugs are known to bind to plasma membraneneurotransmitter transporters, yet their mechanism of action alsoremains incompletely understood. Many currently used antide-pressants are Strongly Serotonergic Reuptake Inhibitors (SSRIs;e.g. fluoxetine/Prozac) and primarily inhibit the serotonin trans-porter (SERT), while others target the noradrenaline transporter(NET) or both NET and SERT (Hamon and Blier, 2013). Most psy-chostimulants, including cocaine, amphetamines and MDMA/ecstasy also interact with neurotransmitter transporters actingeither as inhibitors or, in the case of amphetamines, to promoteefflux (Fleckenstein et al., 2007; Sulzer, 2011). Importantly, drugsthat act to block transport use a mechanism different from thosethat cause efflux and the nature of these differences remains a veryimportant area of investigation (Fleckenstein et al., 2007; Fosteret al., 2013; Kahlig et al., 2005; Sulzer, 2011). The downstreameffects of both antidepressants and psychostimulants and the


way they exert their therapeutic and/or addictive effects remainsobscure. The developmental regulation of transport function andthe potential effects of pharmacologically disrupting this activityis another fascinating topic (Daubert and Condron, 2010). Therapid development of the fly and the relative simplicity of its ner-vous system may be advantageous for these studies (Daubert andCondron, 2010).

A number of current investigations focus on the interplaybetween transporters and regulatory pathways. Transporters areessential elements of a network of interacting genes required forneurotransmitter homeostasis and behavior (see e.g. (Talkowskiet al., 2008)), and a few elegant biochemical studies have high-lighted direct protein–protein interactions and relatively directregulatory processes (Bauer et al., 2012; Binda et al., 2008; Brunket al., 2006; Sager and Torres, 2011; Winter et al., 2005; Yanget al., 2009). However, compared to our knowledge of the complexweb of proteins implicated in receptor-mediated signaling, ourunderstanding of transporter interactions and their regulation islimited.

Pathways critical for regulating the function of neurotransmit-ter transporters include those required for trafficking within thecell. Localization of vesicular transporters to different subsets ofsecretory vesicles has the potential to dramatically influence thesite and amount of presynaptic neurotransmitter release. Some ofthe signals and machinery responsible for this process have beendetermined, but much remains unclear (Colgan et al., 2007; Fosset al., 2013; Grygoruk et al., 2010; Hua et al., 2011; Santos et al.,2013; Yao and Hersh, 2007). Current questions include the physio-logical function of vesicular transporter co-localization to the samevesicles in some cells. This phenomenon has important ramifica-tions for both the possible co-release of neurotransmitters as wellas the regulation of the electrochemical gradients that drive trans-port activity (Guzman et al., 2011; Hnasko and Edwards, 2011).Drosophila VMAT and VGLUT are co-expressed in at least somenerve terminals (Greer et al., 2005) but co-release has not beeninvestigated in the fly. The trafficking of plasma membrane trans-porters through the secretory pathway and their regulated inter-nalization is a particularly active and exciting area of study, anda number of the intrinsic signals required for internalization aswell as the protein kinases that regulate these processes have beenidentified (Bauer et al., 2012; Chen et al., 2013b; Gabriel et al.,2013; Madsen et al., 2012; Sakrikar et al., 2012; Vina-Vilasecaand Sorkin, 2010). The increasingly refined understanding of theseevents may help determine the mechanism of action of psycho-stimulants, as well as providing fundamental information aboutsynaptic physiology.

Some research questions are particularly well suited to fly mod-els. These include structure–function analyses in vivo and the rela-tionship between transporter function and disease processes.Variation in transporter expression may function as markers fordisease and in some cases may play a pathogenenic role (Hahnand Blakely, 2007; Lawal and Krantz, 2013; Yang et al., 2009). Inaddition, risk alleles for human disease have been associated withsome neurotransmitter transporters (Adamczyk et al., 2011;Barwick et al., 2012; Hahn and Blakely, 2007; Hamilton et al.,



MammalianNeuron

InsectNeuron

GlomerulusCortical Rind

Neuropil

Dendrite

Dendrite

A

B

C


2013; Jen et al., 2005; Mazei-Robison et al., 2008; Veenstra-VanderWeele et al., 2012), however the mechanisms by whichthese variants cause downstream phenotypic changes remainspoorly understood. More generally, it remains unclear how varia-tions in transport activity or regulatory processes may effect syn-aptic transmission and behavior. The powerful molecular genetictools available in the fly coupled with their surprisingly complexarray of behaviors provide an excellent platform to address thesequestions.

Although we focus here on Drosophila, many of the same prin-ciples apply to Caenorhabditis elegans, an equally important inver-tebrate model with a similarly large array of powerful molecularand genetic tools. As highlighted below, comparative studies inother insects including locusts, bees and moths represent anothervaluable source of information about the function and regulationof neurotransmitter transporters.

Fig. 2. The morphology and organization of mammalian and insect neurons. Acanonical mammalian (A) and insect neuron (B) are indicated, highlightingdifferences in the location of dendrites: on the cell body of mammalian neuronsversus a single process emanating from the soma of insect neurons that also givesrise to the axon. This morphology allows the characteristic organization of insectneurons into glomeruli (C), or larger but similarly organized ganglia, which includea cortical rind and neuropil as indicated.

4. A brief fly neurobiology primer

To discuss the molecular genetics of fly transporters it is usefulto first briefly describe a few characteristics of fly neurobiology andterms that may be obscure to those outside the Drosophila researchcommunity. After fertilization and egg-laying, embryos developrapidly over the course of 24 h, through 20 relatively distinctstages (Hartenstein and Wodarz, 2013; Menon et al., 2013). Afterhatching from the egg, larvae mature over �5 days through threesuccessive stages known as instars. Following a �5 day pupalstage, the adult or ‘‘imago’’, ecloses from the pupa. A dramatic reor-ganization of the nervous system and body plan occurs duringpupation and includes the addition of adult-specific organs fromdiscreet packets of stem cells known as imaginal discs. Both neu-rons and glia are present in both larval and adult nervous systems.These include cells that make up the glial sheath, which surroundsthe nervous system at both stages and, acts as both a diffusion bar-rier and a site for active xenobiotic removal, analogous to the bloodbrain barrier (BBB) in mammals (DeSalvo et al., 2011; Mayer et al.,2009). The BBB and the glial sheath also express a similar comple-ment of transporters for sugars, mono carboxylic acids and neutralamino acids, supporting the idea that they serve similar functions(R. Bainton, personal communication).

The structure of most neurons in flies and other insects differssomewhat from their canonical form in mammals. In flies, a singleprocess emanates from the soma and gives rise to both dendritesand axons. This structure facilitates the organization of insect neu-rons into glomeruli or ganglia in which both axons and dendritesproject into a central neuoropil, with the somata forming a sur-rounding cortical rind (Fig. 2). Fly neurons support action poten-tials and calcium mediated release at the nerve terminal similarto mammals, but most terminals in the fly possess a characteristicpresynaptic specialization known as a T-bar, around which synap-tic vesicles (SVs) cluster. Large dense core vesicles (LDCVs), whichrelease aminergic neurotransmitters and neuropeptides do notcluster near T-bars and few if any T-bars are found in terminalsthat contain primarily LDCVs either centrally (Karsai et al., 2013)or peripherally (Atwood et al., 1993; Jia et al., 1993).

5. Neuroanatomy

A complete description of fly neuroanatomy is beyond the scopeof this review. However, a brief description of some particularlysalient features may be useful for understanding the effects ofmutations in neurotransmitter transporters. Although variationwithin individual cell clusters exist (Sykes et al., 2004), neuronalsomata and processes are generally localized to stereotypic posi-tions. The embryonic and larval central nervous systems include


the segmented ventral nerve cord and the bulbous, anterior struc-ture variously referred to as ‘‘the larval brain,’’ or the ‘‘fused sub-esophageal and supraesophageal ganglia’’. Since the centralnervous systems of both the larva and adult are small (on the orderof 100 microns), most immunolabeling studies employ confocalimaging of the entire brin. Another common preparation for immu-nolabeling and electrophysiological studies is the larval fillet,which exposes the abdominal musculature and the neuromuscularjunction (NMJ) (Jan and Jan, 1976).

Excellent resources for learning the neuroanatomy of the adultfly are available online http://www.virtualflybrain.org/site/vfb_site/home.htm. Here we describe a few well-characterized struc-tures that may be encountered by the reader in studies ofneurotransmitter transporters (Fig. 3). These include the optic gan-glia (lamina, medulla, lobula and lobula plate) (Melnattur and Lee,2011; Menon et al., 2013) and the antennal lobes (a critical compo-nent of the olfactory system) (Masse et al., 2009; Mu et al., 2012;Wilson, 2013). The function and anatomy of two anatomically dis-tinct structures in the ‘‘central brain’’ have been extensively stud-ied: the mushroom bodies (MBs) shown to be critical for learning,memory and the integration of sensory information (Heisenberg,2003), and the central complex (including the fan shaped body,ellipsoid body, protocerebral bridge and noduli), important forthe coordination of movement (Strauss, 2002) and an increasingnumber of other behaviors (Lebestky et al., 2009; Liu et al.,2012a; Seelig and Jayaraman, 2013; Ueno et al., 2012).

6. A brief fly genetics primer

The growing number of molecular-genetic tools available inDrosophila are described in detail elsewhere (Bier, 2005; Franket al., 2013). Here we provide a brief description of the Gal4/UASexpression system (Brand and Perrimon, 1993), a tool used in anumber of studies on neurotransmitter transporters (see below).In the Gal4/UAS system, a promoter sequence of choice drivesexpression of the yeast-derived Gal4 transcriptional activator(Fig. 4) (Brand and Perrimon, 1993). The Gal4 protein binds to itscognate upstream activating sequence (UAS) to express a cDNAof choice (e.g. for GFP, Fig. 4). Expression of the Gal4 protein in aspecific temporal and spatial pattern dictated by the chosen


http://www.virtualflybrain.org/site/vfb_site/home.htm

http://www.virtualflybrain.org/site/vfb_site/home.htm


SagittalFrontal

Central ComplexMushroom Body

Central Brain Lamina

MedullaLobula Complex

Antennal Lobe

Horizontal B

Thoracic

Optic

Cervical Lobe

Connective

Ganglion

A C

Fig. 3. The neuroanatomy of the adult fly brain. Cartoons show (A) horizontal, (B) frontal and (C) sagittal views of the fly and corresponding diagrams of the central nervoussystem. For simplicity, structures are labeled in one panel but color-coded to allow identification in all panels. Labeled structures include the central brain (orange), bilaterallysymmetric optic lobes, the thoracic ganglion (yellow) and the cervical connective (blue), which contains both ascending and descending fibers. Ganglia within the optic lobesinclude the lamina (blue) and medulla (green) both of which receive projections from photoreceptors in the retina. Cells in both the lamina and medulla project to the lobulaand lobula plate, indicated here as the lobular complex (pink). Major structures within the central brain include the mushroom bodies (purple), required for olfactory learningand memory and the central complex (yellow ellipsoid), which regulates some motor behaviors.

Eye-Specific Yeast Gal4

cDNA

Promoter

TranscriptionStart Site

ProteinGal4

Eye-Specific Trascription

Factor +

X

for Gal4Yeast UAS

cDNA for GFP+

Eye-Specific Promoter

Yeast Gal4cDNA

ProteinGal4

+

ProteinGFP

Eye-Specific Trascription

Factor

for Gal4Yeast UAS

cDNA for GFP

(N0 EXPRESSION)

A B

C

Fig. 4. The Gal4/UAS System. The bipartite Gal4/UAS System employs separatetransgenes to (A) drive expression and (B) allow specific cDNAs to be expressed, e.g.for Green Fluorescent Protein (GFP) as shown here. The Gal4 ‘‘driver’’ consists of acell or tissue specific promoter e.g. for cells in the eye as shown in (A) immediatelyupstream of the cDNA encoding the yeast Gal4 transcription factor. Mating theselines results in a fly that contains both the Gal4 and UAS transgenes (C). In theseprogeny, Gal4 is expressed and binds to its UAS, which in turn drives expression ofGFP in cells defined by the Gal4 promoter.


promoter will thus express the transgene in a highly specific man-ner. RNAi interference constructs can be similarly expressed inspecific cell types using this system. The bipartite nature of thesystem allows any lines containing a Gal4 ‘‘driver’’ transgene tobe paired with any other line containing a UAS transgene.Thousands of Gal4 and UAS lines are available including those rep-resenting specific neurotransmitter systems. These includedVGLUT-Gal4 (Daniels et al., 2008), dVGAT-Gal4 (Fei et al., 2010),TH-Gal4 (Friggi-Grelin et al., 2003), Tdc2-Gal4 (Cole et al., 2005),TrH-Gal4 (Alekseyenko et al., 2010; Park et al., 2006a) andDdc-Gal4 (Li et al., 2000) for glutamate, GABA, dopamine, serotonin,octopamine + tyramine, and dopamine + serotonin neuronsrespectively.


Additional, specialized UAS transgenes may be used to eitheractivate or disrupt the function of specific populations of cells.For example, the fly ortholog of dynamin, shibire (shi), is requiredfor endocytosis as well as other vesicle budding events(Kitamoto, 2001; Koenig and Ikeda, 1989; Poodry et al., 1973).The temperature sensitive (ts) mutant shits can be shifted to anon-permissive temperature to block endocytosis and depletethe pool of SVs (Kitamoto, 2001). Another useful transgene encodestetanus toxin (TNT), which cleaves SNARE proteins and thus blocksexocytosis (Frank et al., 2013). Optogenetic and temperature sensi-tive probes capable of activating or inactivating electrical activityin subpopulations of neurons are also available (Frank et al.,2013). Importantly, both optogenetic probes and TNT will affectexocytosis of both SVs as well as LDCVs, the latter containing bothclassical and peptide neurotransmitters. By contrast, transgenes ormutations that alter the function of neurotransmitter transportersor synthetic enzymes do not share this potentially confoundingeffect. Genetic reagents based on neurotransmitter transportersmay become increasingly useful to specifically assess the affectsof amines, GABA and glutamate in the fly ‘‘connectome’’.

Thousands of mutations and useful chromosomal abnormalitiesare available in the fly (St Johnston, 2013). Remarkably, many ofthe first reported Drosophila mutants such as white are still inuse. Point mutations have been traditionally generated usingchemical mutagens such as the DNA alkylating agent Ethyl MethylSulfate (EMS) (St Johnston, 2013). Inversions, duplications and lar-ger deletions have been traditionally generated using X-rays. Morerecently, mutations as well as small deletions have been generatedusing mobile, transposable elements. Several types of mobile ele-ments have been used, including ‘‘P’’ elements, the first mobile ele-ments to be identified in the fly (Bachmann and Knust, 2008;Engels, 1992). Mobile elements can cause mutations either by‘‘hopping’’ into a gene, or by hopping out. Hopping out can be pre-cise, leaving no trace of the insertion. Mutations are generatedwhen transposons hop out via ‘‘imprecise excision’’, either remov-ing a portion of genomic DNA or leaving behind a portion of thetransposable element. Of note, some types of mobile elements onlygenerate precise excisions, and mutations can be generated onlywhen they hop into a gene. Knock-out technology is also availablein the fly (Rong and Golic, 2000), but the large number of existingmutations (and perhaps newer technologies such as CRISPR/Cas9(Gratz et al., 2013)) may alleviate the need for de novo knockouts.

The nomenclature for Drosophila mutations is somewhat idio-syncratic. Alleles with no detectable function are labeled nullswhereas mutants that possess small or large amounts of residualactivity are known as strong and weak ‘‘hypomorphs’’ respectively(Muller, 1932). The historic precedent of using the phenotype of




the mutation to name the gene can be confusing to those morefamiliar with other genetic systems. In the current molecular age,it may seem odd that a gene required for the transport of redand brown eye pigments is named white.

7. Drosophila neurotransmitters and their metabolism

Drosophila use many of the same neurotransmitters as mammalsincluding GABA, glutamate, acetylcholine and some of the samemonoamines including dopamine, serotonin and histamine. Similarto mammals, GABA is synthesized via Glutamic Acid Decarboxylase(GAD) and GABA functions as an inhibitory transmitter (Lee et al.,2003). Ach is synthesized from choline and an activated acetyl moi-ety via choline acetyl transferase (ChAT) (Kitamoto et al., 1998). Thegenomic organization of the cholinergic locus is phylogeneticallyconserved, with the vesicular acetylcholine transporter (VAChT)contained within an intron of ChAT (Kitamoto et al., 1998). Both glu-tamate and acetylcholine act as excitatory transmitters, but unlikemammals, glutamate rather than ACh is released at the fly NMJ(Jan and Jan, 1976). In addition, glutamate in flies can also functionas an inhibitory transmitter through activation of a glutamate-gatedchloride channels (Liu and Wilson, 2013; Rohrbough and Broadie,2002). Unlike mammals, flies and other insects do not appear to fur-ther process dopamine to noradrenalin or adrenalin, but rather usethe structurally similar neurotransmitters tyramine and octopa-mine (OA) (Roeder, 2005), both classified as trace amines in mam-mals (Borowsky et al., 2001). OA and tyramine contain one ratherthan two hydroxyl (OH) groups on the phenyl ring and thus are clas-sified as phenolamines rather than catecholamines. Unlike DA, syn-thesis of OA and tyramine does not employ tyrosine hydroxylase ordopa decarboxylase. Rather, both are converted from tyrosine bytyrosine decarboxylase (Tdc) (Cole et al., 2005) and in case of OA,an additional hydroxylation step catalyzed by tyramine b hydroxy-lase (TbH) (Monastirioti et al., 1996).

Neurotransmitter metabolism in the fly has not been docu-mented as thoroughly as in mammals. In particular, the role ofmonoamine oxidation remains unclear. Although a monoamineoxidase (MAO) homolog is absent from the fly genome (Roelofsand Van Haastert, 2001), MAO-like activity has been reported(Chaudhuri et al., 2007; Wang et al., 2011) and some drugs activeas MAO inhibitors in mammals also show activity in the fly(Yellman et al., 1997).

Unlike mammals, conjugation to b-alanine plays a role in hista-mine and dopamine metabolism in the fly. Interestingly, some ofsame biosynthetic and conjugation pathways used for cuticle for-mation and pigmentation are also used in neurotransmitter syn-thesis and degradation (Wright, 1987). One of the earliestidentified fly mutants was ebony, which generates a darker cuticlethan wild type (Wittkopp et al., 2003). Ebony has since been shownto encode a b-alanyl-dopamine synthetase and to include both his-tamine and DA as substrates (Borycz et al., 2002; Hovemann et al.,1998; Richardt et al., 2002). In addition to metabolism of DA in thecuticle, ebony may also metabolize DA released as a neurotrans-mitter in the central nervous system (Suh and Jackson, 2007). Inthe visual system, ebony conjugates histamine to alanine to gener-ate carcinine (Borycz et al., 2002). Since ebony is expressed in glia,histamine and its metabolites are presumably transported in andout of both glia and neurons, but the details of this process arepoorly understood (Edwards and Meinertzhagen, 2010).

Based on the localization of the plasma membrane transporters,exocytotically released glutamate and GABA are likely to be takenup primarily into glia in flies, similar to the prominent role of gliain recycling glutamate and GABA in mammals (see below). Locali-zation of DA and 5HT plasma membrane transporters on aminergicneurons suggests, that as is in mammals, these amines are recycledin part into presynaptic cells, although other, less specific transport


pathways for amines are also possible in flies as in mammals(Daws, 2009).

8. Drosophila vesicular neurotransmitter transporters

8.1. dVAChT

VAChT was the first vesicular neurotransmitter transporter tobe molecularly identified in the fly (Kitamoto et al., 2000). dVAChTmutants include dVAChT1 which is embryonic lethal, and theweaker allele dVAChT2 which survives through the second larvalstage but locomotes slower than wild type animals (Kitamotoet al., 2000). Similar to VAChT knock-down mice, heterozygousdVAChT mutants survive to adulthood and show subtle phenotypicdifferences from controls. In particular, electrophysiological analy-sis of adult dVAChT heterozygotes suggest that during periods ofsustained vesicle release, at least one circuit in the adult CNS failsto maintain normal levels of ACh release (Kitamoto et al., 2000).This phenomenon may be related to changes in ACh releaseobserved during periods of sustained release at the cholinergicNMJ of frogs (Naves and Van der Kloot, 1996; Van der Kloot,2003) and in VAChT knock-down mice (Lima et al., 2010;Rodrigues et al., 2013). The mechanisms underlying these changesmay include the presence of variable numbers of VAChT moleculesper SV and a consequent reduction in the amount of transmitterstored in each vesicle (Prado et al., 2013). In addition, it is likelythat under conditions of reduced VAChT expression, some vesiclesare essentially empty (Prado et al., 2013). The contribution of eachmechanism may vary depending on the rate of SV recycling andremains an outstanding unanswered question (Prado et al., 2013).

8.2. dVGLUT

Prior to its molecular characterization, biochemical studies sug-gested vesicular glutamate transport activity was present in the fly(Tabb and Ueda, 1991). Unlike mammals, which have three distinctVGLUT genes, Drosophila contains only a single VGLUT ortholog,dVGLUT (Daniels et al., 2004). dVGLUT is expressed in all glutama-tergic neurons in the larva and adult fly including the glutamater-gic motoneurons innervating the larval NMJ (Daniels et al., 2004,2008). The membrane topology of dVGLUT has been examinedusing a series of epitope tags predicted to reside on the lumenaland cytosolic loops between transmembrane domains as well asthe cytosolic N- and C-termini (Fei et al., 2007). Deletion of thecytosolic C-terminus abrogates lethality caused by dVGLUT over-expression, clearly implicating an important functional role for thisdomain (Grygoruk et al., 2010) and some critical trafficking motifsreside in the C-termini of mammalian VGLUTs (Foss et al., 2013;Voglmaier et al., 2006). However, the baseline localization ofdVGLUT to SVs appears to be surprisingly unaffected by deletionof the C-terminus (Grygoruk et al., 2010) and recent studies ofmammalian VGLUTs have identified trafficking motifs in both theN- and C-termini (Foss et al., 2013; Voglmaier et al., 2006). It ispossible that the contribution of specific domains to VGLUT traf-ficking could vary between cell types or between species.

Post-synaptic excitatory potentials representing individual ves-icles (minis) have been used to examine neurotransmitter contentin those vesicles capable of glutamate storage and release inembryonic hypomorphic dVGLUT mutants (Daniels et al., 2006).Mini frequency was significantly lowered, suggesting that emptyvesicles can fuse with the plasma membrane, consistent with othermammalian studies. More surprisingly, the size of the remainingminis – an indication of SV filling – was not altered in the mutantin comparison to controls, suggesting that a reduced number ofVGLUT molecules, and perhaps only one, may suffice to fill a vesicleunder these conditions (Daniels et al., 2006). Using other




techniques, recent estimates of VGLUT copies per vesicle in wildtype mammalian preparations have ranged from 4 to 14 (Mutchet al., 2011; Takamori et al., 2006). One electrophysiological studyin VGLUT1 knock-out mice suggested a reduction in mini size(Wojcik et al., 2004), while another did not (Fremeau et al.,2004). Unlike flies, the presence of multiple VGLUT isoforms inmammals complicates these analyses (Takamori et al., 2006).Although there is no evidence for oligomerization of vesiculartransporters, it is possible that a single functional unit is composedof multiple molecules. It also possible that multiple copies ofvesicular transporters may be needed to increase the rate of fillingduring rapid recycling, with fewer copies sufficient for neurotrans-mitter release under some conditions (Blakely and Edwards, 2012).These questions remain an important topic in studies of vesiculartransporters.

Over-expression of dVGLUT at high levels using relativelystrong Gal4 drivers is larval lethal. Over-expression using a weakerdriver allows survival through adulthood (Daniels et al., 2011).Interestingly, the surviving adults show large lacunae in theirCNS, possibly the result of glutamate-mediated excitotoxicity(Daniels et al., 2011). Consistent with the observation that dVGLUTover-expression causes functional changes in SV homeostasis, elec-trophysiological studies of dVGLUT over-expression in larva showan increase in quantal size, as well as large spontaneous eventsat the larval NMJ (Daniels et al., 2004, 2011), a phenotype similarto that seen with over-expression of vesicular transporters inmammals (Edwards, 2007; Wilson et al., 2005).

8.3. dVMAT

In contrast to the mammalian genome which contains two dis-tinct VMAT genes (Liu et al., 1992), flies contains only one (Greeret al., 2005). The primary structures of dVMAT and mammalianVMATs are similar throughout the 12 predicted transmembranedomains, the regions likely to be responsible for substrate recogni-tion and transport (Greer et al., 2005). Indeed, neurotransmittersubstrate specificity and relative affinity of dVMAT are generallysimilar to mammalian VMATs. For example, dVMAT is also inhib-ited by the drug reserpine at sub-micromolar concentrations(Greer et al., 2005).

8.4. dVMAT

encodes at least two mRNA splice variants, including dVMAT-Aand -B which differ at their C-termini (Greer et al., 2005). dVMAT-Ais expressed in both larvae and adults in all dopaminergic,serotonergic and octopaminergic cells (Greer et al., 2005). NeitherdVMAT-A nor B appear to be expressed in the otherwisewell-characterized histaminergic photoreceptor cells of the adulteye (Chang et al., 2006), and the vesicular transporter responsiblefor the storage of histamine in the SVs of histaminergic photorecep-tors is not known. It also is not known whether dVMAT is used for DAstorage in other non-neuronal tissue, such as ‘‘crystal cells’’, whichare present in Drosophila hemolymph (blood) and release DA formelanotic scab formation (Galko and Krasnow, 2004; Rizki andRizki, 1984).

The cytosolic C-terminus of dVMAT-A is similar to mammalianorthologs, whereas the C-terminus of dVMAT-B is divergent (Greeret al., 2005). The C-terminus of dVMAT-A, but not dVMAT-B, alsocontains endocytosis motifs active in vitro in S2 cells and in vivo((Greer et al., 2005; Grygoruk et al., 2010) and manuscript underreview). Both dVMAT-A and B contain a large amino terminaldomain that is not conserved in mammals (Greer et al., 2005).Interestingly, this region contains a number of polyproline motifsthat could conceivably play a role in protein–protein interactions,but this has not yet been tested.


In contrast to dVMAT-A and other VMAT orthologs, dVMAT-B isexpressed in glia rather than neurons, and more specifically, in asmall subset of glia in the adult optic lobes where its functionappears to include the storage of histamine (Romero-Calderónet al., 2008). Although mammalian glia likely take up amines vialow affinity mechanisms (Dahlin et al., 2007; Yoshikawa et al.,2013), the expression of a specific amine transporter in glia isclearly unusual. Further studies of dVMAT-B may help determinethe role of glia in the recycling of histamine and perhaps otherbiogenic amines.

The dVMAT mutant shows a number of behavioral deficits con-sistent with the loss of exocytotic amine release in the nervous sys-tem (Chen et al., 2013a; Simon et al., 2009). Similar to mouseknockouts, mutant tissue levels of amines are dramaticallyreduced, presumably due to the degradation of amines that arenot sequestered in secretory vesicles (Fon et al., 1997; Simonet al., 2009). Under standard fly culture conditions, loss of dVMATis lethal, but reducing the density of the culture results in 100% via-bility, thus allowing behavioral assays. dVMAT mutants show a dra-matic decrease in baseline larval locomotion (Simon et al., 2009),mirroring previously demonstrated effects of reserpine(Pendleton et al., 2000). dVMAT mutants also show a reduced star-tle response, reduced male courtship (Simon et al., 2009) andincreased sleep (Nall and Sehgal, 2013). Treatment of adult mutantflies with reserpine also increases sleep (Nall and Sehgal, 2013). Incontrast, amphetamines decrease sleep in flies (Andretic et al.,2005), presumably via release of DA (Pizzo et al., 2013). In eachcase, changes in sleep were distinguished from changes in locomo-tion using previously established criteria (Hendricks et al., 2000;Shaw et al., 2000).

The Gal/UAS system was used to express dVMAT in subpopula-tions of aminergic neurons and thereby determine which aspectsof the mutant phenotype result from loss of specific aminergic cir-cuits (Chen et al., 2013a). Consistent with other reports on thefunction of OA signaling in flies and other insects (Adamo et al.,1995; Bacon et al., 1995; Fox et al., 2006; Koon et al., 2011; Leeet al., 2009; Monastirioti et al., 1996), expression of dVMAT inOA cells was required for rescue of baseline larval locomotion,female fertility, and the initial phase of the startle response(Chen et al., 2013a). In contrast, the later recovery phase of thestartle response depends on serotonergic signaling since expres-sion of dVMAT in 5HT cells rescued this deficit (Chen et al.,2013a). Male courtship could be rescued via expression in DA cells,consistent with recent studies using other genetic reagents todetermine the function of DA circuits in the fly (Alekseyenkoet al., 2010). The restoration of dVMAT function in smaller subsetsof neurons in a null background may be used to map the identity ofaminergic neurons responsible for other complex behaviors.Importantly, relatively broad Gal4 drivers can be used for thesestudies since, in general, dVMAT transgenes will only be active incells in which amines are endogenously synthesized.

Current molecular-genetic techniques in Drosophila are particu-larly well-suited to in vivo over-expression studies, making it possi-ble to determine how increased expression of a vesiculartransporter may affect behavior. Flies over-expressing dVMAT inDA and 5HT cells (Li et al., 2000) survive through adulthood(Chang et al., 2006). The resulting changes in behavior, presumablycaused by an increase in DA and 5HT release, include an increase inmotor activity and a blunted behavioral response to cocaine (Changet al., 2006). Similarly, administration of cocaine results in increasedmotor activity in the fly (Bainton et al., 2000; McClung and Hirsh,1998; Torres and Horowitz, 1998). Similar to amphetamines(Andretic et al., 2005), over-expression of dVMAT in 5HT + DA cellsalso increases male courtship behavior (Chang et al., 2006).

Importantly, the effects of dVMAT and dVGLUT over-expressiondemonstrate that the electrophysiological changes previously seen




with over expression of vesicular transporters in vitro (Pothoset al., 2000; Song et al., 1997; Wilson et al., 2005) can have signif-icant downstream behavioral sequelae in an intact organism. Thisobservation has important ramifications for drugs potentially ableto increase vesicular transporter activity (see Future Directionsbelow).

Over-expression of dVMAT as well as loss of function alleleshave been used to explore the role of DA homeostasis in vivo inneurodegenerative processes relevant to Parkinson’s disease (PD)(Lawal et al., 2010; Sang et al., 2007). DA has a high oxidativepotential and may also conjugate to proteins implicated in geneticforms of PD (Conway et al., 2001; Hastings et al., 1996). Thus, it hasbeen suggested that an increase in VMAT activity may have neuro-protective effects (Guillot and Miller, 2009). This has been testedin vitro in cell cultures using mammalian VMAT and dVMAT(Mosharov et al., 2009; Park et al., 2007), and in vivo in the fly(Inamdar et al., 2013; Lawal et al., 2010; Sang et al., 2007). Over-expression of dVMAT rescues the loss of DA neurons caused byeither a genetic or chemical insult (Inamdar et al., 2013; Lawalet al., 2010; Sang et al., 2007). Conversely, loss of dVMAT activityincreases the toxicity of both genetic and chemical insults to DAcells (Inamdar et al., 2013; Lawal et al., 2010; Sang et al., 2007).Parallel experiments have not yet been reported in rodents. How-ever, a mouse VMAT2 knockdown showed baseline loss of DA cells(Caudle et al., 2007) and some hVMAT2 alleles may decrease therisk of PD in patients (Glatt et al., 2006). It is therefore possible thatover-expression of VMAT2, or drugs that increase VMAT2 activity,could show neuroprotective effects in mammalian models and per-haps patients at risk for PD.

dVMAT-A serves as a useful model for membrane trafficking tosecretory vesicles. Mutation of signals in the C-terminus blocksendocytosis in cultured cells, and also disrupts trafficking to SVsin vivo (Grygoruk et al., 2010). Ongoing experiments will determinehow these and other signals affect trafficking to SVs and LDCVs inneurons. Genetic rescue experiments in which these and otherdVMAT mutant transgenes are expressed in a mutant dVMAT back-ground provide a convenient method for analyzing the in vivoeffects of these alleles without generating a large number ofknock-in lines. Importantly, this type of in vivo structure–functionanalysis provides an opportunity to determine the effects of altereddVMAT trafficking on behavior. Similar studies on Drosophila dopa-mine transporter (dDAT) point mutants have been used to deter-mine the behavioral effects of N-terminal phosphorylation and anhDAT mutant linked to autism (Hamilton et al., 2013; Pizzo et al.,2013, 2014). This method of analysis is likely to useful for the studyof a variety of other transporters (see Future Directions below).

8.5. dVGAT

Similar to mammals, the Drosophila genome contains a singlevesicular GABA transporter gene (dVGAT) (Fei et al., 2010). dVGATappears to be expressed in all GABAergic neurons in the larva, sinceit precisely co-localizes with GABA in the ventral nerve cord, and isalso expressed in most, if not all adult GABAergic neurons (Enellet al., 2007; Fei et al., 2010). It is not clear whether Drosophila, likemammals, use glycine as a neurotransmitter and whether dVGATcould also serve to store glycine. It is also possible that dVGATcould play a role in b-alanine storage or metabolism in the flyalthough this has not been tested. The structure of b-alanine isintermediate between glycine and GABA and mammalian VGAThas been shown to transport b-alanine in vitro (Juge et al., 2013).As noted above, conjugation of b-alanine to histamine is requiredfor histamine recycling in the fly (Borycz et al., 2002), and conjuga-tion of b-alanine is also important for DA metabolism in both thecuticle and the central nervous system (Suh and Jackson, 2007;Wright, 1987).


Mutation of dVGAT causes developmental lethality (Fei et al.,2010). However, inducible expression of a dVGAT transgene hasbeen used to restore dVGAT function during development andthereby allow adult behavioral studies. One adult phenotype dem-onstrated in the conditional rescue line was a surprisingly specificdefect in the detection of small objects in the fly’s visual field (Feiet al., 2010). Other aspects of reduced GABAergic signaling in thisline remain to be explored.

8.6. Portabella

Drosophila and some other insects express portabella, an addi-tional vesicular transporter than appears to be absent from mam-malian genomes (Brooks et al., 2011; Lawal and Krantz, 2013). Thegene name was based on its robust expression in MBs (See Neuro-anatomy above) (Brooks et al., 2011). Prt is expressed in the intrin-sic neurons of the MBs, the Kenyon cells (KCs) (Brooks et al., 2011).Surprisingly, the neurotransmitter stored and released by KCs isnot known. The primary structure of the prt protein is most similarto DVMAT and it is possible that the substrate is also similar toknown monoamines; however, the biosynthetic enzymes for DA,5HT, OA and histamine are not expressed in KCs, suggesting thatprt may transport a novel neurotransmitter (Brooks et al., 2011).In the absence of an identified substrate, a genetic approach wasused to test the hypothesis that prt functions as a transporter.Mutation of residues conserved in prt and VMAT ablated the abilityof a prt transgene to rescue the prt mutant phenotype (Brookset al., 2011). These data support the idea that prt is likely to func-tion as a bone fide transporter and that the substrate may be sim-ilar to known biogenic amines.

Mutation of prt results in a peculiar defect in sexual behavior,characterized primarily by an inability of males to maintain theirposition during copulation (Brooks et al., 2011). The mutants arenonetheless fertile (Brooks et al., 2011). Expression of prt using aGal4 driver that is prominently expressed in MBs restores thesebehavioral defects (Brooks et al., 2011). MBs are best known fortheir role in learning and memory and the prt mutant shows adefect in olfactory learning, however the phenotype is relativelysubtle (Brooks et al., 2011; Kahsai and Zars, 2011; Roman andDavis, 2001). It is possible that compensatory changes in the prtmutant mitigate its effect on learning. Alternatively, the releaseof a peptide, rather than a classical neurotransmitter, may be moreimportant for some functions of KCs (Iliadi et al., 2008; Knapeket al., 2013).

9. Plasma membrane transporters

9.1. dSERT

The Drosophila serotonin transporter (dSERT) was the firstplasma membrane neurotransmitter transporter to be identifiedin the fly (Corey et al., 1994; Demchyshyn et al., 1994). These stud-ies revealed that the substrate specificity of dSERT diverged frommammalian orthologs, including a lower affinity for some antide-pressants such as citalopram but a higher affinity for the mamma-lian DAT antagonist mazindol (Corey et al., 1994; Demchyshynet al., 1994). Transport via dSERT also appeared to show a lessstringent requirement for chloride than hSERT (Demchyshynet al., 1994). Although one report suggested that the affinity ofcocaine for dSERT was significantly higher than hSERT (Coreyet al., 1994), another suggested similar affinities for both orthologs(Demchyshyn et al., 1994). Interestingly, the SERT ortholog that isexpressed in the tobacco hornworm Manduca sexta is more similarto dSERT than hSERT but has an extremely low affinity for cocaineand most antidepressants (Sandhu et al., 2002).




Extensive structure–function comparisons of hSERT and dSERThave been performed using heterologous expression of hSERT/dSERT chimeras in cultured cells. These studies revealed theimportance of a tyrosine residue (Y95 in hSERT, F90 in dSERT) inTM1 for some pharmacologic differences including the differentialbinding to citalopram (Adkins et al., 2001; Barker et al., 1998)Other sites in transmembrane domains 5–9 contribute to theapparent inability of dSERT to transport the neurotoxin MPP+

(Rodriguez et al., 2003). Additional modeling studies have sug-gested an interaction between Y95 and E444 (Combs et al., 2011).

dSERT RNA is expressed late in embryonic developmental (stage15) just before 5HT can be detected (stage 17) (Demchyshyn et al.,1994). In situ hybridization of larva shows a pattern consistentwith the stereotyped localization of serotonergic neurons in larva(Giang et al., 2011) and adult brain (Thimgan et al., 2006). ThedSERT gene is predicted to be less than 4 kB and to encode twonon-coding splice variants http://flybase.org/cgi-bin/gbrowse/dmel/?Search=1;name=FBgn0010414. Northern blots confirm thepresence of at least two bands likely to represent mature mRNAspecies (Romero-Calderón et al., 2007).

A specific antiserum to dSERT has been used to map proteinexpression in embryo, larvae and adults (Giang et al., 2011). All cellbodies in the larva and adult that express dSERT also co-label withan antiserum to 5HT and vice versa (Giang et al., 2011). However,some processes in the adult brain, including those in the MB lobes,express dSERT but do not detectably label for 5HT. The function ofdSERT at these sites is not clear but could conceivably involveuptake of a transmitter other than 5HT (Larsen et al., 2011), andperhaps the substrate of prt (see above).

Immunocytochemistry and fast scan cyclic voltametry havehelped demonstrate the importance of reuptake mechanisms for5HT clearance in Drosophila (Borue et al., 2010, 2009). Pharmaco-logic inhibition of amine uptake increased the time required for5HT clearance (Borue et al., 2010, 2009) in larvae and reducedthe pool available for release following repetitive stimulation(Borue et al., 2010, 2009).

A mutation in dSERT has been generated by Henrike Scholz’s lab(University of Cologne, Germany). A phenotype characterization isnow in progress and will determine how a potential increase inextracellular 5HT might affect fly behavior. Pharmacologic manip-ulation of 5HT release using amphetamine derivatives (fenflura-mine and 3,4-methylenedioxymethamphetamine a.k.a. MDMA or‘ecstasy’) has already demonstrated that increased extracellular5HT causes enlarged varicosities in 5HT processes (Daubert et al.,2010). By contrast, direct application of exogenous 5HT decreasesthe number of varicosities in 5HT processes in the central neuropil(Sykes and Condron, 2005), and loss of 5HT (and DA) synthesis inDdc mutants results in increased branching of serotonergic pro-cesses (Budnik et al., 1989). The mechanisms underlying thesephenomena remain unclear, but support the idea that both exces-sive and inadequate levels of extracellular 5HT, and possiblydSERT, activity could have significant developmental effects(Daubert and Condron, 2010; Lesch and Waider, 2012). Furtherstudies using dSERT transporter transgenes and mutants are likelyto be useful to explore this issue. Importantly, the availability of adSERT mutant will now allow in vivo structure–function studies inwhich specific mutations can be expressed in a loss of functionbackground.

Further studies may also help to determine whether proteinsother than neurotransmitter transporters or cell-specific trans-porter modifiations are required for amine uptake and storage in5HT cells. Mammalian thalamocortical projections appear to takeup and release exogenous 5HT in the absence of cell-autonomoussynthesis (Lebrand et al., 1996, 2006). In contrast, in the fly, exog-enously applied 5HT did not appear to be taken up or stored innon-aminergic cells ectopically expressing dSERT (and dVMAT)


(Giang et al., 2011; Park et al, 2006b). Although speculative, it ispossible that additional factors beyond the transporters them-selves are required for amine uptake and storage.

9.2. dDAT

The Drosophila dopamine transporter (dDAT) was identified viahomology-based cloning and exhibits a kinetic profile similar to thatof hDAT (Porzgen et al., 2001). In situ hybridization experimentsshowed that dDAT expression matched the pattern in larva previ-ously established for DA neurons (Porzgen et al., 2001). Northernblots show a single mRNA species of 3–4 kB (Kume et al., 2005;Romero-Calderón et al., 2007) and in situs of adult heads partiallymatch the localization DA cells, although not all known DA cellswere detectably labeled (Thimgan et al., 2006). As with all otherDAT orthologs, dDAT-mediated transport is sodium dependentand able to support efflux as well as sodium-coupled transport(Porzgen et al., 2001). The affinity of dDAT for cocaine was notedto be relatively low compared to hDAT and dSERT (Porzgen et al.,2001). However, in vivo studies have shown that dDAT is likely tobe required for at least some of the behavioral effects of cocaine(Bainton et al., 2000) and inhibition of dDAT by cocaine prolongsDA uptake in vivo (Makos et al., 2010; Vickrey et al., 2013).

Real time, in vivo measurements of DA have been made withcarbon fiber electrodes using both fast scan cyclic voltametry inlarvae (Vickrey et al., 2009) and amperometry in adults(Berglund et al., 2013; Makos et al., 2010). Both cocaine and nisox-etine block reuptake of endogenous DA in larvae (Vickrey et al.,2009). In the adult fly, clearance of exogenously applied dopaminewas decreased by cocaine, amphetamine, methamphetamine, andmethylphenidate (Ritalin). Additional experiments using a dDATmutant demonstrated that these effects on DA uptake were medi-ated by dDAT, rather than another transporter such as dSERT(Makos et al., 2010).

The dDAT mutant was originally isolated in a screen for sleepdefects and the dDAT gene was renamed fumin (fmn), meaningsleepless in Japanese (Kume et al., 2005). Whereas wild type fliesfollow a diurnal circadian rhythm, fmn mutants are active almostcontinuously over each 24-h period. Moreover, after sleep depriva-tion they show no rebound need for rest (Kume et al., 2005; Uenoet al., 2012; Wu et al., 2008). fmn mutants also show an enhancedresponse to mechanical stimuli (Kume et al., 2005). The pathwaysmediating the sleep defect effect in fmn have been mapped usinggenetic interactions with the DA receptor, DopR (Ueno et al.,2012), which is similar to mammalian DA1 receptors (Kim et al.,2007; Liu et al., 2012b). These data indicate that that DA processesinnervating the adult central complex help promote sleep (Uenoet al., 2012).

Mutations in dDAT have also been used to study the mechanismof action of amphetamines (Pizzo et al., 2013, 2014). By disruptingthe function of membrane rafts in DA cells, the authors elegantlyshowed that localization of dDAT to these sites is required forefflux but not for transport in vivo (Pizzo et al., 2013) consistentwith in vitro studies (Cremona et al., 2011). These studies alsodemonstrated that the behavioral effects of amphetamines in thefly are mediated through DA neurons rather than another aminer-gic cell type (Pizzo et al., 2013, 2014). Further genetic rescue exper-iments used expression of a dDAT phosphoacceptor site mutant inthe dDAT loss of function background to show that phosphoryla-tion of the dDAT N-terminus is required for efflux in vivo (Foget al., 2006; Khoshbouei et al., 2004; Pizzo et al., 2013). In addition,a dominant negative form of CamKII was used to demonstrate thespecific requirement of this kinase for phosphorylation-mediatedefflux through dDAT (Pizzo et al., 2014).

Another elegant study of dDAT used genetic rescue of the mutantto test the behavioral effects of a rare de novo, non-synonymous


http://www.flybase.org/cgi-bin/gbrowse/dmel/?Search=1;name=FBgn0010414




mutation (T356M) detected in a patient with autism (Neale et al.,2012). Importantly, the rarity of some de novo mutations in manygenes can make it difficult to conclusively evaluate theirphenotypic impact on the basis of human genetic studies alone.Thus, additional models to test the potential effect(s) of suchmutants are quite useful. When expressed in cultured cells, theT356M mutation caused sustained constitutive efflux in theabsence of amphetamines. When expressed in vivo in a wild typedDAT background, the mutant caused a genetically dominantincrease in locomotion in adult flies similar to the effects of thedDAT loss of function fmn mutant (Hamilton et al., 2013). Thesedata strongly suggest that physiologically significant effects canresult from the T356M mutation and that this allele is unlikely torepresent an innocuous polymorphism unrelated to disease.

Risk alleles associated with other transporters may also beinvestigated in this way. To our knowledge, only one mouse modelcontaining a risk allele in hSERT for Attention Deficit HyperactiveDisorder (ADHD) has been generated (Veenstra-VanderWeeleet al., 2012). The phenotypic effects of other possible risk allelesin SERT are not known (Ye and Blakely, 2011) and might beexplored in the fly.

10. Octopamine uptake and metabolism in flies vs. other insects

OA is sometimes referred to as the invertebrate noradrenaline,since its structure differs by only a single ring oxygen and it servessome similar functions (Roeder, 2005). OA is a substrate of dVMAT(Greer et al., 2005) and the fly expresses an adrenergic-like OAreceptor gene OAMB and three b-adrenergic-like receptors Octb1R, Oct b2R and Oct b3R (Evans and Maqueira, 2005). However,a plasma membrane OA transporter has not been identified inthe fly. Surprisingly, a high affinity OA transporter has been iden-tified in the cabbage looper Trichoplusia ni (T. ni) and several otherinsect species (Donly et al., 2007; Malutan et al., 2002), but isabsent in Drosophila and other Dipteran species as well as bees(Hymenoptera) (Donly and Caveney, 2005). T. ni also express a highaffinity DA transporter (Gallant et al., 2003). TriDAT and TriOATshow similar affinities for DA, but OA and tyramine inhibit TrnOATto a greater extent than TrnDAT (Gallant et al., 2003). In theabsence of an OAT ortholog, it remains unclear how OA is recycledin Drosophila. It is conceivable that dDAT functions as both an OAand DA transporter in the fly but this remains speculative.

10.1. dEAAT1

Drosophila EAATs were independently identified by three differ-ent groups (Besson et al., 1999; Kawano et al., 1999; Seal et al.,1998). Drosophila EAAT1 (CG3747) shares 41 and 35% amino acididentity, respectively, with human EAAT1 and EAAT2 (Bessonet al., 1999). dEAAT2 is also similar, but current data suggest thatit may not function as a glutamate transporter as we discuss below.We note that the naming of the dEAATs was based on their order ofidentification rather than correspondence to specific mammalianorthologs. dEAAT1 is sodium-dependent and shows high affinityfor L-glutamate, as well as L- and D-aspartate (Seal et al., 1998).The sodium:glutamate stoichiometry of the homologous TriEAAT1is 2:1 (Donly et al., 1997), similar to mammalian EAATs (Wadicheet al., 1995). Also, similar to some mammalian EAATs (Fairmanet al., 1995) dEAAT1 shows loosely coupled chloride channel-likeactivity (Seal et al., 1998).

Four non-coding dEAAT1 mRNA splice variants are predicted,but their functional relevance is not known http://flybase.org/cgi-bin/gbrowse/dmel/?Search=1;name=FBgn0026439. In the embryoand larva, dEAAT1 mRNA appears to be confined to the nervoussystem and robustly expressed in glia, with little if any expression


in neurons (Besson et al., 1999; Freeman et al., 2003; Soustelleet al., 2002; Stacey et al., 2010). dEAAT1 is co-expressed with thewell-characterized glial marker repo but some repo-positive cellsdo not express dEAAT1 (Soustelle et al., 2002). dEAAT1 expressionin adult glia has been confirmed using a dEAAT1 antiserum (Rivalet al., 2006) in addition to a dEAAT1-Gal4 driver (Rival et al., 2004;Stacey et al., 2010). dEAAT1-Gal4 expressing cells co-label for themarker Prospero, indicating that a large majority of dEAAT1-expressing cells are of the anterior LG subtype of glia (Staceyet al., 2010). Surprisingly, dEAAT1 is not expressed in glia (or neu-rons) at the larval glutamatergic NMJ (Rival et al., 2006). Rather,uptake of glutamate at the larval NMJ is likely to be mediated bythe cystine/glutamate exchanger encoded by the gene genderblind(Grosjean et al., 2008).

The reduction of dEAAT1 expression in adult glia using RNAinterference (RNAi) was reported to shorten life span, increase sen-sitivity to oxidative stress and cause neural degeneration (Rivalet al., 2004), however, RNAi experiments in a later report showedless severe effects (Rival et al., 2006). In these later experiments,electrophysiological recordings from adult flight muscles showedbroadening of peaks representing individual depolarization events,consistent with the idea that decreased glutamate uptake can havesignificant post-synaptic effects in flies as well as mammals (Rivalet al., 2006).

Mutations in the dEAAT1 gene were recently reported and arelethal at approximately 48 h post-hatching (Stacey et al., 2010).Two mutant alleles were identified, including dEAAT1SM1 and dEA-AT1SM2, both likely to be null alleles since the start codon and thefirst two transmembrane domains were deleted and no expressionof dEAAT1 mRNA was detected. Additionally, the phenotypes of thehomozygous mutants were equivalent to each other and to hetero-zygotes containing a mutant allele over a large chromosomal dele-tion (Stacey et al., 2010). We note that this type of genetic assay,rather than RNA or protein expression, is the gold standard fordetermining whether a Drosophila mutant is a true null allele.

The survival of the dEAAT1 mutants as 1st instar larvae alloweda further phenotypic analysis (Stacey et al., 2010). Unlike wild typelarvae that move almost continuously, dEAAT1 homozygousmutants were nearly immobile with occasional slow contractionsof the body wall musculature (Stacey et al., 2010). In contrast tofindings in RNAi studies (see above), no obvious morphologic orneurodegenerative defects were detected in the mutant (Staceyet al., 2010).

Additional functional studies of dEAAT1 may help determinehow glutamatergic signaling is regulated and the potential effectsof dysregulation. Mutations in human EAAT1 have been identifiedin a rare form of episodic ataxia (de Vries et al., 2009; Jen et al.,2005) and in collaboration with another lab (Joanna Jen, UCLA)we are currently testing the potential pathogenic effects of thesemutations in Drosophila.

10.2. dEAAT2

Despite the similarity between dEAAT1 and dEAAT2, the lattermay not function as a high affinity glutamate transporter (Bessonet al., 2000, 2005, 2011). Similar to other EAAT orthologs, dEAAT2transports aspartate, but its comparative affinity for glutamate isrelatively low (Besson et al., 2000). Surprisingly, EAAT2 also trans-ports taurine (aminoethane sulfonic acid), a b-amino acid whosephysiological role in all species remains poorly understood(Besson et al., 2005, 2011). Some structures in the insect brain suchas the MBs are labeled by antisera to taurine (Bicker, 1991;Strausfeld et al., 2003) and a role for taurine as a neurotransmitterhas been suggested, but this remains incompletely defined (Wuand Prentice, 2010). Rather, taurine may function primarily as anosmolyte in the CNS and other tissue (Chesney et al., 2010;






Schaffer et al., 2010). In mammals, taurine uptake is mediated by adistinct taurine transporter (Pramod et al., 2013), which appears tobe absent in the fly and other insects (Besson et al., 2011). Consis-tent with an in vivo role for dEAAT2 in taurine uptake, mutations ofdEAAT2 exhibit dramatically decreased tissue content of taurine(Besson et al., 2011). The mutants also showed mild defects inthe behavioral response to saline and propionic acid, but photo-taxis and locomotion were similar to wild type controls; levels ofaspartate and glutamate were also similar to controls (Bessonet al., 2011).

In the embryo, dEAAT2 mRNA is expressed in sensory organs inthe peripheral nervous system and midline glia (Freeman et al.,2003; Soustelle et al., 2002). An antibody to dEAAT2 is not avail-able but a dEAAT2-GAL4 construct has been used to localize expres-sion in conjunction with a UAS-GFP marker. In contrast to in situdata, expression using the Gal4 driver was predominantly neuronal(Besson et al., 2011). However, the authors acknowledged that the4.5-kb promoter fragment used to make the dEAAT2-GAL4 trans-gene could be incomplete and additional studies might uncoverexpression in some subsets of glia.

10.3. dGAT

A Drosophila GABA transporter (GAT) cDNA was identified priorto the sequencing of the fly genome (Neckameyer and Cooper,1998) and corresponds to the single GAT ortholog present in thegenome (CG1732). In parallel, similar GAT orthologs were identi-fied in the tobacco hornworm M. sexta (Mbungu et al., 1995) (Mas-GAT) and the cabbage looper moth T. ni. (TrnGAT) (Gao et al., 1999).All three insect orthologs are similar in primary structure to mam-malian GAT-1. The pharmacology of cloned MasGAT and TrnGAThave been investigated in vitro using heterologous expression sys-tems and, similar to mammalian orthologs, both are sensitive tothe mammalian GAT inhibitor DL-2,4-diaminobutyric acid (DABA)(Gao et al., 1999; Mbungu et al., 1995). In contrast to mammalianGATs, neither MasGAT nor TrnGAT were significantly inhibited byb-alanine. Nipecotic acid showed partial inhibition at high concen-trations but significantly lower affinity for insect versus mamma-lian GATs (Gao et al., 1999; Mbungu et al., 1995).

The effects of both DABA and nipecotic acid have been tested inDrosophila membranes derived from homogenized embryos, lar-vae, pupae and adult flies (Leal et al., 2004; Leal andNeckameyer, 2002). The homogenates showed saturable uptakeof tritiated GABA and inhibition by DABA and nipecotic acid; how-ever, the required concentrations of inhibitors were higher thanthose used in assays of cloned transporters (Leal et al., 2004; Lealand Neckameyer, 2002). Feeding DABA to larva and adults or feed-ing nipecotic acid to adults disrupted several aspects of motorbehavior (Leal et al., 2004; Leal and Neckameyer, 2002). At leastsome affects of DABA and nipecotic acid, such as disruption of neg-ative geotaxis in the adult were reversible over the course of2 days, which might prove useful for further behavioral studies.Although most insect GABA receptors (including RDL) are insensi-tive to the GABAA receptor antagonist bicuculline (Buckinghamet al., 2005) the behavioral effects of DABA could be reversed withthis drug (Leal and Neckameyer, 2002). It was also surprising thatthe GABA analog gabapentin rescued the effects of DABA (Leal andNeckameyer, 2002), since the phenotype caused by blockade ofGABA reuptake might be expected to be enhanced by a GABAergicagonist. A dGAT mutant would be useful to further explore theseeffects, but has not yet been identified.

dGAT expression is likely to be restricted to glia. In the adult fly,in situ hybridization showed an mRNA pattern similar to that ofthe glial marker repo (Thimgan et al., 2006). Two possible splicevariants have been predicted but both are the same predicted sizehttp://flybase.org/reports/FBgn0039915.html. To our knowledge, a


specific antiserum to Drosophila GAT is not available. Antisera tomammalian GATs 1–3 have been used in Drosophila as probesand showed three distinct patterns, but their relationship to theexpression of dGAT is not clear. By contrast, a highly specific anti-serum to MasGAT has been generated (Oland et al., 2010; Umeshand Gill, 2002) and used for both light microscopy and ultrastruc-tural studies. A detailed study of the antennal lobe showed thatMasGAT localizes to neuronal processes early in development(Oland et al., 2010) but labeling appeared to be confined to gliain the adult. Consistent with the idea that insect GATs in generalmay primarily localize to glia in adults, uptake of GABA into theoptic ganglia of both Drosophila and larger flies (Musca) appearedto be primarily glial (Campos-Ortega, 1974) and in situ hybridiza-tion of dGAT also appeared to localize to glia rather than neurons(Thimgan et al., 2006).

11. Choline transport in other insects

Unlike most other neurotransmitters, acetylcholine is degradedextracellularly to choline prior to transport. Choline is then recy-cled by a specific high-affinity transporter that has been exten-sively studied in vertebrates (Parikh et al., 2013; Traiffort et al.,2013). A possible ortholog of the mammalian choline transporter(ChT1) is present in the Drosophila genome (see Table 2) but hasnot been investigated. An ortholog from the cabbage lopper(TrnChT) has been characterized and unlike mammalian orthologs,is weakly inhibited by Hemicholinium-3 (McLean et al., 2005).Interestingly, mammalian ChT resides primarily on synaptic vesi-cles at baseline (Blakely and Edwards, 2012). It is not known if thisis also true in insects.

12. Additional SLC6 members

The Drosophila SLC6 family of sodium-dependent plasma mem-brane transporters contains several additional members whosefunction remains unclear. These include the gene inebriated (ine)(Soehnge et al., 1996; Stern and Ganetzky, 1992). A mutation atthis locus was independently isolated in a screen for defects inthe electroretinogram (ERG), the electrical field potential acrossthe retina and downstream neurons in the optic ganglia (Pak,1975; Wu and Wong, 1977). The characteristic oscillations in themutant led to its designation as ‘‘receptor oscillation A’’, or rosA(Burg et al., 1996; Pak, 1975; Wu and Wong, 1977). rosA/inemutants also show reduced on and off transients in the ERG sug-gesting impaired signaling of photoreceptors to neurons immedi-ately downstream in the optic ganglia (Burg et al., 1996; Pak,1975; Wu and Wong, 1977). However, electrophysiological defectsin rosA/ine are not confined to the eye and include potentiation oflarval motoneuron depolarization (an increase in long term facili-tation) (Huang and Stern, 2002). These observations are consistentwith the idea that rosA/ine may generally function to regulate neu-ronal excitability.

A homolog of ine has been cloned from M. sexta (Chiu et al.,2000) and expression in oocytes was used to test a variety ofpotential substrates but to date, the substrates of Manduca andDrosophila ine remain unclear (Chiu et al., 2000) (however seebelow). Ine mRNA and protein appear to be broadly expressed inboth Drosophila and Manduca and unlike some other members ofSLC6, expression is not confined to the nervous system but alsoprominent in the gut and malpighian tubule, the ‘‘fly equivalent’’of the mammalian kidney (Burg et al., 1996; Chiu et al., 2000). Inthese structures, ine has been proposed to function as an osmolytetransporter (Huang et al., 2002).

It has been suggested that ine could have a function in recyclinghistamine via transport of the histamine metabolite carcinine(Stuart et al., 2007). Carcinine is generated by ebony, which is


http://www.flybase.org/reports/FBgn0039915.html



expressed in a subset glia that surround photoreceptor terminalsand functions to conjugate histamine to b-alanine (Stuart et al.,2007). Several tantalizing findings support the idea that ine couldbe involved in recycling histamine and perhaps function as a carc-inine transporter (Gavin et al., 2007). For example, feeding fliescarcinine phenocopies ine (Gavin et al., 2007). These effects areconsistent with the idea that loss of ine increases extracellularcarcinine and that excess extracellular carcinine is the cause ofthe ine/rosA mutant phenotype.

Since histamine is released from photoreceptor cells but metab-olized in glia, recycling is likely to follow a fairly circuitous path,similar to that proposed for glutamate recycling through glia.Therefore, similar to System A and N in the glutamate:glutaminecycle, additional transporters are likely to be involved in histaminerecycling. Some of these may be novel members of SLC6. In addi-tion to ine, insects express other distinct subfamilies of SLC6including 6 insect-specific Nutrient Amino Acid Transporters(iNATs) (Boudko et al., 2005). Another 5 genes whose functionremains obscure form another distinct subfamily of transportersin SLC6 (Romero-Calderón et al., 2007). mRNA expression of twomembers in this group (CG13794 and 5) as well as CG4476 aredown-regulated by the loss of eye tissue, consistent with a possiblefunction in the visual system (Romero-Calderón et al., 2007). Fur-thermore, mutation of CG4776 causes a defect in phototacticbehavior (Romero-Calderón et al., 2007). We suspect thatCG4476, CG13794 CG13795 and perhaps other novel members ofSLC6 could participate in recycling histamine in the fly and otherinsects, but additional experiments are clearly needed to test thisidea.

13. Future directions

13.1. Structure–function studies in vivo

As described elsewhere in this issue, experiments using cul-tured cells have been invaluable for exploring the functional conse-quences of specific mutations in neurotransmitter transporters. Inparallel, mouse knockouts and mutations in model organisms suchas C. elegans and Drosophila have demonstrated the physiologicalimportance of specific neurotransmitter transporters for a varietyof processes. We suggest that flies and other model organisms willbe very important for combining these strategies and that struc-ture–function studies of neurotransmitter transporters in vivo willbe an important future direction.

One use of this strategy is the expression of risk alleles forhuman disease in vivo. For rare or de novo mutations the relation-ship between a particular polymorphism and the disease pheno-type can be difficult to ascertain. In the absence of a pedigree orthe association of a phenotype with multiple de novo events, it isoften impossible to determine if a given polymorphism or copynumber variant has a relationship to the phenotype of the patient.In some cases, it may be possible to demonstrate molecular or cel-lular phenotypes for a candidate gene, as shown for disease-asso-ciated mutations in ion channels. However, it is also possiblethat the cellular phenotype of a disease allele may not reflect itspreviously defined physiological function, e.g. a cellular phenotypeof a rare mutation associated with autism spectrum disorders(ASDs) is dendritic retraction (Krey et al., 2013). This phenotypewas not predicted from the known function of the gene as a cal-cium channel subunit and does not appear to be associated withalterations in calcium flux (Krey et al., 2013).

The potential pathogenic mechanisms associated with poly-morphisms in neurotransmitter transporters are also likely to becomplex, and it may be difficult to predict how a subtle changein the structure of a neurotransmitter transporter could increase


risk for a complex illness such as autism or ADHD. As discussedabove, the potential use of flies to examine these questions isexemplified by the analysis of a dDAT allele associated with autism(Hamilton et al., 2013). We anticipate that similar studies will bevery useful to explore the consequences of other risk alleles suchas those in SERT linked to autism. To date only one of the SERTmutants has been tested in mice (Ye and Blakely, 2011;Veenstra-VanderWeele et al., 2012). Generating mutant lines inflies or worms may be used for detailed mechanistic studies, oralternatively, large panels of potentially relevant polymorphismsmay be screened in model invertebrates to determine which onesshould be studied in more detail in mammals.

More generally, structure–function studies in vivo will be usefulto determine the downstream consequences of other mutations inneurotransmitter transporters. Mutants predicted to affect trans-port activity, post-translational regulatory processes or traffickingcan be easily expressed in a loss of function mutant backgroundto determine their effects on synaptic transmission and behavior.As noted above, this strategy has already been used to determinethe in vivo importance of phosphoacceptor sites in dDAT (Pizzoet al., 2013).

We believe that this strategy will be particularly useful tounderstand the functional importance of transporter trafficking.A large number of studies have already dissected some of the sig-nals and pathways required for the internalization of vesiculartransporters and their targeting to secretory vesicles (reviewed in(Fei et al., 2008)). Similarly, a number of studies have exploredthe signals that allow internalization of plasma membrane trans-porters (Eriksen et al., 2010; Quick, 2006; Robertson et al., 2009;Sager and Torres, 2011; Steiner et al., 2008). However, it is notknown how disruption of these pathways might affect the functionof the nervous system as a whole. We are currently investigatingthese questions using trafficking mutants in dVMAT. We havefocused on dVMAT in part because it localizes to two distinct typesof vesicles, SVs, required for rapid release at the nerve terminal andLDCVs, thought to be required for neuromodulatory signaling. Thecontribution of amine release from SV versus LDCVs for specificbehaviors remains surprisingly obscure. We are using mutationsthat shift DVMAT between SVs and LDCVs to explore this question(manuscript under review). We suggest that a similar analysis maybe useful to explore the functional significance of mutations thatdisrupt the internalization of plasma membrane transporters.

13.2. Forward Genetic Screens for Interacting Genes and Proteins

Most psychiatric illnesses are likely to follow a complex patternof inheritance with large numbers of genes contributing to thephenotype, and additional environmental factors also playing arole (Sullivan et al., 2012). Genetic modifiers alter the phenotypeof the ASD mutation in SERT when expressed in mice, but the iden-tities of these interacting genes are not known (Kerr et al., 2013).We suggest that ‘‘modifier’’ a.k.a. ‘‘enhancer/suppressor’’ screensin model invertebrates such as C. elegans and Drosophila representa powerful and essentially untapped method to identity specificgenes that interact with risk alleles in SERT, and more generallyto identify genes that interact with neurotransmitter transporters.

Modifier screens may be used to identify new loci that interactwith any type of mutation, provided it generates a genetically trac-table phenotype. In common genetic parlance, the mutation is usedto first generate a ‘‘sensitized genetic background’’ which may con-sist of an anatomic or behavioral defect, or simply a decrease insurvival. A panel of mutations in other genes – so-called ‘‘sec-ond-site mutations’’ – are then crossed into the sensitized geneticbackground. The resulting progeny are screened to determinewhether the phenotype of the parent strain – the sensitized geneticbackground – has been modified by any of the additional




mutations. The defect seen in the parent strain may be worsened or‘‘enhanced’’. Alternatively, the defect might improve, and the phe-notypic defect ‘‘suppressed’’.

Classic screens for molecules that modulate the function of theoncogene ras provide an important example of this strategy in flies(Gaul et al., 1993). Suppression or enhancement of defects in Dro-sophila eye development caused by mutations in ras were used toidentify genes relevant to ras and ras-dependent pathways.Although eye development in flies was used as a screening pheno-type, the molecules that emerged from these screens were not spe-cific for eye development or Drosophila but rather representedgeneral components of the ras signaling pathway. Flies and wormsmay similarly be used to screen for genes that modulate the func-tion of neurotransmitter transporters. The first step will be to iden-tify a genetically tractable phenotype caused by mutation of thetransporter in order to generate a sensitized genetic background.Importantly, the nature of the phenotype need not be particularlyinteresting, just consistent, robust and easily scored.

In C. elegans, mutations in VAChT/unc-17 have already beenused to screen for interacting loci (Mathews et al., 2013;Sandoval et al., 2006). One screen identified a mutant allele of syn-aptobrevin and a second screen identified the novel gene sup-1(Mathews et al., 2013; Sandoval et al., 2006). The nature of thesesecond site mutant alleles suggest that in both cases the interac-tions are likely to be mediated by direct protein–protein bindingto VAChT. However, this need not always be the case. One advan-tage of this type of forward genetic screen is that it can detect indi-rect interactions. Another advantage is that it is not strictlyhypothesis based. The authors of the VAChT interaction studiesdid not know that they would detect synaptobrevin, much lesssup-1 (Mathews et al., 2013; Sandoval et al., 2006). Similarly, wesuggest that identification of molecules that indirectly influencethe function of neurotransmitter transporters in ways that we areunable to predict will be a very important future use for Drosophila.

Forward genetic screens in C. elegans have also been used toidentify novel genes that regulate DA signaling and could poten-tially regulate dopamine transport. Mutations in the C. elegansortholog DAT-1 generate a genetically tractable phenotype termedSwimming-Induced Paralysis (SWIP) (Hardaway et al., 2013;McDonald et al., 2007). Some DAT-1 mutations that cause SWIPdecrease DAT-1 localization to the synapse, and the SWIP pheno-type is thought to result from activation of extrasynaptic DA recep-tors by DA spillover (Hardaway et al., 2013; McDonald et al., 2007).Thus, screens for mutations that modify SWIP have the potential toidentify novel genes required for DAT trafficking as well as otheraspects of DAT activity. Although a classical modifier screen usinga weak allele of DAT-1 has not yet been reported, a recent studyreported a screen for genes that would mimic or phenocopy theDAT-1 mutant and cause SWIP. In addition to new alleles of DAT-1, the screen identified two novel second-site mutations, one ofwhich appears to genetically interact with DAT-1 (Hardawayet al., 2013). It is possible that modifiers of Drosophila fmn couldbe similarly identified using sleep or the response to ampheta-mines as a screening phenotype.

13.3. Drug screens

Although still a nascent field, a number of labs have begun touse flies as an in vivo platform for drug screens (Tickoo andRussell, 2002). One strategy, based on classical forward geneticscreens is to select a phenotype of interest, e.g. sleep (Nall andSehgal, 2013) or feeding (Gasque et al., 2013), then screen for drugsthat are responsible for or enhance that phenotype. Some of theidentified targets of these screens may be neurotransmitter trans-porters, e.g. the VMAT inhibitor reserpine was identified in a screenfor small molecules that increase sleep in flies (Nall and Sehgal,


2013). A more efficient strategy to identify agents that affect thefunction of a given neurotransmitter transporter may be to screenfor drugs that modify the phenotype caused by mutation or over-expression of the transporter, akin to screens to identify geneticmodifiers. Similar to genetic modifier screens, this strategy againtakes advantage of the fact that all of the regulatory machinery isin place, and drug targets may be novel elements of the regulatorymachinery rather than the transporter itself. We suggest that thisapproach might expand the way we think about drugs that affecttransporters. In addition, we suggest that this represents a poten-tial way to identify drugs that may increase transport activity.There are few if any drugs that make neurotransmitters transport-ers work more effectively. Targeting upstream proteins that inhibittransporter function could accomplish this.

We have taken this approach to identity new molecules thatmight potentiate the function of VMAT in vivo (Lawal et al.,2014). Using a partially functional dVMAT mutant background,we screened larvae for drugs that would increase signaling in theoctopaminergic circuit required for baseline larval locomotion.The results of this screen yielded several molecules that a prioriwould not be classified as aminergic drugs and are unlikely todirectly bind dVMAT (Lawal et al., 2014). Rather, we suggest thatsome of the identified drugs may act on upstream targets to indi-rectly increase VMAT activity, and we are currently testing thishypothesis. We suggest that similar screens might be used to iden-tify drugs that modify the function of other neurotransmittertransporters.

14. Abbreviations and conventions

For Drosophila genes we follow the fly genetics convention ofdesignating the gene in italics and the protein in non-italic text.For other genes we have attempted to maintain the conventionused in their system of origin.

Acknowledgements

We thank Henrike Scholz (University of Cologne, Germany),Roland Bainton (UCSF, USA) and the anonymous reviewers forhelpful comments on the manuscript. This work was supportedby National Institute of Mental Health R01 MH076900, NationalInstitute of Environmental Health (NIEHS) R01 ES015747, an Inde-pendent Investigator Award from The Brain and Behavior ResearchFoundation, and the Joanne and George Miller and Family EndowedChair in Depression Research at the UCLA Brain Research Institute(D.E.K.) with additional funding from an the NIEHS Center Grant(P01 ES016732; PI: M.F. Chesselet), and the NIEHS UCLA traininggrant in Molecular Toxicology and the Parkinson’s Disease Founda-tion (PDF-SFW-1336) (C.A.M.).

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drosophila melanogaster as a genetic model system to study...

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