Activating properties of cocaine and cocaethylene in a behavioral preparation of Drosophila...

Activating Properties of Cocaineand Cocaethylene in a Behavioral

Preparation of Drosophila melanogasterGERMAN TORRES* AND JUDITH M. HOROWITZ

Behavioral Neuroscience Program, Department of Psychology, State University of New York at Buffalo,Buffalo, New York 14260

KEY WORDS behavior; psychostimulant drugs; dopamine; glutamate; invertebrates

ABSTRACT The use of Drosophila as a model to study the behavioral consequencesof stimulant drugs was analyzed in an active preparation of decapitated Drosophila.Application of cocaine and cocaethylene to discrete nerve cord cells regulating motorprograms of behavior produced striking patterns of behavioral activity in a concentration-related manner. In general, intense circling behavior and significant wing buzzingactivity were distinguishable behavioral markers in flies treated with mM concentra-tions of cocaine or cocaethylene. The significant changes in motor behavior induced bystimulant drugs in decapitated flies were not reproduced by the application of apomor-phine, a direct dopamine (DA) agonist, or octopamine, a naturally occurring transmitterin arthropods. Because both cocaine and cocaethylene interfere with DA reuptake inmammals, we characterized the role of DA receptors mediating increased stereotypy andmotor behavior in flies. Coadministration of SCH-23390, a specific D1 receptor antago-nist, significantly attenuated the behavior-activating properties of cocaine and cocaethyl-ene in this active experimental preparation. Therefore, the receptor protein mediatingthe behavioral responses to stimulant drugs in Drosophila is pharmacologically similarto the mammalian D1 subtype. In rats, cocaine- and cocaethylene-induced behavioralactivity is complex, with increasing evidence that the D1 receptor interacts significantlywith N-methyl-D-aspartate (NMDA) receptor pathways to produce an altered behavioralphenotype. To further characterize additional receptor subtypes targeted by the actionsof cocaine and cocaethylene, we pretreated flies with MK-801 and dextromethorphan.Both of these drugs are potent, selective noncompetitive NMDA receptor antagonists.Interestingly, MK-801 and dextromethorphan profoundly reduced the behavior-activating properties of cocaine and cocaethylene in Drosophila. Therefore, as in rats, theNMDA (and D1) receptor pathways in this arthropod represent obligatory targets for thebehavioral effects of stimulant drugs. Synapse 29:148–161,1998. r 1998 Wiley-Liss, Inc.

INTRODUCTION

Cocaine is a powerful stimulant drug that has promi-nent effects on behavior. Administration of this drug torats and humans produces significant behavioralchanges, including locomotor activity, sensitization,tolerance, and dependence (Koob, 1996; Woolvertonand Johnson, 1992). The mechanisms underlying thebehavioral actions of cocaine have been shown to de-pend, to a large extent, on the dopamine (DA) system.More specifically, cocaine and related stimulant drugsblock the DA reuptake transporter, thereby potentiat-ing dopaminergic transmission across the synapse(Giros et al., 1996; Kuhar et al., 1991). Increasedsynaptic activity leads to the interaction of DA withseveral membrane-bound receptors, particularly with

the D1 receptor subtype. Subsequently, the D1 receptorinteracts with Gs molecules, resulting in the activationof adenylyl cyclase, which in turn increases cAMPproduction (Gingrich and Caron, 1993). This intracellu-lar signal transduction pathway appears to contributegreatly to the stimulatory actions of cocaine; therefore,understanding the relationship of potential cellularsignal changes to behavior is of significant interest. Tothis end, intense research has been directed at under-standing cocaine-induced changes in DA transmission

*Correspondence to: German Torres, Ph.D., Behavioral Neuroscience Program,Department of Psychology, State University of New York at Buffalo, Buffalo, NY14260. E-mail: [email protected]

Contract grant sponsor: Research Foundation of the State University of NewYork.

Received 5 September 1997; accepted 20 November 1997.

SYNAPSE 29:148–161 (1998)

r 1998 WILEY-LISS, INC.

because this catecholamine is heavily involved in thecontrol of motor function (Koob, 1996). However, if weare trying to understand and relate basic gene functionto cocaine-induced behavior, a relatively simple animalmodel that is both amenable to studies of genetics andmotor behavior must be found. In this regard, Dro-sophila melanogaster fills these criteria well. First,there is a large body of knowledge concerning itsgenetics and biochemistry. Second, the thoracic andabdominal ganglia of this arthropod harbor severalmotor programs related to grooming, walking, andrighting (Strauss and Heisenberg, 1993, and referencestherein). And third, DA and its receptors are discretelyexpressed in the Drosophila central nervous system (forreviews see Fryxell, 1995; Walker and Holden-Dye,1989).

Although the central and peripheral nervous systemsof Drosophila melanogaster are complex, they neverthe-less contain significantly fewer neurons and receptorsthan do their vertebrate counterparts (e.g., mammals).This fact alone provides a practical and suitable modelto study in detail the mechanisms by which stimulantdrugs, like cocaine, promote behavioral activity. Further-more, the ubiquity of DA cell clusters in Drosophila notonly points to an evolutionary preservation of thistransmitter across different animal lineages, but also tothe possibility that DA may function in a similarfashion in both insects and vertebrates. In this context,DA and enzyme proteins required for its biosynthesishave been identified in adult brain and thoracic paren-chymal cells of Drosophila (Budnik and White, 1988;Lundell and Hirsh, 1994; Nassel and Elekes, 1992).Moreover, dopamine-sensitive adenylyl cyclase activityand active cAMP metabolism have been observed inthis insect as well (Davis and Kiger, 1981; Lane andKalderon, 1993). Finally, and of major significance tothe present study, functional DA receptors are ex-pressed in the nervous system of Drosophila. Forinstance, transmembrane receptors with high aminoacid sequence homology to D1-like DA receptors ofvertebrates have been cloned and found to be localizedto a variety of Drosophila parenchymal cells, includingbrain nerve cells (Feng et al., 1996; Han et al., 1996;Sugamori et al., 1995). The functional characteristics ofthe aforementioned receptors are, by and large, similarto those of vertebrate D1 receptors in that cAMP isgenerated in response to DA application, and that thisresponse is blocked by D1 receptor antagonists such asflupenthixol and SCH-23390 (Feng et al., 1996; Han etal., 1996). The spatial presence of functional D1 recep-tors in Drosophila, therefore, establishes this animal asan attractive model in which to study basic behavioralprocesses produced by cocaine exposure.

To our knowledge, no published reports exist describ-ing the behavioral consequences of cocaine in Dro-sophila. The reasons for this are obvious. Given itssmall size and neuroepithelia covering brain-sensitive

neurons precludes any attempt to inject cocaine di-rectly into the insect central nervous system or thoracicregion. However, a recent report appears to alleviatethis problem significantly by providing a behaviorallyactive preparation of Drosophila that could be used fordirect application of cocaine to DA-containing neuronsof the nerve cord. This preparation takes advantage ofthe fact that Drosophila, when decapitated, retainstriking and viable motor behaviors for several hours(or days) after surgery (see Yellman et al., 1997). Moreimportantly, when the catecholamine, DA, and otherbiogenic amines are applied to the exposed nerve cord,significant motor behaviors are spontaneously dis-played by the flies (Yellman et al., 1997). We have usedthis in vivo preparation to determine whether cocaineapplication to the nerve cord results in increasedbehavioral activity. In addition, such a preparation ofDrosophila was used to determine whether cocaine-induced behavioral activity is mediated by homologueD1 receptors of those operant in mammals. Finally, wetested the possibility that glutamate receptors [of theN-methyl-D-aspartate (NMDA) subtype] could also beinvolved in the motor stimulant actions of cocaine indecapitated flies. This latter hypothesis is of interestand potential importance because the locomotor-activating properties of cocaine in rats are reported toalso be mediated by functional NMDA receptors (Kali-vas and Alesdatter, 1993; Karler et al., 1989; Wolf andJeziorski, 1993). Although Drosophila neurons exhibitglutamate-mediated synapses, there is little or noevidence for NMDA receptors in this invertebrate ani-mal (Walker and Holden-Dye, 1989). The demonstra-tion of pharmacologically-defined NMDA receptors inDrosophila nerve cord cells could be utilized as entrypoints to analyze the functional relationship betweenexcitatory amino acid transmission and the stimulantactions of cocaine.

MATERIALS AND METHODSDrosophila Stock

Wild-type melanogaster flies (of the Canton-S strain;kindly donated by Dr. L. M. Hall) were raised at roomtemperature (21oC) on standard food media and main-tained on a 12:12 light:dark cycle (lights on at 0700A.M.). For all experiments, 5- to 10-day-old flies wereused. Both male and females flies were used as wefailed to detect gross gender-dependent differences incocaine-induced behavioral activity. All behavioral test-ing was conducted between 1200–1500 h of the lightcycle.

Decapitation and BehavioralPreparation Procedures

Decapitation of Drosophila was accomplished as de-scribed by Yellman et al. (1997) with some minormodifications. Briefly, flies were anesthetized by chill-

149EFFECTS OF COCAINE AND COCAETHYLENE IN DROSOPHILA

ing them at 4oC for approximately 2 minutes. With asharp steel-blade razor, a rapid cut was made betweenthe subesophageal section of the head and the protho-racic neuromere. The flies recovered quickly, and imme-diately showed a strong righting reflex. Once the ani-mals showed an upright posture, they were gentlyplaced (with the aid of a fine paintbrush) onto glass-dishes (12 3 12 cm). The dishes were placed overgraph-paper squares arranged numerically, so thateach fly was assigned to a particular number. Theanimals were allowed to recover from the surgery for 15minutes at room temperature. As described by Yellmanet al. (1997), only flies showing an upright postureposition and grooming responses following tactile stimu-lation of the legs were used in the experiments. Inaddition, only decapitated flies devoid of any remnanthead tissue still attached to the prothoracic region wereselected for further experimentation.

Drugs

The following drugs were used for the behavioralexperiments in Drosophila melanogaster: cocaine HCl;the benzazepine, R(1) -SCH-23390; the noncompetitiveNMDA receptor antagonist, (1) -MK-801 (dizocilpinemaleate); dextromethorphan HB (an allosteric antago-nist at NMDA receptors); the phenolamine, (1)-octopamine HCl; the DA receptor agonist, R(-)-apomor-phine HCl, and the local anesthetic, lidocaine HCl. Allthese drugs were purchased from Research Biochemi-cals (Natick, MA). Cocaethylene fumarate (an activecocaine metabolite derived from the combined ingestionof cocaine and ethanol) was obtained from the NationalInstitute on Drug Abuse (Rockville, MD). All of theaforementioned drugs were dissolved in distilled water,except for apomorphine, which was dissolved in 1%ethanol. Unless otherwise noted, cocaine, cocaethylene,octopamine, apomorphine, and lidocaine were adminis-tered at a concentration of 1 mM; whereas SCH-23390,MK-801, and dextromethorphan were applied at aconcentration of 5 mM. All drugs were prepared freshprior to use.

Behavioral Activity Measurements

Decapitated flies were randomly assigned to control(water-treated animals) or cocaine-treated groups (or inseparate experiments, to cocaethylene-treated groups).For the characterization of receptor subtypes mediat-ing the effects of stimulant drugs in Drosophila, a seriesof two drugs were administered to the flies. The firstdrug was either water, SCH-23390, MK-801 or dextro-methorphan. One minute later, a second drug, cocaineor cocaethylene, was administered. All of the aforemen-tioned compounds were applied to the exposed nervecord via a micropipette at a volume of 0.5 µl. To ensureoptimal behavioral results, contact between a drugdroplet and the anterior aspect of the Drosophila notum

was maintained for about 5 seconds. It should be notedthat in most cases, behavioral activity in flies wasgenerated immediately upon contact with cocaine orcocaethylene droplets.

Insect behavior was visually rated for 1 minutebefore water or cocaine (or cocaethylene) applicationand 1 minute after appropriate drug treatment by anobserver ‘blind‘ to the experimental conditions. For thereceptor characterization experiments, behaviors of theflies were rated blindly 1 minute before the first andsecond drug application and for 1 minute thereafter. Tomeasure behavioral activity in Drosophila, a ratingscale was designed to be sensitive to significant changesin motor behavior produced by 1 mM cocaine or cocaeth-ylene. This rating scale provides an estimate of increas-ing behavioral activity ranging from in-place activity toself-grooming to intense circling behavior (i.e., stereo-typy). Such a behavioral rating scale was also designedto approximate a scale widely used in rat studies, whereexploratory and stereotyped behaviors are estimated toyield discrete behavioral changes after cocaine or coca-ethylene exposure (Baumann et al., 1993; Horowitz etal., 1997a; Kalivas et al., 1988). At every observationtime, a single score was given on a scale from 1 to 7,where:

1 5 Inactive or still2 5 In-place activity (grooming, front-/hind-leg move-

ment)3 5 Spontaneous behavioral activity (locomotion within

a 5-second period)4 5 A combination of locomotor and wing activity (. 5-

second duration)5 5 Continuous leg movement, grooming and wing

activity for 5–10 second6 5 Continuous circling behavior and wing buzzing

activity (. 15-second duration)7 5 Abrupt cessation of all behavior, including loss of

the righting reflex, and contracted legs

To measure the behavioral consequences of cocaine orcocaethylene in decapitated Drosophila further, sequen-tial video frames of fly motor behavior were analyzedprior to and following application of the above stimu-lant drugs to exposed nerve cord cells. These data weresubsequently compared with those derived from thevisual observations made by an observer unaware ofthe specific drug treatments. Such comparisons yieldedno significant differences in the behavioral rating ascocaine- or cocaethylene-induced behaviors were foundto be extremely robust and consistent across experimen-tal groups.

Scanning Electron Microscopy Procedures

At the end of each experimental session, the insectswere sexed under magnification by examining thegenital organs for dark bristles. A subset of the behavior-

150 G. TORRES AND J.M. HOROWITZ

ally studied flies (n 5 5 per group) was immediatelyplaced in a 4% glutaraldehyde-0.1 M Na1 cacodylatefixative solution and prepared for scanning electronmicroscopy. Briefly, entire decapitated and intact Dro-sophila bodies were postfixed in 4% OsO4 in 0.1 M Na1

cacodylate. After several rinses in buffer, the bodieswere dehydrated in graded series of ethanol and criti-cally point dried. Subsequently, the flies were mountedon stubs, sputter-coated with gold, and observed in aHitachi (Danbury, CT) S-800 Field Emission ScanningElectron Microscope operating at 10 kv.

Data Analysis

Behavioral activity in response to cocaine or cocaeth-ylene exposure in flies was analyzed by repeated mea-sures ANOVAs for drug and time effects. Differencesbetween specific experimental groups across observa-tion times were subsequently determined by Newman-Keuls posthoc test comparisons with statistically signifi-cant differences defined as P , 0.05. Statistical analyseswere performed on an IBM-compatible computer withstatistical program software (Jandel Scientific, CorteMadera, CA; SigmaStat). Data are presented as themean 6 SEM of behavioral scores. The number of fliesranged from 10 to 32 flies per experimental group for atotal of 377 flies tested.

RESULTSEffects of Cocaine and Cocaethyleneon Behavioral Activity in Drosophila

A well-established manifestation of stimulant drugexposure in mammals is an increase in motor behaviorthat can culminate in identifiable stereotypies. Todetermine whether stimulant drugs evoke similar be-havioral responses in decapitated flies, we appliedcocaine and cocaethylene to exposed nerve cord cells ofDrosophila. For illustration purposes, Figure 1C showsan electron photomicrograph of a section through acommon level of the nerve cord depicting the drugapplication site. Immediately upon contact with 1 mMcocaine, a set of complex behavioral responses wasgenerated in flies. These responses were largely charac-terized by significant motor activity, circling behavior,and intense wing buzzing. In sharp contrast, fliesexposed to water alone showed absolutely no behavioralresponses (P , 0.0001 when compared with cocaine-treated flies; see Fig. 2). Motor activities evoked bycocaine were comprised of specific bouts of locomotion,foreleg hyperactivity, and elevation of the body byoutstretched fore- and mid-legs. In most cases, thesemotor activities culminated in the insects falling ontotheir backs with extended wings and leg tremors. Thesestriking behaviors lasted for at least 1–2 minutes aftercocaine administration and in some cases for protractedperiods of time. It should be noted that most of thecocaine-treated flies recovered from this state and

responded vigorously to re-application of 1 mM cocaineby exhibiting all of the aforementioned behavioralresponses with no apparent diminution in intensity ormagnitude of stereotyped behaviors (data not shown).

Cocaine HCl elicited strong behavioral activity fromDrosophila in a concentration-dependent manner. Forinstance, cocaine at a concentration of 1 µM failed toproduce significant changes in motor activity. Indeed,when data collected from these animals (n 5 6) werecompared with data of insects (n 5 10) exposed to aninnocuous water solution, no significant statistical ef-fects were obtained between the two experimentalgroups (means 6 SEM: 1.5 6 0.22 for 1 µM cocaine-treated flies and 1.1 6 0.10 for water-treated flies; P .0.05). Provided the concentration of cocaine was 0.5mM or greater, significant behavioral responses wereinduced in decapitated flies. In this context, applicationof 1 mM cocaethylene fumarate to exposed nerve cord ofDrosophila also produced striking circling behavior andintense wing buzzing (Fig. 2). As with cocaine, thecircling behavior elicited by cocaethylene was character-ized by rapid rotation of the body with concomitantin-place wing buzzing activity. This stereotyped behav-ior was discernible only in flies treated with stimulantdrugs and provided the most distinguishable behav-ioral response in cocaethylene-treated flies. Further-more, a direct comparison of behaviors between cocaine-exposed and cocaethylene-exposed flies revealed nomarked differences in the relative potency of the behav-ioral value of these two drug congeners (P . 0.05; seeFig. 2). This is consistent with the fact that cocaethyl-ene is equipotent to cocaine with respect to inducingbehavioral activity in rats and nonhuman primates(Jatlow et al., 1991; Katz et al., 1992).

It should be noted that the behavior-activating prop-erties of cocaine and cocaethylene were not replicatedby the application of lidocaine HCl to Drosophila, asthis local anesthetic failed to generate significant boutsof behavioral activity. For example, flies (n 5 18)treated with lidocaine (molar equivalent of 1 mMcocaine) exhibited behavioral rating scores (means 6SEM) of 1.2 6 0.09 that were not markedly different(P . 0.05) from behavioral rating scores of 1.2 6 0.08obtained at t 5 0 min or behavioral scores obtainedfrom water-treated flies (see Fig. 2). Therefore, thepossibility of an anesthetic effect permeating our activebehavioral preparation of Drosophila may be excludedfrom consideration.

Effects of Apomorphine and Octopamineon Behavioral Activity in Drosophila

In general, our results from the previous set ofexperiments indicate that stimulant drugs that inter-fere with DA reuptake, such as cocaine and cocaethyl-ene, increase behavioral activity in Drosophila. We nextexamined whether agents that directly activate D1

receptors (i.e., apomorphine) or endogenous transmit-


Fig.1. Representative scanning electron photomicrographs depict-ing (A) an intact fly, (B) a decapitated fly and (C) surface features ofthe prothoracic neuromere (T1) with an emphasis (arrow) of the dorsalportion, or notum. Stimulant drugs were applied to the notum asdroplets. It should be noted that not all decapitated Drosophilaresponded to the stimulatory actions of cocaine or cocaethylene. Forinstance, out of 97 flies tested with cocaine HCl, 16 (or ,16%) of the

flies failed to show intense circling behavior and significant wingbuzzing activity. The reason for this is not known but may be due to (1)individual differences and/or (2) proteolytic activity that leads tocoagulation and melanization at the site of injury. Perhaps localizedblood clotting initiated by injury forms melanotic masses at the level ofthe notum, thereby preventing cocaine droplets from gaining access todrug-sensitive nerve cells. Scale bar for A and B 5 600 µm.


ters (i.e., octopamine) that play a clear stimulatory rolein invertebrates would generate profound behavioralactivation similar to that observed for cocaine andcocaethylene.Application of apomorphine (molar equiva-lent of 1 mM cocaine) to exposed nerve cord cells ofDrosophila failed to induce significant changes in motorbehavior (Fig. 3). Typical behaviors generated by apo-morphine consisted primarily of discrete fore- andhind-leg grooming (i.e., a rating score of 2) that wereslow and deliberate in their onset. As expected, decapi-tated flies exposed to water alone showed absolutely nosalient bouts of behavioral activity (Fig. 3). From theseresults we conclude that apomorphine, a mixed D1/D2

agonist, is not as powerful as cocaine (or cocaethylene)in producing a robust sequelae of motor activities andstereotypies in decapitated Drosophila. The reason forthis may be related to the fact that low concentrationsof apomorphine activate DA autoreceptors, therebyincreasing the inhibitory actions of such receptors onDA release (Bradbury et al., 1984; Di Chiara et al.,1976).

Octopamine, the invertebrate counterpart of norepi-nephrine, is found in arthropod nerve cells where itmediates neuromuscular transmission (Evans andO’Shea, 1977). This transmitter acts on membrane-

bound receptors through activation of adenylyl cyclaseand cAMP metabolism (Guillen et al., 1989). Applica-tion of octopamine (molar equivalent of 1 mM cocaine)to exposed nerve cord cells of decapitated Drosophilagenerated mild grooming by the fore- and hind-legs(i.e., a rating score of 2). Octopamine at this concentra-tion, however, produced no significant locomotor activ-ity and was devoid of the pronounced stimulatorybehaviors evidenced after cocaine or cocaethylene expo-sure (Fig. 3). In fact, behavioral rating scores assignedto octopamine-treated flies were not markedly different(P . 0.05) than those observed at t 5 0 min (pre-exposure levels) or than behavioral rating scores ofwater-treated flies. Therefore, the striking behavioralresponses generated by stimulant drugs appear to bespecific to the DA system and such a specificity addssignificance to our hypothesis that D1 receptors arelikely to mediate the stimulatory consequences of co-caine application in Drosophila melanogaster.

Effects of SCH-23390, MK-801 andDextromethorphan Agents on Cocaine-Induced

Behavioral Activity in Drosophila

The D1 receptor subtype interacts with Gs complexesto induce second messenger pathways in different

Fig.2. Effects of cocaine (1 mM) and cocaethylene (1 mM) onbehavioral activity in decapitated Drosophila. Each bar represents themean 1 SEM of behavioral rating scores before (t 5 0 min) and afterdrug application. Note the incidence of behavioral activity produced bythe aforementioned stimulant drugs. Behavioral activity was assessedduring 1 minute of visual observation using a graded scale designed toestimate increasing behavioral activity and stereotypy. ANOVAs withone repeated measure showed a significant main effect of drugtreatment (F2,37 5 67.9; P , 0.0001) and a significant main effect of

time (F1,37 5 369.0; P , 0.0001) as well as a significant drug treatmentby time interaction (F2,37 5 87.0; P , 0.0001). Subsequent pairwisemultiple comparison procedures (Newman-Keuls method) were per-formed to determine at which times cocaine- or cocaethylene-treatedflies differed (** P , 0.01) from water-treated flies. Profile analyses ofwater-exposed flies showed no significant effect of treatment or timefactors. Group n values: water, n 5 10; cocaine, n 5 20; cocaethylene,n 5 10.


cellular environments. The gene coding sequence of thisparticular receptor occurs ubiquitously throughout theanimal phyla, suggesting therefore minimal rate ofsequence change over the course of evolution. In mam-mals, D1 receptors exhibit a high affinity for benzaz-epine ligands such as SCH-23390, thereby precludingDA from binding to its postsynaptic membrane site(Civelli et al., 1993). Because cocaine interferes indi-rectly with DA transmission, we tested the possibilitythat blockade of Drosophila putative D1 receptors withSCH-23390 would prevent the stimulatory actions ofcocaine administration. As shown in Figure 4A, thebenzazepine agent SCH-23390, at a concentration of 5mM, effectively inhibited cocaine-induced behavioralactivity in decapitated flies. Such an inhibition wasdose-dependent, as SCH-23390 concentrations below 5mM were ineffectual in preventing bouts of locomotoractivity, circling behavior, and intense wing buzzing incocaine-treated flies. For instance, application of 1 mMSCH-23390 to Drosophila (n 5 16) 1 minute prior to thesubsequent application of cocaine (1 mM) resulted inbehavioral rating scores of (means 6 SEM) 4.1 6 0.5that were not significantly different (P . 0.05) frombehavioral rating scores of 4.6 6 0.4 assigned to flies(n 5 17) administered with water and then cocaine HCl.

In flies, increased locomotor behavior typically elic-ited by cocaethylene exposure was also prevented by 5mM SCH-23390 (Fig. 4B). These findings suggest,

therefore, that D1 receptors (like those identified in thevertebrate brain) mediate the activating properties ofstimulant drugs in decapitated Drosophila. It is note-worthy that when the above selective benzazepinereceptor antagonist was applied to nerve cord parenchy-mal cells, a profound akinesic state was observed in allflies tested. In general, the treated insects were foundto be unresponsive to tactile stimulation and werelargely devoid of spontaneous behavioral activity, likeself-grooming. In addition, the flies assumed a rigidposture position (i.e., immobility or freezing) that wasreminiscent of that assumed in rodents administeredwith the neurotoxin 1,2,3,6-tetrahydro-1-methyl-4-phenylpyridine (MPTP). In some cases, the treated flieslost their balance and displayed a rhythmic tremorpattern in fore- and hind-legs. These results suggestthat functional D1 receptors harbored within thoracicganglia mediate fine-tuning behaviors such as groom-ing and the righting reflex.

In the rat brain, DA and glutamate-mediated syn-apses are coincident on the same neurons and, inparticular, pharmacological manipulation of these syn-apses significantly affect locomotor stimulation inducedby cocaine and other stimulant drugs (Jeziorski et al.,1994; Karler et al., 1989). The specific purpose of thisexperiment was, therefore, to determine whether thebehavior-activating properties of cocaine and cocaethyl-ene in Drosophila would be blocked by excitatory

Fig.3. Effects of apomorphine and octopamine on behavioral activ-ity in decapitated Drosophila. Each bar represents the mean 6 SEM ofbehavioral rating scores before (t 5 0) and after drug application. Bothapomorphine (1 mM) and octopamine (1 mM) failed to produce strikingbehavioral responses similar to those generated by cocaine andcocaethylene exposure. Behavioral activity was assessed during 1

minute of visual observation using a graded scale designed to estimateincreasing behavioral activity in flies. ANOVAs with one repeatedmeasure revealed no significant differences in drug treatment (P 50.25), time (P 5 0.60), or treatment by time interaction (P 5 0.73).Group n values: water, n 5 10; apomorphine, n 5 16; octopamine,n 5 16.


amino acid receptor antagonists. As illustrated in Fig-ure 5A, pretreatment of decapitated flies with 5 mMMK-801 markedly reduced (P , 0.01) the behavioralactivity typically associated with cocaine administra-tion. This behavioral blockade was concentration-dependent, as flies applied with 1 mM MK-801 1minute prior to cocaine (1 mM) continued to expressintense circling behavior and wing buzzing activity. Forexample, decapitated flies (n 5 21) pretreated withMK-801 at a concentration of 1 mM followed by cocaineexhibited behavioral rating scores of (means 6 SEM)4.3 6 0.4 that were not significantly different (P . 0.05)

from behavioral rating scores of 5.0 6 0.3 assigned toflies (n 5 30) treated with water and cocaine HCl. Tofurther characterize the participation of NMDA recep-tors for the expression of cocaine-induced behavioralactivity, we pretreated flies with 5 mM dextro-methorphan. This drug acts as an NMDA antag-onist and also prevents the behavioral consequences ofcocaine administration in rats (Pulvirenti et al., 1997).As observed with MK-801, the incidence of intensebehavioral activity in Drosophila applied with cocainewas significantly reduced (P , 0.01) by pretreatmentwith dextromethorphan (Fig. 6A). It should be noted

Fig.4. Effects of SCH-23390 on (A) cocaine- and (B) cocaethylene-induced behavioral activity in decapitated Drosophila. Each barrepresents the mean 6 SEM of behavioral rating scores before (t 5 0),after pretreatment (t 5 1), and after stimulant drug application (t 52). The D1 receptor antagonist was applied 1 min before the adminis-tration of cocaine or cocaethylene. Note that SCH-23390 (5 mM)profoundly inhibited the behavior-activating properties of cocaine andits ethyl metabolite, cocaethylene. By a two-way repeated measureANOVA, the main effect of cocaine treatment was significant (F2,112 536.1; P , 0.0001) as was the main effect of time (F2,112 5 57.1; P ,

0.0001). The drug and time interaction was also significant (F4,112 541.0; P , 0.0001). A similar statistical trend was observed forcocaethylene in terms of drug treatment (F2,84 5 23.1; P , 0.0001),time effect (F2,84 5 40.0; P , 0.0001), and drug by time interaction(F4,84 5 21.4; P , 0.0001). Subsequent pairwise multiple comparisonprocedures (Newman-Keuls method) were performed to determine themarked (* P , 0.05) inhibitory effect of SCH-23390 on cocaine- andcocaethylene-induced behavioral activity. Group n values: water 1water, n 5 10; water 1 cocaine, n 5 17; water 1 cocaethylene, n 5 10;SCH-23390 1 cocaine, n 5 32; SCH-23390 1 cocaethylene, n 5 25.


that both MK-801 and dextromethorphan (at equalmolar dosage) produced similar reductions in behav-ioral activity in flies when administered 1 minute priorto cocaine.

We next examined whether MK-801 and dextrometho-rphan would block the stimulatory actions of cocaethyl-ene in an active preparation of Drosophila. As withcocaine, the two NMDA receptor antagonists effectivelyreduced the expression of hyperactive behavior typi-cally associated with cocaethylene exposure (Fig. 6B).Over the concentration range studied (5 mM), MK-801

and dextromethorphan, when administered in conjunc-tion with water, depressed spontaneous behavioralactivity and caused mild ataxia in decapitated flies.Therefore, antagonists of NMDA receptors that medi-ate excitatory amino acid synapses block the inductionof behavioral activity to stimulant drugs. Congruently,the results described here demonstrate two additionalimportant findings: (1) that (ionotropic) NMDA recep-tors are present in thoracic ganglia of Drosophila, and(2) NMDA receptors coordinate fine-tuning of motorprograms in behavioral networks in invertebrates.

Fig.5. Effects of MK-801 on (A) cocaine- and (B) cocaethylene-induced behavioral activity in decapitated Drosophila. Each barrepresents the mean 6 SEM of behavioral rating scores before (t 5 0),after pretreatment (t 5 1), and after stimulant drug application (t 52). The NMDA receptor antagonist was applied 1 minute before theadministration of cocaine or cocaethylene. In general, MK-801 (5 mM)substantially decreased the stimulatory effects of cocaine and its ethylmetabolite, cocaethylene. ANOVAs with one repeated measure showeda significant main effect of cocaine treatment (F2,104 5 34.7; P ,0.0001) and a significant main effect of time (F2,104 5 45.0; P , 0.0001)as well as a significant drug treatment by time interaction (F4,104 5

35.1; P , 0.0001). A similar significant profile analysis was observedfor cocaethylene in terms of drug treatment (F2,68 5 11.0; P , 0.0002),time effect (F2,68 5 22.1; P , 0.0001), and drug treatment by timeinteraction (F4,68 5 13.6; P , 0.0001). Subsequent pairwise multiplecomparison procedures (Newman-Keuls method) were performed todetermine the substantial (* P , 0.05) inhibitory effect of MK-801 oncocaine- and cocaethylene-induced behavioral activity. Group n val-ues: water 1 water, n 5 10; water 1 cocaine, n 5 30; water 1cocaethylene, n 5 15; MK-801 1 cocaine, n 5 15; MK-801 1 cocaethyl-ene, n 5 12.


DISCUSSION

The present experiments demonstrate that cocaineand cocaethylene independently produce significantincreases in behavioral activity in an active prepara-tion of Drosophila melanogaster. In addition, we showthat the stimulatory actions of these drugs depend onpharmacologically-defined D1 and NMDA membrane-bound receptors presumably localized to discrete tho-racic neurons. In general, these findings indicate thatcocaine and other stimulant drugs generate motorprograms of activity that are common to all animals

studied thus far, and more importantly, generation ofsuch motor programs of activity are mediated by homolo-gous, highly conserved cellular receptor subtypes. Theseoutstanding experimental features make Drosophilaan attractive model in which to study basic mecha-nisms specifying changes in (1) behavior activity; (2)signal transduction pathways and; (3) gene expressionassociated with the development of a drug-dependentphenotype.

Administration of cocaine to rats, nonhuman pri-mates, and humans often results in stereotypy and

Fig.6. Effects of dextromethorphan (DEX) on (A) cocaine- and (B)cocaethylene-induced behavioral activity in decapitated Drosophila.Each bar represents the mean 6 SEM of behavioral rating scoresbefore (t 5 0), after pretreatment (t 5 1), and after stimulant drugapplication (t 5 2). The NMDA receptor antagonist (and also avoltage-dependent Ca21 channel blocker) was applied 1 minute beforethe administration of cocaine or cocaethylene. DEX (5 mM) inhibitedbehavioral activity that was induced by cocaine or cocaethylene inflies. By a two-way repeated measure ANOVA, the main effect ofcocaine treatment was significant (F2,98 5 3.84; P , 0.0282) as was themain effect of time (F2,98 5 28.1; P , 0.0001). The drug and time

interaction was also significant (F4,98 5 10.0; P , 0.0001). A similarstatistical trend was observed for cocaethylene in terms of drugtreatment (F2,72 5 35.1; P , 0.0001), time effect (F2,72 5 48.4; P ,0.0001), and drug by time interaction (F4,72 5 34.0; P , 0.0001).Subsequent pairwise multiple comparison procedures (Newman-Keuls method) were performed to determine the significant (* P ,0.05) inhibitory effect of DEX on cocaine- and cocaethylene-inducedbehavioral activity. Group n values: water 1 water, n 5 10; water 1cocaine, n 5 30; water 1 cocaethylene, n 5 10; DEX 1 cocaine, n 5 12;DEX 1 cocaethylene, n 5 19.


increased motor behaviors. The mechanism by whichcocaine promotes behavioral activity in mammals re-sides in its ability to block the DA reuptake transporter,a Na1/Cl2 dependent protein that mediates the re-moval of DA molecules from the synapse (Amara, 1995;Giros et al., 1996). As a result of this blockade, thetemporal and spatial activity of DA is dramaticallyincreased, thereby facilitating the binding of this mol-ecule to well-defined D1 receptors (Civelli et al., 1993;Gingrich and Caron, 1993). In this report, we show thatapplication of cocaine to decapitated flies also results instriking bouts of behavioral activity. In addition, weshow that application of cocaethylene, another drugthat interferes with DA reuptake in humans (Hearn etal., 1991), results in distinguishable behavioral re-sponses in Drosophila. Cocaethylene is a cocaine me-tabolite synthesized in mammalian hepatic cells follow-ing the simultaneous coingestion of cocaine and ethanol(Boyer and Petersen, 1992; Dean et al., 1991). Ofsignificant interest, cocaethylene blocks the DA trans-porter but, unlike cocaine, has little or no effect on theserotonin (5-hydroxytryptamine; 5-HT) transporter(Bradberry et al., 1993; Hearn et al., 1991). Thisparticular characteristic of cocaethylene points to thepossibility that both cocaine and cocaethylene are ableto affect the DA reuptake transporter of Drosophila,selectively, to produce an altered behavioral phenotype.However, to the best of our knowledge, there are noindications of DA transporter transcripts in nerve cellsof the thoracic dopaminergic pathway. Therefore, weare unable to explain fully how cocaine and cocaethyl-ene produce profound behavioral activity in decapi-tated flies. One possibility may be that Drosophilaexpress distinct genes encoding transporter proteinsthat mobilize DA and other monoamines very efficientlyacross the synapse. In fact, a selective 5-HT transporterthat is cocaine-sensitive has been recently cloned inDrosophila (Corey et al., 1994; Demchyshyn et al.,1994). This finding lends support to the possibility thatcocaine and cocaethylene generate motor activity inflies through the blockade of a biogenic amine trans-porter that lacks relative substrate and inhibitionselectivity. The topographic expression of 5-HT trans-porter mRNA in perikarya in each thoracic ganglion ofDrosophila (Demchyshyn et al., 1994) adds furthercredence to the above possibility. Therefore, a Dro-sophila 5-HT transporter that has a high substratespecificity for 5-HT, but shares a pharmacological pro-file similar to the mammalian DA transporter, may bethe transporter molecule regulating dopaminergic toneand subsequent behavioral function in cocaine- andcocaethylene-treated flies.

The thoracic central nervous system of Drosophilaharbors both DA- and 5-HT-containing neurons (Bud-nik and White, 1988; Budnik et al., 1989). These cellgroups, in conjunction with brain neurons of the centralcomplex, control certain features of insect motor behav-

ior such as righting, grooming, and walking (Straussand Heisenberg, 1993). Neural cluster circuits contain-ing DA are found near the prothoracic neuromere ofDrosophila, thereby providing a topographic relation-ship between neurons that regulate motor activity andsite of administration for cocaine and cocaethylenesolutions (see Fig. 1 for details). Although not wellcharacterized in terms of dendritic synaptic contactsand terminal axonal arbors, the dopaminergic circuit offlies consists of descending and ascending axonal fibersthat connect subesophageal and thoracic ganglia in arather circuitous trajectory (Nassel and Elekes, 1992).In this study, administration of stimulant drugs todecapitated flies was made to the exposed nerve cord atthe level of the prothoracic neuromere. Therefore, it islikely that both cocaine and cocaethylene solutions areeither diffusing or are transported to thoracic targetneurons via descending axonal fibers that emanatefrom cell bodies in the subesophageal ganglion. Thishypothetical mode of diffusion of stimulant drugs isderived entirely from that proposed by Hirsh’s group toexplain how exogenous applied amines reach neuronsof the nerve cord (Yellman et al., 1997). This mode ofdiffusion would also account, in part, for the relativelyhigh concentrations of cocaine and cocaethylene neededin the present experiments to elicit behavioral re-sponses in decapitated flies. For example, mM concen-trations of stimulant drugs may be required because ofpotential Wallerian degeneration of descending axonalfibers after severance from their perikarya. However,axonal segments in various invertebrate cells degener-ate very slowly and maintain axoplasmic transportintegrity for several days after severance (Tanner et al.,1995). Because the flies in our experiments were usedimmediately after decapitation, this possibility may beexcluded from consideration. Alternatively, the rela-tively high concentrations of cocaine and cocaethylenerequired in this study may be related to nonspecificchanges in sensitivity of DA postsynaptic receptorsresulting from DA denervation or depletion, or both.Such changes (i.e., down-regulation) may reflect themovement of receptors into or out of neuronal mem-branes to return their function to normal levels. Regard-less of the underlying mechanisms for the high concen-trations of stimulant drugs used here, our resultsclearly show that cocaine and cocaethylene stimulatemotor programs of activity in flies devoid of any braintissue, including the central complex. Therefore, it islikely that thoracic neurons of Drosophila harbor motorprograms that regulate specific patterns of behavior.Interestingly, the spinal cord of a decerebrated verte-brate also responds to DA agonists despite the fact thatit is the functional D1 receptors in the substantia nigrathat regulate muscle tone activity (Double and Crocker,1995). The similarity of behavioral responses betweeninsect and mammal lineages is of significant interestbecause it suggests a conserved genetic linkage or


relationship of certain features of autonomic function.In principle, this linkage or evolutionary relationshipcould be useful for identifying nerve cord cell types thatmay have a similar biochemical and functional activityin arthropods and rats. For instance, one can speculatethat DA cells in the prothoracic neuromere of Dro-sophila may have an equivalent match with small,intensely fluorescent (SIF) cells of vertebrates. In thiscontext, SIF cells (1) synthesize DA; (2) are found insympathetic ganglia, including the superior cervicalganglia of rats where they both receive and providesynapses; and (3) mediate vertebrate autonomic ner-vous system function (Matthews, 1989). Thus, it istempting to draw a parallel between the two homolo-gous DA-cell types.

Cocaine and cocaethylene have a striking effect onbehavioral activity in flies. In rats, these behavioraleffects appear to be mediated by functional D1 (and D2)receptors as these effects can be attenuated by selectiveDA receptor antagonists (Horowitz et al., 1997b; Hy-man, 1996; Koob, 1996). Here we demonstrate thatblockade of DA receptors in Drosophila by SCH-23390,a selective antagonist at the mammalian D1 receptor,significantly attenuates the behavior-activating proper-ties of cocaine and cocaethylene. This suggests that thereceptor mediating the behavioral responses to stimu-lant drugs in flies is pharmacologically similar to themammalian D1 subtype. Our findings are limited to theeffects of SCH-23390, and, therefore, we cannot excludethe possibility of this benzazepine ligand blocking otherDA-like receptors involved in Drosophila motor behav-ior. Nevertheless, the fact that SCH-23390 blocks thebehavioral consequences of cocaine and cocaethylene isconsistent with the notion that mammalian DA recep-tors are sensitive to this class of ligand. The apparentdependence of stimulant drugs on D1 receptor functionin flies raises the possibility of a prototypic DA receptorlocalized to a defined region of the prothoracic neuro-mere. However, the cDNAs that encode D1-like recep-tors in Drosophila appear to be expressed exclusively inbrain tissue (Feng et al., 1996; Han et al., 1996) and,therefore, they may be excluded from consideration. Anadditional D1-like receptor recently cloned from Dro-sophila may also be excluded as the receptor required toinitiate patterns of behavioral activity by cocaine asthis novel receptor displays a poor affinity for SCH-23390 (Sugamori et al., 1995). We are then left with thepossibility that D1-like receptors in fly prothoracic cellsare similar, but not identical to D1 receptor subtypesfound in brain neurons. Discrete substitutions of amino-acids in a receptor gene sequence can significantlymodify the pharmacological profile of closely-relatedreceptors; therefore, differences in binding affinity be-tween D1-like receptors in Drosophila are not unex-pected. Regardless of the receptor subtype mediatingthe stimulant effects of cocaine and cocaethylene indecapitated flies, our results clearly show that a D1-like

receptor represents an obligatory target for these behav-ioral effects. This hypothesis is further corroborated bythe fact that octopamine, which acts via non-DA recep-tors (Bodnaryk, 1979), failed to generate comparablesets of behavioral patterns as those ascribed in fliesafter cocaine administration. This finding suggests,therefore, that the behavioral effects of stimulant drugsare unrelated to changes in the octopamine system ofDrosophila.

Virtually all signal transmission at neuronal syn-apses is mediated by glutamate. This excitatory aminoacid molecule acts in brain cells by binding to ionotropicor metabotropic transmembrane-bound receptors. Iono-tropic receptors are subdivided into NMDA receptorsand alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropio-nate (AMP)-kainate receptors according to their selec-tive agonists (Nakanishi, 1992, and references therein).In rats, stimulation of NMDA receptors is one of thecrucial pathways by which cocaine and other drugs ofabuse lead to specific neuroadaptive phenomena. Theseinclude sensitization to cocaine, opiate tolerance, andabstinence and ethanol withdrawal (Koob, 1996; Taba-koff and Hoffman, 1996). Conversely, blockade of ratNMDA receptors by coadministration of MK-801 signifi-cantly mitigates the locomotor-activating properties ofcocaine and amphetamine (Kalivas and Alesdatter,1993; Karler et al., 1989; Wolf and Jeziorski, 1993). Theexact mechanisms for the apparent synergy betweenNMDA and DA receptors in the rat brain are unknown.It may be that neurons targeted by the actions ofcocaine express both types of receptor molecules, anotion consistent with present models of drug addiction(Nestler, 1995; Hyman, 1996; Torres and Horowitz,1996). A second possibility is that the effects of cocaineare mediated by local NMDA and DA neural circuitinteractions (Hyman, 1996). In either case, NMDAreceptors appear to be crucial for some of the behavioralconsequences of cocaine administration in rats. Interest-ingly, here we describe that an NMDA-like receptor alsorepresents an obligatory target for the activating prop-erties of cocaine (and cocaethylene) in Drosophila. Asblockade of putative NMDA receptors by MK-801 suc-ceeded in mitigating the behavioral changes associatedwith cocaine exposure in decapitated flies, it is tempt-ing to draw a parallel between rats and flies in terms ofintegrative NMDA receptor function. Indeed, this paral-lelism is further corroborated by the fact that dextro-methorphan, a noncompetitive NMDA receptor antago-nist and a blocker of voltage-dependent Ca21 channels(Carpenter et al., 1988), also prevented the stimulatoryeffects of cocaine (and cocaethylene) in decapitatedflies. These data suggest, therefore, that the Drosophilaexcitatory pathway cascade indirectly initiated by appli-cation of cocaine to prothoracic nerve cells activatesboth D1 and NMDA-like receptors to produce an alteredbehavioral phenotype. If this suggestion is substanti-ated by future experimental work, invertebrate NMDA-


like receptors would appear to be transmembraneproteins regulating intracellular signals that ulti-mately give rise to specific motor patterns of behavior.It is of interest to note that Drosophila neurons respondto glutamate, where it appears to act as a motorexcitatory transmitter (for review see Walker andHolden-Dye, 1989). However, there is little or no evi-dence for an NMDA receptor subtype in this or otherarthropods. Using a pharmacological approach charac-terized from cocaine-challenged Drosophila, we demon-strate that NMDA-like receptors show remarkablefunctional similarities to receptor homologues studiedin rats. This suggests, but does not prove, that (1)NMDA-like receptors are present in Drosophila; (2)NMDA-like receptors may work in synergy with puta-tive D1 receptors to activate motor programs of behav-ior in response to cocaine exposure; and (3) the strikingparallels that appear to exist between DrosophilaNMDA-like receptors with NMDA receptors in rats,point to a common ancestral origin of essential aspectsof motor behavior. Thus, as in mammals, behavioralactivation by cocaine or cocaethylene in Drosophilacould be due to a summation of effects at multiplereceptor targets. Of interest, the involvement of both D1

and NMDA-like receptor systems in cocaine-treatedflies may help explain an apparent paradoxical issue inthe present experiments, namely that of the effects ofapomorphine. As previously shown, apomorphine, adirect DA receptor agonist failed to produce behavioraleffects similar to those observed after cocaine or cocae-thylene administration. The results obtained from theNMDA-related experiments suggest that this discrep-ancy may reflect the inability of apomorphine to stimu-late NMDA receptors as well. Similarly, our resultsappear to confirm that application of low doses ofapomorphine to Drosophila activate DA autoreceptors,which in turn inhibit DA release. Less DA content atthe synapse would reduce behavioral activity mediatedby putative D1 (and NMDA) postsynaptic receptorpathways.

In conclusion this study, in conjunction with that ofothers (Yellman et al., 1997), establishes an activepreparation of decapitated Drosophila as a viable experi-mental system in which to investigate the pharmacol-ogy of DA and glutamate synapses. In addition, thisstudy is, to our knowledge, the first to use this activepreparation to ascertain the behavioral consequences ofcocaine and cocaethylene administration. Because theinitial cellular steps in the mechanisms mediatingdrug-induced behavioral activity appear to be similar ininsects and mammals, Drosophila could be used toanswer many fundamental questions about the cir-cuitry that regulates motor programs of behavior. Fi-nally, given the conservation of cellular function at themolecular level in all animals, this active preparationcould be used to study changes in signaling pathways,gene expression, and protein synthesis that inevitably

occur after cocaine exposure. Such molecular changesand their relationship to behavior should provide novelinsights into the neurobiology of drug addiction.

ACKNOWLEDGMENTS

This work was supported in part by a ResearchFoundation Award of the State University of New Yorkto German Torres. We thank Dr. Mark B. Kristal for hiscomments on early versions of this manuscript. Theauthors thank the National Institute on Drug Abuse forproviding cocaethylene fumarate. We also thank, at theState University of New York at Buffalo, the Micro-scopic Imaging Facility (Alan Siegel) for scanning elec-tron microscopy services, and the South Campus Instru-mentation Center (Peter J. Bush) for video andcomputer-based imaging analysis.

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