Methamphetamine causes anorexia in Drosophila melanogaster, exhausting metabolic reserves and contributing to mortality

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1 The Journl of Toxicologicl Sciences (J. Toxicol. Sci.) Vol.37, No.4, , Originl Article Methmphetmine cuses norexi in Drosophil melnogster, exhusting metolic reserves nd contriuting to mortlity Kent R. Wlters, Jr. 1, S. Indu Rupssr 2, R.J. Cody Mrkelz 3,4, Andrew D.B. Lekey 3,4, Willim M. Muir nd Brry R. Pittendrigh 1 1 Deprtment of Entomology, 16/18 Morrill Hll, University of Illinois, Urn, IL 6181, USA 2 Deprtment of Moleculr nd Integrtive Physiology, University of Illinois, Urn, IL 6181, USA 3 Deprtment of Plnt Biology, University of Illinois, Urn, IL 6181, USA 4 Institute for Genomic Biology, 142 Institute for Genomic Biology, 126 W Gregory Dr, University of Illinois, Urn, IL 6181, USA Deprtment of Animl Sciences, Room G4, Lily Hll, Purdue University, West Lfyette, IN 4796, USA (Received April 19, 212; Accepted My 28, 212) ABSTRACT Methmphetmine (MA) ppers to produce neurotoxic effects, in prt, through disruptions of energy metolism. A recent study of the whole-ody proteome of Drosophil melnogster showed mny chnges in energy metolism-relted proteins, leding us to hypothesize tht MA toxicity my cuse whole-ody disruptions of energy metolism. To test this, we monitored the response of energy reserves nd other metolites to MA-exposure with nd without the ddition of dietry glucose. We lso monitored chnges in feeding ehvior, locomotor ctivity nd respirtion rtes ssocited with MA-exposure to investigte how MA ffects energy lnce. We oserved tht glycogen nd triglyceride levels decresed drmticlly within 48 hr of MA-exposure, indicting strongly negtive cloric lnce. Behviorl ssys reveled tht MA-treted flies decresed food consumption y 6-8% nd exhiited 2-fold increse in locomotion. Cloric expenditure decresed with MA-exposure, pprently due to compenstory decrese in resting metolism, showing tht norexi ws the primry driver of the negtive cloric lnce. Additionlly, we oserved tht glucose supplementtion of MA-contining diet incresed glycogen reserves y 44% t 48 hr, leding to commensurte increse in survivorship. We conclude tht dietry sugr supplementtion enhnces survivorship y prtilly compensting for decresed cloric intke resulting from MA-induced norexi. The oservtion tht MA produces similr ehviorl chnges in Drosophil nd humns, i.e. incresed locomotor ctivity nd norexi, further supports the use of Drosophil s model orgnism for the study of the effects of MA. Key words: Methmphetmine, Toxicity, Feeding ehvior, Anorexi, Locomotor ctivity, Metolic rte INTRODUCTION Methmphetmine (MA) is proly the most widely used synthetic stimulnt in the world nd ppers to e incresing in populrity (EMCDDA, 29), with some Europen countries reporting tht MA is the most widespred illicit drug fter cnnis. In ddition to eing psychoctive stimulnt, MA lso produces numerous toxic side-effects tht hve importnt implictions for humn helth, including incresed oxidtive stress (Cuells et l., 1994; Ymmoto nd Zhu, 1998), hert filure (Hong et l., 1991; Yu et l., 23), neurotoxicity leding to poptosis (Cuells et l., 1994; Ymmoto nd Zhu, 1998; Stephns et l.,1998; Dvidson et l., 21), nd ltertion of energy metolism in the rin (Stephns et l., 1998; Chn et l., 1994; Wng et l., 24; Virmni et l., 22). In mny cses, the moleculr underpinnings of how MA induces these toxic effects re not completely understood nd cnnot redily e studied in humns for ethicl resons. Results from Sun et l. (211) showed tht Drosophil melnogster is useful model system in which to study Correspondence: Kent R. Wlters, Jr. (E-mil: krwltersjr@gmil.com)

2 774 K.R. Wlters et l. the toxic effects of MA. In comined proteomic, trnscriptomic, nd metolomic study, MA produced similr toxicologicl effects in Drosophil to those oserved in humns (Sun et l., 211), suggesting the involvement of evolutionrily conserved pthwys. Furthermore, Drosophil hs mny ttriutes tht mke it n extremely ttrctive model orgnism, including well chrcterized nd functionlly nnotted genome (Roy et l., 21; Celniker et l., 29; Adms et l., 2); lrge numer of genetic tools, such s ility to selectively knock down the expression of the mjority of the known genes, using RNAi, in tissue- nd developmentl stte-specific mnner (Dietzl et l., 27); nd Drosophil is redily mintined in lortory culture. In ddition, reserchers hve estlished ehviorl iossys tht mesure feeding, locomotor ctivity nd ggression in Drosophil (Wong et l., 28, 29; Kume et l., 2; Dnkert et l., 29), which re useful for understnding the iologicl responses to prticulr drug/toxin. The toxic effects of MA re est understood in the rin, where there is compelling evidence tht toxicity rises, in prt, from the ility of MA to disrupt norml metolism. Depletion of ATP following MA exposure is correlted to MA-induced neurotoxicity in the stritum nd impeding glucose (Glc) metolism prior to MAtretment potentites this toxicity (Chn et l., 1994). Moreover, supplementtion with sustrtes or cofctors of energy metolism llevites the effects of MAinduced neurotoxicity (Stephns et l., 1998; Virmni et l., 22). In humn sujects, MA produced pronounced nd long lsting suppression of Glc metolism in the stritum nd thlmus (Wng et l., 24). As thlmic metolism reounded with time, corresponding improvements were lso oserved in neuropsychologicl function. All of these studies support the role of MA s metolic toxin in the rin. Chnges in the whole-ody proteome oserved y Sun et l. (211) suggest tht the metolic chnges produced y MA my not e limited to the rin. Crohydrte trnsporters nd mny glycolytic enzymes, including hexokinse, the rte-limiting step of glycolysis, were found to e up-regulted y ~1-fold in whole ody homogentes (Sun et l., 211). Furthermore, the ddition of dietry crohydrtes incresed survivorship of MA exposed flies. While these oservtions suggest tht MA my disrupt norml energy metolism, they do not elucidte how MA ffects energy metolism, leding us to formulte two competing hypotheses concerning the mjor driver of the system. One possiility, the metolic hypothesis, is tht MA directly disrupts metolism y ltering the expression of metolic genes nd/or y intercting directly with metolic proteins, which is supported y the oserved 1-fold increses in crohydrte trnsporters nd glycolytic proteins (Sun et l., 211). In contrst, ccording to the ehvior hypothesis, MA chnges ehviors, such s feeding ehvior nd locomotor ctivity, leding to disruption of the energy lnce. The ltter hypothesis is supported y oservtions in humns nd other mmmls where MA leds to norexi (Cole, 1967; Evns, 1971; Mdden et l., 2) nd chnges in locomotor ctivity (Wllce et l., 1999). In this study, we monitored the response of energy reserves nd other metolites to MA-tretment to test for chnges in energy lnce. We lso mesured ehviorl/physiologicl prmeters, such s feeding ehvior, locomotor ctivity nd respirtion rte, to test the metolic nd ehvior hypotheses. Methods nd Mterils Fly rering conditions nd virgin collection Drosophil melnogster (w 1118 ) stocks ttined from Dr. Mish Ludwig (University of Chicgo) were rered on Formul 24-4 instnt diet (Crolin Biologicl Supply Co., Burlington, NC, USA) in ottles t 2ºC nd % reltive humidity on 12 hr photoperiod. All flies used in this study were -dy old virgin mles. To collect virgin flies, dult flies were removed from the ottles on the evening prior to collection, nd the following morning the new dults were nesthetized y CO 2. Virgin flies were visully identified nd were not nesthetized for longer thn 4 min to limit CO 2 -induced stress. Virgin mles were trnsferred to 1 ml vils (2 individuls per vil) nd mtured for d prior to experimentl tretments. Biossy procedures Ten -dy-old virgin mle flies were plced in 2. ml microcentrifuge tues contining 4 mg of reconstituted Formul 24-4 instnt Drosophil diet in the ottom, nd the top of ech tue ws plugged with cotton. Flies were riefly nesthetized with CO 2 to fcilitte trnsfer unless otherwise noted. Flies were plced in one of the four tretments tht differed only in the finl composition of the diet: (1) Control- diet reconstituted with distilled H 2 O; (2) MA- diet supplemented with.6% MA; (3) MA+glc- diet supplemented with.6% MA nd 3% Glc; nd (4) Glc- diet supplemented with 3% Glc. Flies were trnsferred into 2 ml tues in the morning (2-4 hr fter lights on). The tues contining the flies were kept inside lrge plstic pill ox, which ws closed fter 48 hr to prevent excessive desicction.

3 77 Methmphetmine lters feeding nd locomotor ehvior in Drosophil Survivorship ssys Flies were hndled nd mintined ccording to the guidelines previously outlined in the iossy procedures. Survivorship, which ssessed under dissecting scope y the presence/sence of movement, ws monitored t two-hr intervls from 7 m to 9 pm for seven dys. Trehlose ssys After 12 hr, 24 hr, nd 48 hr of tretment using iossy procedures outlined ove, flies were nesthetized with CO 2, trnsferred to pre-weighed 1. ml tue, weighed to the nerest.1 mg, nd flsh frozen in liquid nitrogen. For the hr time-point, the -dy-old virgin flies were weighed nd flsh frozen without eing plced in the 2 ml tues. Flies were stored t -8ºC until metolites could e extrcted. For the metolite extrction, 2 µl of ice-cold 1% ethnol were dded to the flies, which were then homogenized for 3 min using sterile pestle driven y ttery-powered pestle motor. The smples were plced in heting lock t 8ºC for 1 min, fter which 4 µl of % (v/v) methnol ws dded. The smples were centrifuged t 1, g for 1 min to sediment insolule deris. The superntnt from ech ws trnsferred to new tue nd ws concentrted to dryness using SpeedVc. Ech smple ws reconstituted with µl of distilled wter. The stndrd protocol from the spectrophotometric trehlose kit (Megzyme Interntionl Irelnd Ltd., Wicklow, Irelnd, UK), including the orohydride reduction, ws scled down ten-fold to determine trehlose concentrtions in 96-well plte reder ssy. The spectrophotometric ssy used trehlse to convert trehlose into glucose, which ws converted in two successive rections to 6-phosphogluconte. The conversion of the cofctor for this ltter rection (NADP to NADPH), which ws stoichiometric to the mount of trehlose, ws mesured t 34 nm using Synergy HT pltereder (BioTek Instruments, Winooski, VT, USA). This ssy ws found to give results similr to HPLC detection of trehlose (Holovti nd Acker, 27). Ech smple ws run in duplicte nd the trehlose concentrtion ws determined from stndrd curve on ech plte. Methmphetmine ELISA The metolite extrcts prepred for trehlose nlysis were nlyzed for MA content using MA direct ELI- SA kit (Immunnlysis Corportion, Pomon, CA, USA). Reconstituted smples were diluted from 1- to 2,- fold with 1 mm phosphte uffer, ph 7., so tht MA concentrtions were etween 1- ng/ml. Plin diet nd Glc-treted control smples were diluted 1-fold. A stndrd curve ws run on ech plte. In ddition, smples from ll four tretments were spiked with known mounts of MA stndrd to test for interference from the metolite extrcts. Glycogen nd totl Glc nlysis The tretment of flies for the glycogen nlysis only vried from the trehlose ssy (see ove) with respect to how flies were processed fter storge. Following storge t -8ºC, the flies were homogenized in 1 µl of distilled wter for 3 min using ttery powered pestle motor. The homogente ws centrifuged t 4ºC for min t 1, g to sediment deris. Glycogen stndrds (2 µg/ml, 4 µg/ml, 6 µg/ml, nd 8 µg/ml) were treted identiclly to superntnt from the smples. To determine totl Glc concentrtions, Glc-contining compounds, including trehlose nd glycogen, were enzymticlly hydrolyzed, nd the resulting Glc concentrtion ws mesured. Trehlose ws presumly hydrolyzed y endogenous trehlse; we confirmed tht trehlose ws not detectle t the end of the incution nd tht trehlose ws detectle if the homogente ws het treted t 8ºC following homogeniztion (dt not shown). Fifteen µl of mm cette uffer, ph 4., contining.2 units of myloglucosidse, ws dded to 3 µl of superntnt from ech smple (dpted from Roehrig nd Allred (1974)). The totl volume ws mde up to 7 µl with distilled wter nd the resulting solution ws incuted t ºC for 2 min. An dditionl 7 µl of distilled wter were dded to ech smple fter incution. All smples were lso incuted with cette uffer from which myloglucosidse omitted to permit the clcultion of totl glycogen. Glc concentrtions were determined using Glc oxidse (GO) ssy kit (Sigm, St Louis, MO, USA) (Roehrig nd Allred, 1974). The ssy ws scled down 2-fold from the kit instructions for 96-well plte formt. Triglyceride nlysis Flies were prepred nd frozen in mnner identicl to the trehlose nd glycogen ssys (see ove). After storge t -8ºC, lipids were extrcted nd quntified ccording to Al-Anzi nd Zinn (21) with slight modifictions. Briefly, flies were crushed in 2 µl of chloroform/methnol (2:1) nd were incuted t room temperture for one hour with gentle gittion. The smples were evported to dryness in fume hood nd the resulting pellet ws resuspended in 2 µl chloroform/methnol per 1 mg fresh mss. Two µl of superntnt from ech smple ws spotted on silic gel TLC plte. The pltes were run with moile phse of hexne nd diethyl ether

4 776 K.R. Wlters et l. (4:1) nd were visulized y dipping in ceric mmonium molydte solution (see Al-Anzi et l. (29) for recipe) followed y heting t 9ºC for 1-2 min. Bnd intensity ws quntified using NIH ImgeJ softwre. Two replicte pltes (technicl replictes) were run for ech timepoint nd ech plte contined four iologicl replictes for ech of the four tretments, s well s series of stndrds (Fig. 1D). Feeding experiments Flies were fed Formul 24-4 instnt diet reconstituted with 2.% solution of lue dye, which is neither ssimilted nor degrded, in order to mesure gut cpcity nd gut throughput (dpted from Wong et l. (28)). The iossy procedures outlined ove were used with two exceptions. First, 2.% (w/v) lue dye #1 ws dded to the diet in ech of the four tretments. Second, in ddition to eing trnsferred with CO 2 nesthesi (stndrd procedure), flies were lso trnsferred into the 2 ml tues using n spirtor (without nesthesi) to permit comprison etween the two methods. After 24 hr, the flies were trnsferred to pre-weighed clen 1. ml microcentrifuge tues (flies were visully inspected for superficil lue dye, soiled individuls were excluded), were immeditely weighed nd flsh frozen in liquid nitrogen. The flies were ground y hnd in 2 µl of distilled wter using lue pestle. The smples were centrifuged for 1 min t 1, g. After which, the superntnt ws removed nd filtered through.2 µm filter. An dditionl 4 µl of distilled wter were dded to the smple prior to nlysis. The sornce of the lue dye relesed from the flies, mesure of gut cpcity (Wong et l., 28), ws mesured t 63 nm nd the concentrtion ws determined from stndrd curve. The effect of MA nd sucrose octcette (SOA), itter nontoxic tstnt, on dietry preference ws mesured in two-choice test. Ten flies were strved for 24 hr prior to the experiment with ccess only to wter. The flies Reltive nd intensity A C12 M12 M+G12 G12 Reltive nd intensity B C24 M24 M+G24 G24 Reltive nd intensity C C48 M48 M+G48 G48 D solvent front C48 M48 M+G48 G48 Std } triglycerides origin Fig. 1. Triglyceride content s function of tretment durtion. Triglyceride content ws mesured fter 12 hr, 24 hr nd 48 hr for ten virgin mle -dy-old Drosophil dults tht were plced in 2 ml microcentrifuge tues contining either undulterted 4-24 instnt medium, medium supplemented with.6% methmphetmine, medium supplemented with.6% methmphetmine nd 3% glucose or medium supplemented with 3% glucose (n = 4, ± S.E.M.). Sttisticl comprisons of men triglyceride content for 12 hr, 24 hr nd 48 hr time-points re presented in A, B nd C, respectively. Ordinte xes lels in A, B, C nd D use letters to indicte tretment (e.g., C = plin diet control, M = MA-contining diet, M+G = diet supplemented with MA nd Glc, nd G = Glc supplemented control) nd numers to indicte time. Distinct lowercse letters represent different men vlues (p <., Tukey s MCT). D shows TLC plte with four replictes for ech tretment t 48 hr. For the triglyceride stndrd, 2 µg, 4 µg, 8 µg nd 1 µg of triglyceride were spotted from left to right.

5 777 Methmphetmine lters feeding nd locomotor ehvior in Drosophil were then trnsferred without nesthesi into n ren (Suppl. Fig. 1) contining one of the following pired diet options: either (i) control diet leled with 2.% (w/v) red food coloring nd.6% MA-contining diet leled with 2.% (w/v) lue food coloring or (ii) 1.2% SOA-contining diet leled with 2.% (w/v) red food coloring nd.6% MA-contining diet leled with 2.% (w/v) lue food coloring. The flies were plced in the drk during the ssy to prevent color from confounding the results. After 9 min, the flies were weighed, flsh frozen, nd stored t -2 C. The flies were ground y hnd in 2 µl of distilled wter using lue pestle. The smples were centrifuged for 1 min t 1, g. After which, the superntnt ws removed nd filtered through.2 µm memrne. An dditionl µl of distilled wter ws used to rinse the filter memrne. The sornce of the lue nd red dyes in ech smple ws mesured t 63 nm nd nm, respectively. The concentrtion of ech ws determined from stndrd curve. The effect of MA nd sucrose octcette (SOA) on short term (6 hr) nd long term (24 hr) food consumption ws mesured in slightly different method thn descried ove to llow for short term mesurements. Thirty mg of dye-leled diet, which ws either undulterted, or supplemented with.6% MA or 1.2% SOA ws plced on polypropylene mesh support inside clen 2. ml microcentrifuge tue (Suppl. Fig. 2). Flies were trnsferred y spirtion into the tue nd were held in there for either 6 hr or 24 hr, t which point they were trnsferred to pre-weighed clen 1. ml microcentrifuge tues. After which the polypropylene support ws removed nd the inside of the tue ws rinsed with 2 µl of distilled wter. For the 24 hr tretment, the rinste ws diluted n dditionl 4-fold for nlysis. The lue dye present in the ccumulted excret on the sides of the tues ws quntified to estimte reltive gut throughput/ feeding rtes of the tretments. The dye ws quntified s descried in the previous prgrph. Metolomic nlysis Between 9 nd 11 flies were plced in vil contining Formul 24-4 medi supplemented with 3% nturl undnce Glc. After 24 hr, the experimentl flies were trnsferred to new vil of medi contining.6% MA nd 3% [1,2-13 C 2 ]Glc or contining just 3% [1,2-13 C 2 ]Glc for the control flies. After 24 hr on the medi supplemented with 13 C-leled Glc (+/-.6% MA), the flies were nesthetized with CO 2, consolidted into 1. ml microcentrifuge tue, weighed to the nerest tenth of milligrm, nd flsh frozen with liquid nitrogen. Metolites were extrcted immeditely fter flsh freezing. For the metolite extrction, µl of ice-cold 1% ethnol were dded to the flies nd then homogenized for min using sterile lue pestle driven y ttery-powered pestle motor. After homogeniztion, n dditionl µl of ethnol were dded nd mixed thoroughly. From the smple, µl were trnsferred to new 1. ml tue, nd oth tues were plced in heting lock t 8ºC for 1 min, fter which 1 ml of % (v/v) methnol ws dded to ech. The smples were centrifuged t 13, x g for 1 min to sediment insolule deris. The superntnt from ech ws trnsferred to new tue. Smples were stored t -8ºC until they could e further processed. Metolite profiling of the extrcts ws conducted using GC-MS (Gs Chromtogrphy-Mss Spectroscopy) s descried previously y Rupssr (28) with the following modifictions. Smples of 2-ml extrcts, 1 reps ech for the tretment nd control, were vcuum-dried (Speed- Vc, ThermoFinnign, Wlthm, MA, USA). Ten microliters of internl stndrd (1 mg Melissic cid in 1 ml pyridine) were dded to ech of the dried extrcts nd vcuum-dried gin. The dried smples were derivtized with µl of methoxmine hydrochloride (2 mg/ml in pyridine) for 1 hr t C, followed y the ddition of 1 µl N-methyl-N-(trimethylsilyl)trifluorocetmide (MST- FA+1% TMCS: trimethylchlorosilne) nd further derivtiztion t C for 3 min. GC-MS nlysis of 2 µl of ech derivtized smple ws performed using 689N GC nd 973N mss detector (Agilent Technologies, Snt Clr, CA, USA). Smples were nlyzed under electron impct ioniztion. The GC-MS interfce temperture ws 31 C, nd the ion source ws kept t 22 C. The dt system used ws Chem Sttion (Agilent Technologies) with Amdis softwre used for chromtogrphic pek deconvolution nd spectrl extrction. The spectrl identifiction nd nlysis were done s reported y Rupssr (28). Locomotion experiments Flies were individully plced in 6. cm x 3 mm glss tues (length x inner dimeter, trnsferred with CO 2 nesthesi or y spirtor for comprison), contining 24-4 instnt diet +/-.6% MA. To ssess ctivity, the numer of times tht ech fly crossed the infrred em ws recorded t 1 min intervls using Trikinetics (Wlthm, MA, USA) Drosophil ctivity monitoring system (Kume et l., 2). Accumulted ctivity ws logged for 16 individuls per tretment for up to 48 hr. Respirometry experiments Rtes of CO 2 relese y respirtion were ssessed over oth short (min) nd long (hr) timescles. Short-

6 778 K.R. Wlters et l. term respirtion rtes were mesured using closed gs exchnge system. This comprised mchined luminum smple chmer tht hd nickel/teflon coting to prevent unnecessry d/sorption of CO 2 nd wter vpor, nd LI-84 CO 2 /H 2 O Anlyzer (LI-COR, Lincoln, NE, USA) with. l min -1 pump to mix the gs within the system. For twenty four hours prior to the mesurements, fifteen flies were held under humidity controlled conditions in nylon mesh g ( cm x 2. cm x 1 cm, L x W x H) contining 1 mg of 24-4 instnt medi +/-.6% MA. Rtes of CO 2 relese y -6 replicte tches of flies from ech tretment were mesured t 2 C over period of two min. A negtive control ws lso tested demonstrting tht there ws no significnt CO 2 relese from mesh gs contining food tht ws fed upon y flies, ut not flies themselves. Long-term rtes of respirtion were determined y holding ten flies in 2-ml glss vil contining 3 mg of instnt diet +/- the ddition of.6% MA nd mesuring the increse in [CO 2 ] t 21. C over -6 hr. Flies were cclimtized in the glss vils for 18 hr prior to mesurements with the vil opening plugged with cotton. During mesurements the vil ws then seled y gs impermele cp with ruer septum (Ntionl Scientific Compny, Rockwood, TN, USA). Respirtion rtes were clculted y compring the [CO 2 ] in 6 replicte vils from the MA nd control tretments to the [CO 2 ] in negtive control vils (from which the flies were removed immeditely efore seling the vil) t the end of the -6 hr incution period. [CO 2 ] ws mesured y injecting three 1 µl liquots of gs from ech smple vil into the closed gs exchnge system descried ove. After the respirometry experiments, flies were homogenized in 3 µl of mm Tris uffer, ph 7.4, nd solule protein content of ech smple ws nlyzed with Coomssie plus (Brdford) ssy kit (Pierce Biotechnology, Rockford, IL, USA). Sttisticl nlyses Typiclly, dt were nlyzed seprtely y time-point using one-wy ANOVA. Afterwrds, if sttisticl significnce ws oserved, individul mens were compred in pir-wise mnner using Tukey s MCT. In instnces where experiments were performed oth with nd without CO 2 nesthesi, dt were nlyzed using twowy ANOVA, which permitted simultneous testing of effects the trnsfer method nd tretment. In experiments with only two tretments nd one time point, such s the respirometry experiments, the dt were nlyzed y the Student s t-test. Becuse the locomotor ctivity dt were counts, heterogeneous vrince ws ssumed, i.e. we Percent survivorship Fig Time (h) Glucose ffects ility to survive methmphetmine exposure. Ten virgin mle -dy-old Drosophil dults were plced in 2 ml microcentrifuge tues contining either undulterted 4-24 instnt medium (lck dimonds), medium supplemented with.6% methmphetmine (light grey squres), medium supplemented with.6% methmphetmine nd 3% glucose (drk grey tringles) or medium supplemented with 3% glucose (lck circles). The men survivorship (± S.E.M., n = 6) is presented for ech time point. expected lrger numers to hve lrger vrince. A squre root trnsformtion did not fix the prolem, thus we used t-test sed on unequl vrince, nd the Cochrn nd Stterthwite pproximte degrees of freedom with unequl confidence intervls were clculted nd significnce determined. Results Supplementtion with dietry glucose forestlled MA-induced mortlity The LT for MA-exposed flies ws 8. hr (77.4 hr hr, 9% CI) (Fig. 2). Supplementtion of the MAcontining diet with 3% Glc incresed the LT to hr (112. hr hr, 9% CI) (p <.). The LT for the plin diet nd Glc-treted controls ws not determined ecuse survivorship ws greter thn 8% fter one week. Crohydrtes reserves decresed stedily with MA-exposure nd were uffered y ddition dietry glucose Totl Glc showed continul decrese with incresing durtion of MA-exposure (Fig. 3). At 12 hr, MA-tretment reduced totl Glc/mg fresh mss (totl Glc includes free Glc, in ddition to Glc lierted from enzymtic hydrolysis of glycogen nd trehlose) y 27% reltive to the control (p <.1). By 24 hr, MA-tretment decresed totl

7 779 Methmphetmine lters feeding nd locomotor ehvior in Drosophil µg totl glucose/mg fresh mss A Time (h) C c c C24 MA24 MA+Glc24 Glc24 µg totl glucose/mg fresh mss B C12 D c c MA12 MA+Glc12 Glc12 c C48 MA48 MA+Glc48 Glc48 Fig. 3. Totl glucose levels (~totl crohydrtes) s function of tretment durtion. (A) Men totl glucose content ws mesured fter 12 hr, 24 hr nd 48 hr for ten virgin mle -dy-old Drosophil dults tht were plced in 2 ml microcentrifuge tues contining either undulterted 4-24 instnt medium (lck dimonds), medium supplemented with.6% methmphetmine (light grey squres), medium supplemented with.6% methmphetmine nd 3% glucose (drk grey tringles) or medium supplemented with 3% glucose (dt not shown in A) (n = 4, ± S.E.M.). Sttisticl comprisons of men totl Glc levels for 12 hr, 24 hr nd 48 hr time-points re presented in B, C nd D, respectively. Ordinte xes lels in B, C nd D use letters to indicte tretment (e.g., C = plin diet control, M = MA-contining diet, M+G = diet supplemented with MA nd Glc, nd G = Glc supplemented control) nd numers to indicte time. Distinct lowercse letters represent different men vlues (p <., Tukey s MCT). Glc y over 28% (p =.2). After 48 hr, MA-tretment reduced totl Glc levels y 46% (p <.1). The MA+Glc-tretment incresed totl Glc reserves (p =.14) reltive to the MA-treted flies t 48 hr (Fig. 3D), showing tht dietry crohydrtes re le to uffer crohydrte reserves in the presence of MA. While the sme trend in the dt ws oserved t 12 hr nd 24 hr (Figs. 3B,C), tht is to sy MA+Glc-treted flies exhiited higher crohydrte reserves thn those treted with MA lone, the differences etween the MAtretment nd MA+Glc-tretment were not sttisticlly significnt. Chnges in glycogen levels with MA-exposure mirrored those oserved in totl Glc levels. Glycogen levels showed stedy declines over time in the MA-treted flies reltive to the control, decresing y ~22% (p =.324), 31% (p =.39), 48% (p =.2) fter 12 hr, 24 hr, nd 48 hr, respectively (Fig. 4). When Glc ws dded to the MA-tretment, glycogen reserves were incresed y ~44% t 48 hr (p =.49) reltive to MA-tretment lone (Fig. 4D). Trehlose levels decresed, ut stilized with continuing MA-exposure Trehlose levels trended lower fter 12 hr of MA-tretment, ut did not decrese significntly reltive to the control (p =.1427) (Fig. B). Interestingly, the MA+Glctretment incresed the mount of trehlose reltive to flies treted with MA lone (p =.214). However, trehlose levels in the MA+glc tretment were sttisticlly the sme s those in the plin diet nd Glc-treted controls (p =.6889 nd p =.9633, respectively). At 24 hr of tretment, the MA-tretment decresed trehlose y 22% reltive to the control (p =.2)

8 78 K.R. Wlters et l. µg glycogen/mg fresh mss A Time (h) µg glycogen/mg fresh mss B C12 M12 M+G12 G12 µg glycogen/mg fresh mss C C24 M24 M+G24 G24 µg glycogen/mg fresh mss 3 D c C48 M48 M+G48 G48 Fig. 4. Glycogen s function of tretment durtion. (A) Glycogen content ws mesured fter 12 hr, 24 hr nd 48 hr for ten virgin mle -dy-old D. melnogster dults tht were plced in 2 ml microcentrifuge tues contining either undulterted 4-24 instnt medium (lck dimonds), medium supplemented with.6% methmphetmine (light grey squres), medium supplemented with.6% methmphetmine nd 3% glucose (drk grey tringles) or medium supplemented with 3% glucose (dt not shown in A) (n = 4, ± S.E.M.). Sttisticl comprisons of men glycogen levels for 12 hr, 24 hr nd 48 hr timepoints re presented in B, C nd D, respectively. Ordinte xes lels in B, C nd D use letters to indicte tretment (e.g., C= plin diet control, M = MA-contining diet, M+G = diet supplemented with MA nd Glc, nd G = Glc supplemented control) nd numers to indicte time. Distinct lowercse letters represent different men vlues (p <., Tukey s MCT). (Fig. C). The MA+Glc-tretment no longer incresed trehlose levels reltive to MA-treted flies (p =.836) s it hd done t 12 hr. In ddition, trehlose levels in oth the MA- nd MA+Glc-tretments were lower thn those of either of the control tretments (p <.44). After 48 hr similr pttern ws oserved. MA-tretment reduced trehlose levels y 2% (p =.6) reltive to the control nd no difference ws oserved etween the MA- nd MA+Glc-tretments (p =.2126) (Fig. D). However, trehlose levels in the MA+glc-tretment trended higher nd were not significntly lower thn the Glctreted control (p =.7). Triglyceride reserves re rpidly depleted with MA-exposure Triglyceride levels decresed drmticlly with MAexposure. At 12 hr of MA-exposure, there ws no significnt difference mong ny of the tretments (p =.296, F-vlue = 1.38, d.f. = 3, one-wy ANOVA), even though triglyceride levels in MA-treted flies trended ~2% lower (Fig. 1A) thn those in the control. However, t 24 hr triglycerides in MA-treted flies decresed y more thn % reltive to the untreted control (p =.33) (Fig. 1B), nd similr decrese ws oserved in MA+Glc-tretment. At 48 hr, we oserved tht triglyceride levels decresed in the MA-treted flies y 87% (p <.1) (Fig. 1C). No significnt difference ws oserved in the triglyceride levels of the MA-treted nd the MA+Glc treted flies; however, triglyceride levels in MA+Glc-treted flies trended higher t ll time-points. Free mino cids trended towrds decresed concentrtions with MA-exposure All of the free mino cids oserved in metolomic nlysis, including glutmine, glycine, lnine, glutmic cid, sprtic cid, nd serine, trended towrd decresed

9 781 Methmphetmine lters feeding nd locomotor ehvior in Drosophil µg trehlose/mg fresh mss A Time (h) µg trehlose/mg fresh mss B C12 M12 M+G12 G12 µg trehlose/mg fresh mss C C24 M24 M+G24 G24 µg trehlose/mg fresh mss D C48 M48 M+G48 G48 Fig.. Trehlose s function of tretment durtion. (A) Trehlose content ws mesured fter 12 hr, 24 hr nd 48 hr for ten virgin mle -dy-old Drosophil dults tht were plced in 2 ml microcentrifuge tues contining either undulterted 4-24 instnt medium (lck dimonds), medium supplemented with.6% methmphetmine (light grey squres), medium supplemented with.6% methmphetmine nd 3% glucose (drk grey tringles) or medium supplemented with 3% glucose (dt not shown in A) (n = 4, ± S.E.M.). Sttisticl comprisons of men trehlose levels for 12 hr, 24 hr nd 48 hr time-points re presented in B, C nd D, respectively. Ordinte xes lels in B, C nd D use letters to indicte tretment (e.g., C= plin diet control, M = MA-contining diet, M+G = diet supplemented with MA nd Glc, nd G = Glc supplemented control) nd numers to indicte time. Distinct lowercse letters represent different men vlues (p <., Tukey s MCT). concentrtion in MA-exposed flies with the exception of serine (Tle 1). However, only two of these, glutmine nd glycine, were found to decrese significntly with MA exposure (Tle 1). Dietry nd endogenous crohydrtes showed opposing trends Three dditionl crohydrtes/ sugr lcohols detected in metolomic nlysis were found to chnge significntly with MA-exposure (Tle 1). Both endogenous compounds, nmely glycerol 3-phosphte nd trehlose, were found to decrese. Glycerol 3-phosphte, precursor for lipid iosynthesis, ws reduced in the MA+Glctreted flies y more thn % reltive to the Glc-treted control (p =.34). Trehlose lso decresed with MAexposure (p =.2), confirming the results of the spectrophotometric ssys (Fig. C). A diet-derived crohydrte, [1,2-13 C 2 ]Glc, incresed in the MA+Glc-treted flies y 6% (p =.2) reltive to the Glc-treted control (Fig. 6). Thus, endogenous crohydrtes decresed with MA-exposure, while those derived from the diet incresed. Locomotor nd feeding ehviors chnge in response to MA Locomotor ctivity incresed with MA-tretment from 6 hr-12 hr (p =.18), 12 hr-24 hr (p =.29) nd 24 hr-48 hr (p =.4) fter the onset of tretment (Fig. 7A). However, locomotor ctivity did not increse during the first 6 hr (p =.2) time in, indicting tht MA dministered in the diet requires severl hours to increse locomotor ctivity. Note tht MA-treted flies mintined the sme circdin rhythm s the controls, ut were generlly more ctive. Fig. 7B shows flies trnsferred into the

10 782 K.R. Wlters et l. Tle 1. The men reltive undnce (ritrry units, n = 1) of selected metolites detected y GC-MS for MA+Glc- nd Glc-treted Drosophil fter 24 hr of tretment MA+Glc tretment Glc control tretment Men SEM Men SEM p-vlue Glycerol 3-phosphte * FDR Glycine * FDR Glutmine * FDR Trehlose * FDR Sucrose * Glucose 6-phosphte NS Alnine NS Glutmic cid NS Succinic cid NS Methionine or IAA NS Lctic cid NS Fructose 6-phosphte NS Asprtic cid NS Riose -phosphte NS Fructose NS Serine NS * Metolites differed significntly etween the two tretments ccording to Student s t-test (p <.). FDR Metolites differed significntly etween the two tretments with flse discovery rte (FDR) of %. NS Metolites did not differ significntly etween tretments (Student s t-test (p >.)). Count Intensity (ritrry units) Fig Unleled (m) m+1 Leled (m+2) Glucose isotopic distriution. Men reltive undnce of glucose isotopomers (± S.E.M.) detected y GC-MS for MA+[1,2-13 C 2 ]Glc- (grey rs) nd [1,2-13 C 2 ]Glctreted (lck rs) Drosophil fter 24 hr of tretment. The first x-xis lel (from left to right) indictes unleled/nturl undnce Glc with mss of m. m+1 indictes glucose tht contins single 13 C nd m+2 indictes the distriution of douly 13 C-leled Glc, which ws lmost exclusively otined from the fly diet. Asterisks indicte tht MA+Glc-treted is different thn control (p =.2, Student s t-test, n = 1). *** locomotor ssy without CO 2 nesthesi ct qulittively the sme s those trnsferred with nesthesi. In seprte locomotor ctivity experiment, the presence of MA in the diet incresed locomotor ctivity regrdless of the presence of Glc. When compred to the MA-tretment, MA+Glc-treted flies were sttisticlly the sme cross ll time ins (p >.294) (Suppl. Fig. 3). Furthermore, oth the MA+Glc- nd Glc-tretments showed significntly more ctivity tht the control flies t from 6 hr-12 hr (p <.414), 12 hr-24 hr (p <.47) nd 24 hr-48 hr (p <.89) fter the onset of tretment. In oth cses, locomotor ctivity in the first time in did not differ from the control (p >.4428). All flies, regrdless of tretment, spent most of their time on the sides of the 2 ml tue nd MA(+Glc)-treted flies were no more likely to spend time on the food surfce thn the control flies (dt not shown), suggesting tht the mount of lue dye in the excret ccumulted on the side of the tue is proportionl to the excretion rte. Dietry tretment (presence or sence of MA) ws the most importnt determinnt of feeding ehvior (p <.1, two-wy ANOVA) (Fig. 8). The mount of excret ccumulted over 24 hr, proxy for feeding rte, significntly decresed (p <.1, Tukey s MCT) in

11 783 Methmphetmine lters feeding nd locomotor ehvior in Drosophil Rolling verge of ctivity counts/1 min in Rolling verge of ctivity counts/1 min in Fig Time (h) (p=.2) (p=.18) (p=.29) (p=.4) A B Time (h) Locomotor ctivity s function of tretment durtion. The locomotor ctivity of five-dy-old virgin mle Drosophil dults ws monitored fter the trnsfer of single fly with CO 2 nesthesi (A) or y spirtor (B) to 3 mm tue. Ech tue contined either undulterted diet (lck trce) or diet supplemented with.6% MA (light grey trce) (n = 16). The rolling verge of five time ins is presented to reduce vriility. For A, the ctivity dt ws divided into four six-hour time ins nd t-test sed on unequl vrince ws used to clculte p-vlues (locted t the top of the pnel). oth MA- nd MA+Glc-treted flies reltive to either control (Fig. 8C). However, the quntity of food contined in the gut did not chnge with tretment (p =.3988, ANOVA) (Fig. 8A). When flies were trnsferred without nesthesi, similr qulittive trends were oserved in ccumulted excret; however, the method of trnsfer (i.e., CO 2 nesthesi vs. spirtion) did significntly ffect feeding ehvior (twowy ANOVA, p =.329). Similr to results ttined with CO 2 nesthesi, ccumulted excret decresed for MAtreted flies when compred to tht of the plin diet controls (p =.2, Tukey s MCT) (Fig. 8D). In ddition, ccumulted excret for MA+Glc-treted flies decresed reltive to the plin diet control (p =.11) nd trended lower reltive Glc-treted control (p =.87). The most striking difference etween the two trnsfer methods ws tht ccumulted excret trended higher y 7% nd 4% for Glc nd plin diet controls trnsferred without nesthesi, respectively, indicting tht CO 2 nesthesi suppressed feeding ehvior. No interction ws oserved etween the method of trnsfer nd the dietry tretment (two-wy ANOVA, p =.2436). When mesured following trnsfer of flies without nesthesi, the quntity of food contined in the gut ws found to increse significntly for flies co-treted with MA nd Glc reltive to those treted with MA lone (p =.366) (Fig. 8B). The sme trend ws oserved following CO 2 nesthesi, ut the difference ws not significnt (Fig. 8A). Drosophil preferred not to et diet contining MA nd SOA. When given the choice etween control diet nd diet supplemented with.6% MA, flies consumed 14-fold more of the former thn the ltter (Student s t-test, p <.1) (Fig. 9A). In contrst, when given the choice etween MA- or SOA-supplemented diet flies consumed similr mounts of ech (Student s t-test, p =.62) (Fig. 9B). In the first 6 hr, version to these compounds led to decresed consumption of food contining MA or SOA when compred to the control (p <.21, Tukey s MCT) (Fig. 9C) with consumption of oth compounds eing the sme (p =.8921, Tukey s MCT). However, y 24 hr consumption of food dulterted with SOA, incresed nd ws no longer different from the control (p =.1367, Tukey s MCT) (Fig. 9D). In contrst, consumption of MA-contining food remined suppressed y more thn 6% reltive to the control (p =.4) nd y more thn % reltive to SOA-contining diet (p =.76). Levels of MA were higher in flies odies when Glc ws dded MA levels, mesured in whole-ody metolite extrcts, were significntly higher for MA+Glc-treted flies reltive those mesured on MA-treted flies (Suppl. Fig. 4) (two-wy ANOVA, p =.6). However, MA concentrtions in the flies odies did not chnge with the durtion of exposure (two-wy ANOVA, p =.166), nor ws ny time y tretment interction oserved (two-wy ANOVA, p =.136). MA-exposure decresed rtes of respirtion Respirtion rtes per unit fresh weight trended lower with MA exposure when ssessed in short-term incutions (-13%, p =.66, Student s t-test, control n = 6, MA tretment n = ). Since we expected MA-tretment to increse respirtion due to incresed locomotor ctivity, we decided to pursue long-term incution to void ny

12 784 K.R. Wlters et l. µg diet/mg fresh mss µg egested diet /cm²/mg fresh mss A C C24 M24 M+G24 G24 c C24 M24 M+G24 G24 c µg diet/mg fresh mss µg egested diet /cm²/mg fresh mss B D C24 M24 M+G24 G24 C24 M24 M+G24 G24 Fig. 8. MA-tretment nd trnsfer method ffect feeding ehvior. The men gut contents of ten virgin mle -dy-old Drosophil dults ws mesured 24 hr fter plcement in 2 ml microcentrifuge tues contining medi supplemented with 2.% lue dye. There were four tretments: plin medium (C24), medium supplemented with.6% methmphetmine (M24), medium supplemented with.6% methmphetmine nd 3% glucose (M+G24) or medium supplemented with 3% glucose (G24) (n =, ± S.E.M.). Flies were trnsferred to the 2 ml tues oth with (A) nd without (B) the use of CO 2 nesthesi. The reltive mount of egested dye ws lso mesured for ech of the four tretments with (C) nd without (D) the use of CO 2 nesthesi. Distinct lowercse letters represent different men vlues (p <., Tukey s MCT). hndling-induced perturtions of ehvior tht could led to spurious results. In the long-term incutions, respirtion rtes decresed from 3.7 ±.2 ml CO 2 g fresh mss -1 h -1 (men ± S.E.M.) for the control flies to 2.9 ±.1 ml CO 2 g fresh mss -1 h -1 (-2%, p =.47, Student s t-test, n = 6) with MA-tretment. Furthermore, when CO 2 production ws normlized to totl solule protein, MAtretment similrly decresed rtes of whole-ody respirtion from the control level of 178 ± 11 ml CO 2 g -1 h -1 to 14 ± 6 ml CO 2 g -1 h -1 (-18%, p =.29, Student s t-test, n = 6). Similr results in oth the short-term nd longterm incutions gve us confidence tht the results were not n rtifct of ltered ehvior during the mesurement process. Discussion Our study clerly demonstrtes tht MA hs profound effects on energy metolism in Drosophil. Triglycerides nd glycogen re the two predominnt energy storge molecules in nimls nd oth decresed stedily with continued MA-exposure over 48 hr period (Figs. 1 nd 4), suggesting tht MA dministered in the diet resulted in negtive cloric lnce. The lrge decrese in oserved levels of glycerol 3-phosphte (Tle 1), n essentil precursor in triglyceride iosynthesis, in conjunction with decresed levels of triglycerides suggests tht lipid iosynthesis ws drmticlly reduced, supporting the cse for cloric deficit. Moreover, the oservtion tht trehlose, the mjor lood sugr in insects (Bedford, 1977; Wytt, 1961), decresed with MA-exposure (Fig., Tle 1) is consistent with the response to strvtion in other insects (Wytt, 1961; Jones et l., 1981; Duchteu nd Florkin, 199). The stiliztion of trehlose levels fter 12 hr of MA exposure (Fig. ), when compred to the continul decrese of glycogen levels (Fig. 4), suggests tht trehlose levels in the hemolymph were ctively regulted t the expense of the mjor storge crohydrte, which is consistent with the physiologiclly importnt role of tre-

13 78 Methmphetmine lters feeding nd locomotor ehvior in Drosophil µg ingested food/ mg fresh mss µg egested food/ mg fresh mss C A µg ingested food/ mg fresh mss C M SOA M 3 D p<.1 µg egested food/ mg fresh mss C6 M6 SOA6 C24 M24 SOA24 B p=.62 Fig. 9. Aversive compounds nd food consumption. In two-choice experiments, flies were given the choice etween red-dye leled control diet nd lue dye-leled diet contining.6% MA (C) or red dye-leled diet contining 1.2% SOA nd lue dye-leled diet contining.6% MA (D). Flies were llowed to feed for 9 min, fter which they were homogenized nd dye ws quntified. (n =, ± S.E.M.). In ddition, the mount of lue dye egested y virgin mle -dy-old Drosophil dults mesured 6 hr nd 24 hr (A nd B, respectively, n =, ± S.E.M.) fter plcement in 2 ml microcentrifuge tues contining one of the following: plin medium + 2.% lue dye (C), medium supplemented with.6% methmphetmine nd 2.% lue dye (M) or medium supplemented with 1.2% sucrose octcette nd 2.% lue dye (SOA). Distinct lowercse letters represent different men vlues (p <., Tukey s MCT).The numer component of ech column lel indictes the tretment time (e.g., M6 indictes 6 hr of exposure to MA-contining diet) (n =, ± S.E.M.). hlose s lood sugr (Lee nd Prk, 24). Decresed lood sugr hs lso een ssocited with MA-exposure in mmmlin models for the study of MA (Mcmhon et l., 1971). Finlly, Sun et l. (211) previously reported tht MA-exposure decresed trehlose levels in Drosophil. Decresed concentrtions of glutmine nd glycine (Tle 1) my lso reflect negtive cloric stte. Tissuespecific decreses in free mino cids hve een shown to occur during short-term strvtion in thorcic muscles of the tsetse fly (Glossin swynnertoni) (Bursell, 196), nd in humn muscles (Hmmrqvist et l., 2). In fct, the mino cid proline cn e utilized s n energy source to power insect flight muscle [37, 38] (Brosemer nd Veerhdrpp, 196; Scktor nd Wormser-Shvit, 1966) nd glutmine cn e directly oxidized y cells from the fll rmyworm (Spodopter frugiperd) in culture (Neermnn nd Wgner, 1996). However, studies tht monitored free mino cids in the hemolymph of insects hve shown tht concentrtions remin constnt or increse with strvtion (Wytt, 1961; Po-Chedley, 198), presumly to uffer energy metolism. In this study, since we evluted whole ody extrcts, we oserved the net chnge for mino cids contined in oth the intrcellulr nd extrcellulr comprtments. Our oservtion tht free mino cid concentrtions decresed (Tle 1) is consistent with others oservtions tht most free mino cids re intrcellulr (Hnzl et l., 1992; Bjerke nd Zchrissen, 1997) nd this pool of mino cids typiclly decreses with strvtion (Bursell, 196; Hmmrqvist et l., 2). However, since glutmine nd glycine hve importnt roles outside of energy metolism, including protein iosynthesis, osmotic regultion (Woodring nd Blkeney, 198), nd mintining reduced glutthione pools (glutmine specific) (Roth et l., 22), the oserved chnges my reflect

14 786 K.R. Wlters et l. chnges other thn energy sttus. The negtive cloric lnce in MA-exposed flies ppers to e driven predominntly y norexi, supporting the ehvior hypothesis. We oserved tht mount of excret ccumulted in 24 hr, proxy for the mount of food consumed, decresed y ~6-8% when MA ws dded to the diet (Figs. 8, 9D), indicting 6-8% reduction in cloric intke. We cn reject the metolic hypothesis ecuse if metolic chnges were cusing the oserved depletion of glycogen nd triglycerides, then we would expect to oserve n increse, not decrese, in metolic rte, ssuming tht metolism t the orgnisml level remins eroic. Perhps more prsimonious explntion for the over-expression of glycolytic proteins oserved y Sun et l. (211) is tht glycolytic proteins, re highly susceptile to cronyltion rising from drug-induced oxidtive stress (Englnd et l., 24). Thus, these proteins re up-regulted to compenste for decresed enzymtic ctivity resulting from cronyltion. Additionlly, the oserved over-expression of crohydrte trnsporters oserved y Sun et l. (211) proly rises from norexi-induced strvtion, since reduced levels of crohydrtes led to the up-regultion of crohydrte trnsporters (Ferrris nd Dimond, 1992; Ngmtsu et l., 1994). Our dt indicte tht itter tste of MA, per se, did not cuse prolonged norexi, since flies hituted to itterness. Nïve flies strongly preferred to et undulterted diet over tht contining MA (Fig. 9A), which is reported to hve itter tste (Scielli et l., 211), reveling tht flies cn detect MA nd find it versive. When given the choice etween SOA, itter non-toxic tstnt, nd MA, flies showed no preference for either compound nd generlly te much less thn when undulterted diet ws ville (Figs. 9A, B). In the short term, the presence of either MA or SOA in the diet led to decresed food consumption (Fig. 9C). However, flies redily hituted to itterness, leding to incresed consumption of SOA-contining food over the first 24 hr (Fig. 9D). In contrst, flies did not hitute to MA nd continued to exhiit pronounced norexi. MA-induced norexi could e ttriuted to conditioned version to MA resulting from post-ingestive toxic effects of the drug nd/or my rise from the ility of MA to suppress ppetite. The method of drug dministrtion (dietry) does not llow us to independently evlute these two possiilities nd oth re supported y the literture. For instnce, Arvich nd Sclfni (198) concluded tht rts lost weight when itter quinine ws incorported in their diet, not ecuse of the itterness of the compound, ut rther ecuse of its toxicity. In fct, rts lerned to prefer SOA over itter quinine even though nïve rts preferred the ltter. In support of the second possiility, ppetite suppression is commonly oserved side effect of MA in humns nd in mmmls regrdless of the method of dministrtion (Cole, 1967; Evns, 1971; Mdden et l., 2). We hypothesized tht MA-induced increses in locomotor ctivity tht we oserved in Drosophil (Fig. 7) would worsen the cloric deficit y incresing cloric expenditure. Incresed locomotor ctivity is lso known side effect of MA consumption in mmmls (Wllce et l., 1999) nd in humns where MA is tken, in prt, for its stimulnt effects (Ntionl Institutes on Drug Ause, 21). However, the metolic contriution of incresed locomotion in Drosophil ws proly smll since flies were lrgely restricted to wlking in the iossys, nd the contriution of wlking to the overll metolic rte is modest. In fct, wlking t n verge speed is predicted to increse the metolic rte in Drosophil y only -1% over routine metolism (Berrign nd Prtridge, 1997). Therefore, the incresed locomotor ctivity oserved in the MA-treted flies might e expected to increse the metolic rte y roughly -1% over tht of the control flies. In contrst to our hypothesis, we oserved tht the overll metolic rte for MA-treted flies tended to decrese, indicting tht MA decresed the resting metolic rte. The decresed metolic rte my e relted to MA-induced strvtion, since strvtion is known to decrese the reltive metolic rte in insects y 1-9% when compred to non-strved individuls (Scrier nd Slnsky, 1981). Importntly, our dt re consistent with the overll the cloric lnce eing strongly negtive, since cloric intke decresed y 6-8%, ut the compenstory decrese in resting metolism ws only 1-2%. With the discovery tht MA-induced mortlity ppered to e medited y ehviorl chnges, we decided to perform key ehviorl ssys with nd without CO 2 nesthesi (flies were routinely nesthetized with CO 2 to fcilitte hndling during experimentl setup) ecuse cute CO 2 exposure hs een shown to produce ehviorl effects lsting t lest 24 hr (Brron, 2). We wnted to ensure tht tretment (MA exposure), not trnsfer method (CO 2 nesthesi vs. spirtion), ws the primry driver of the oserved ehviorl chnges. A two-wy ANOVA reveled oth trnsfer method nd tretment ffect feeding ehvior, with tretment eing the primry driver of differences in food consumption (p <.1). The primry effect of the trnsfer method ws tht CO 2 nesthesi reduced the feeding rte nd/or delyed onset of feeding (Figs. 8C, D). However, in ll the feeding nd