fly stocks were raised on standard cornmeal / agar food at 25c and 60% relative humidity .
the tdc2-gal4 , np7088 , uas - shi ( carrying insertions on the first and third chromosome ) and uas - dtrpa1 flies are described .
the tdc2-lexa : vp16 transgenic line was generated by cloning the same regulatory region as described previously into the pbs_lexa::vp16_sv40 vector .
transgenic flies were raised by standard procedures and lines were screened for those with appropriate expression .
the lexaop - dtrpa1 was constructed by subcloning a noti and xhoi restriction site- flanked dtrpa1 cdna from pox - dtrpa1 ( paul garrity , brandeis university ) into the plot transformation vector .
the uas - oamb[2861gd ] and uas - oct2r[104524kk ] were obtained from the vdrc .
0104-gal4 , 0665-gal4 , 0891-gal4 and 0273-gal4 flies refer to pbac{it.gal4}0104 , pbac{it.gal4}0665 , pbac{it.gal4}0891 and pbac{it.gal4}0273 and were generated within the framework of the insite project .
the lexaop - rcd2::mrfp , uas - brp::gfp , and uas - denmark flies are described .
the uas - gcamp3.0 flies are described . to generate flies to block or stimulate oa neurons we crossed uas - shibire or uas - dtrpa1 female flies to tdc2-gal4 males .
6 - 8 day old flies were tested . to block refined subsets of oa neurons we crossed uas - shibire female flies to male np7088/cyo , 0665-gal4 or 0891-gal4/tm3 males , and only flies negative for cyo or tm3 were assayed . to stimulate tdc2 neurons in the th mutant background , th /fm7i - gfp;uas - dtrpa1 females
were crossed with th ; tdc2-gal4 males and only progeny negative for fm7i - gfp were assayed . to stimulate tdc2 neurons in the dumb mutant background , uas - dtrpa1 ; dumb / tm3 females
were crossed with tdc2- gal4;dumb males and only dumb homozygous flies were assayed . to stimulate 0273-gal4 or 0104-gal4 pam - da neurons ,
uas - dtrpa1 female flies were crossed to 0273-gal4 or 0104-gal4/tm6b male flies . to block 0104-gal4 da
neurons uas - shibire females were crossed to 0104-gal4/ tm6b males . to stimulate 0273-gal4 or 0104-gal4 neurons in the th mutant background , th / fm7i;uas - dtrpa1 females were crossed with th;0273-gal4/tm6b or th;0104-gal4/tm6b males respectively and progeny negative for fm7i and tm6b were assayed .
to stimulate tdc2 neurons in the oa2 , oamb and oct2r mutant backgrounds uas - dtrpa1 ; oa2/tm3 or uas - dtrpa1;oct2r / tm6 or uas - dtrpa1;oamb / tm6 females were crossed to tdc2- gal4;oa2/tm3 or tdc2-gal4;oct2r / tm3 or
the oct2r insertion is homozygous lethal so only heterozygous oct2r/+ flies were assayed . to express oamb in 0104-gal4 neurons , we crossed uas - oamb[2861gd ] females to 0104-gal4/ tm6b males and only flies lacking tm6b were assayed . to express oct2r in mb - mp1 neurons while stimulatingtdc2 neurons , we crossed c061-gal4;mbgal80;tdc2-lexa
/ tm3 females to uas - oct2r[104524kk];lexaop - dtrpa1/tm3 males . to express shibire in mb - mp1 neurons while stimulating tdc2 neurons , we crossed
c061-gal4;mbgal80;tdc2-lexa / tm3 females to uas - shi;lexaop - dtrpa1/tm3 males . to stimulate tdc2 neurons in the npfr1 mutant background female uas - dtrpa1;npfr1 flies
heterozygous control flies were generated by crossing the respective uas - transgene flies with wild - type flies .
for the oa2 , oct2r , oamb , dumb and npfr1 experiments , tdc2-gal4;[mutant ] or uas - dtrpa1;[mutant ] flies were flies mutant at the same locus to generate heterozygous transgene controls within the relevant homozygous mutant background .
controls for mb - mp1 neuron manipulation were generated by crossing c061-gal4;mbgal80;tdc2-gal4/tm3 or uas - shi;lexaop - dtrpa1/tm3 or uas - oct2r[104524kk];lexaop - dtrpa1/ tm3 females to wild - type flies .
mixed sex populations were tested together in all behavior experiments unless genotype required sorting single sexes .
hungry state experiments involved food depriving flies for 1820 h before training in milk bottles containing a damp filter paper . to test flies in the satiated state , flies were food - deprived 14 - 16 h , then transferred into fresh bottles containing food to satiate 4 h before training . the olfactory appetitive paradigm was performed as described with the following modifications : for neural blockade experiments using uas - shi , flies were incubated at 31c for 30 min prior to and during training and testing for 3 min memory .
for permissive temperature experiments flies were kept at 23c at all times . for memory implantation experiments using uas - dtrpa1 ,
flies were presented with one odour at the permissive temperature 23c for 2 min in filter paper - lined tubes .
they were then transferred into a new prewarmed filter paper - lined tube and immediately presented with a second odour at the activating 31c for 2 min .
flies were then returned to 23c and tested for immediate memory . to test 3 h memory flies
were trained as above and stored in plastic vials containing dampened filter paper until testing . for 24 h memory experiments ,
flies were trained as above and stored in food vials for 3 h followed by 21 h of food - deprivation before testing .
odours were 3-octanol ( 9.2 l in 8 ml mineral oil ) or 4-methylcyclohexanol ( 18 l in 8 ml mineral oil ) .
the performance index ( pi ) was calculated as the number of flies running toward the conditioned odour minus the number of flies running toward the unconditioned odour divided by the total number of flies in the experiment .
a single pi value is the average score from flies of the identical genotype tested with each odour .
overall analyses of variance ( anova ) were followed by planned pairwise comparisons between the relevant groups with a tukey hsd post - hoc test .
to visualize native gfp , mrfp or mcherry adult female flies were collected 2 - 10 days after eclosion ( 1 day for grasp flies ) and brains were dissected in ice - cold 4% paraformaldehyde solution in pbs [ 1.86 mm nah2po4 , 8.41 mm na2hpo4 , 175 mm nacl ] and fixed for an additional 60 - 120 min at room temperature under vacuum .
samples were washed 310 min with pbs containing 0.1% triton - x100 ( pbt ) , and 2x in pbs before mounting in vectashield ( vector labs ) . for immunohistochemistry , brains were fixed and washed as described above , followed by overnight incubation on a shaker in 10% normal goat serum ( ngs ) at 4c . for staining using the th antiserum ,
brains were dissected in ice - cold pbs , then fixed in undiluted bouin s solution for 20 minutes .
samples were washed 310 min with pbs , then 210 min with pbt and incubated in 10% ngs overnight as above . the anti - th antiserum was added to a final dilution of 1:300 , together with anti - nc82 antibody ( mouse igg1 ) and anti - gfp antibody ( mouse igg2a ) to a final dilution of 1:200 each and incubated for 3 days .
after washing with pbt , alexa594-coupled donkey anti - rat , alexa488 goat anti - mouse igg2a , and alexa649 goat anti - mouse igg1 antibodies were added 1:200 for two days , followed by washing and embedding as described above .. an anti - tyrosine hydroxylase ( th ) antibody raised in rabbit ( ab152 , millipore ) was added to a final dilution of 1:200 and kept in same conditions for another three days .
after washing with pbt , alexa594-coupled goat anti - rabbit antibody ( a-11037 , invitrogen ) was added 1:200 for one more night , followed by washing and embedding as described before .
imaging was performed on a zeiss lsm 5 pascal confocal microscope and a leica tcs sp5 x. images were processed in amira 5.2 ( mercury systems ) . in some cases ,
debris on the brain surface and/or antennal and gustatory nerves were manually deleted from the relevant confocal sections to permit construction of a clear projection view of the z - stack .
up to 7 day old uas - gcamp3.0;0104-gal4 flies were anaesthetised on ice and waxed to a custom imaging chamber .
the head capsule was opened under 800 l of sugar - free hl3-like saline , and the whole preparation transferred under a slicescope microscope ( scientifica ) .
epifluorescence images were acquired using a pike ccd camera ( allied ) at a rate of 3 images / s at one set gain .
the spontaneous baseline gcamp3.0 response was imaged for 30 s , then either 100 l saline or 100 l mianserin ( 12.5 mm , sigma - aldrich , filtered ) were added to the bath .
after another 30 s , 100 l octopamine ( 50 mm , sigma - aldrich ) or 100 l octopamine ( 50 mm ) + mianserin ( 12 mm , filtered ) were added as before , to reach a final bath concentration of 5 mm octopamine and 2.45 mm mianserin , respectively .
after registration of images ( stackreg plugin ) a standardized region of interest ( roi ) was centered within the area of the mb  lobe tip ( fig .
intensity tables were exported to excel and the f / f was calculated , with an f consisting of the averaged first 24 images .
respective peak intensities within 5 s after saline / octopamine / mianserin application were selected and compared to other groups for significant differences .
total rna from adult fly heads was isolated with trizol ( invitrogen ) and cleaned with rneasy micro kit ( qiagen ) with dnase i treatment .
rna ( 200 ng ) was reverse transcribed using the high capacity cdna reverse transcription kit ( applied biosystems ) and random primers . the cdna was used for quantitative real - time pcr with abi prism 7000 sequence detection system ( applied biosystems ) with standard cycling parameters ( 2 min at 50 c , 10 min at 95 c , and 45 alternate cycles of 15 s at 95 c and 60 s at 60 c ) .
the pcr mixture contained taqman gene expression master mix and the appropriate gene expression assay ( applied biosystems ) .
gapdh ( ab : dm01841185_m1 ) was used as endogenous control for normalization ( ct value ) . the decrease in expression ( ct value )
fly stocks were raised on standard cornmeal / agar food at 25c and 60% relative humidity .
the tdc2-gal4 , np7088 , uas - shi ( carrying insertions on the first and third chromosome ) and uas - dtrpa1 flies are described .
the tdc2-lexa : vp16 transgenic line was generated by cloning the same regulatory region as described previously into the pbs_lexa::vp16_sv40 vector .
transgenic flies were raised by standard procedures and lines were screened for those with appropriate expression .
the lexaop - dtrpa1 was constructed by subcloning a noti and xhoi restriction site- flanked dtrpa1 cdna from pox - dtrpa1 ( paul garrity , brandeis university ) into the plot transformation vector .
the uas - oamb[2861gd ] and uas - oct2r[104524kk ] were obtained from the vdrc .
0104-gal4 , 0665-gal4 , 0891-gal4 and 0273-gal4 flies refer to pbac{it.gal4}0104 , pbac{it.gal4}0665 , pbac{it.gal4}0891 and pbac{it.gal4}0273 and were generated within the framework of the insite project .
the lexaop - rcd2::mrfp , uas - brp::gfp , and uas - denmark flies are described .
to generate flies to block or stimulate oa neurons we crossed uas - shibire or uas - dtrpa1 female flies to tdc2-gal4 males .
6 - 8 day old flies were tested . to block refined subsets of oa neurons we crossed uas - shibire female flies to male np7088/cyo , 0665-gal4 or 0891-gal4/tm3 males , and only flies negative for cyo or tm3 were assayed . to stimulate tdc2 neurons in the th mutant background , th /fm7i - gfp;uas - dtrpa1 females
were crossed with th ; tdc2-gal4 males and only progeny negative for fm7i - gfp were assayed . to stimulate tdc2 neurons in the dumb mutant background , uas - dtrpa1 ;
dumb / tm3 females were crossed with tdc2- gal4;dumb males and only dumb homozygous flies were assayed . to stimulate 0273-gal4 or 0104-gal4 pam - da neurons ,
uas - dtrpa1 female flies were crossed to 0273-gal4 or 0104-gal4/tm6b male flies . to block 0104-gal4 da
neurons uas - shibire females were crossed to 0104-gal4/ tm6b males . to stimulate 0273-gal4 or 0104-gal4 neurons in the th mutant background ,
th / fm7i;uas - dtrpa1 females were crossed with th;0273-gal4/tm6b or th;0104-gal4/tm6b males respectively and progeny negative for fm7i and tm6b were assayed . to stimulate tdc2 neurons in the oa2 , oamb and oct2r mutant backgrounds uas - dtrpa1 ; oa2/tm3 or uas - dtrpa1;oct2r
/ tm6 or uas - dtrpa1;oamb / tm6 females were crossed to tdc2- gal4;oa2/tm3 or tdc2-gal4;oct2r / tm3 or tdc2-gal4;oamb
/ tm3 males , respectively . only flies homozygous for oa2 and oamb flies were assayed .
the oct2r insertion is homozygous lethal so only heterozygous oct2r/+ flies were assayed . to express oamb in 0104-gal4 neurons , we crossed uas - oamb[2861gd ] females to 0104-gal4/ tm6b males and only flies lacking tm6b were assayed . to express oct2r in mb - mp1 neurons while stimulatingtdc2 neurons , we crossed c061-gal4;mbgal80;tdc2-lexa
/ tm3 females to uas - oct2r[104524kk];lexaop - dtrpa1/tm3 males . to express shibire in mb - mp1 neurons while stimulating tdc2 neurons , we crossed
c061-gal4;mbgal80;tdc2-lexa / tm3 females to uas - shi;lexaop - dtrpa1/tm3 males . to stimulate tdc2 neurons in the npfr1 mutant background female uas - dtrpa1;npfr1 flies
heterozygous control flies were generated by crossing the respective uas - transgene flies with wild - type flies .
for the oa2 , oct2r , oamb , dumb and npfr1 experiments , tdc2-gal4;[mutant ] or uas - dtrpa1;[mutant ] flies were flies mutant at the same locus to generate heterozygous transgene controls within the relevant homozygous mutant background .
controls for mb - mp1 neuron manipulation were generated by crossing c061-gal4;mbgal80;tdc2-gal4/tm3 or uas - shi;lexaop - dtrpa1/tm3 or uas - oct2r[104524kk];lexaop - dtrpa1/ tm3 females to wild - type flies .
mixed sex populations were tested together in all behavior experiments unless genotype required sorting single sexes .
hungry state experiments involved food depriving flies for 1820 h before training in milk bottles containing a damp filter paper .
to test flies in the satiated state , flies were food - deprived 14 - 16 h , then transferred into fresh bottles containing food to satiate 4 h before training .
the olfactory appetitive paradigm was performed as described with the following modifications : for neural blockade experiments using uas - shi , flies were incubated at 31c for 30 min prior to and during training and testing for 3 min memory .
for permissive temperature experiments flies were kept at 23c at all times . for memory implantation experiments using uas - dtrpa1 ,
flies were presented with one odour at the permissive temperature 23c for 2 min in filter paper - lined tubes .
they were then transferred into a new prewarmed filter paper - lined tube and immediately presented with a second odour at the activating 31c for 2 min .
flies were then returned to 23c and tested for immediate memory . to test 3 h memory flies
were trained as above and stored in plastic vials containing dampened filter paper until testing . for 24 h memory experiments ,
flies were trained as above and stored in food vials for 3 h followed by 21 h of food - deprivation before testing .
odours were 3-octanol ( 9.2 l in 8 ml mineral oil ) or 4-methylcyclohexanol ( 18 l in 8 ml mineral oil ) .
the performance index ( pi ) was calculated as the number of flies running toward the conditioned odour minus the number of flies running toward the unconditioned odour divided by the total number of flies in the experiment .
a single pi value is the average score from flies of the identical genotype tested with each odour .
overall analyses of variance ( anova ) were followed by planned pairwise comparisons between the relevant groups with a tukey hsd post - hoc test .
to visualize native gfp , mrfp or mcherry adult female flies were collected 2 - 10 days after eclosion ( 1 day for grasp flies ) and brains were dissected in ice - cold 4% paraformaldehyde solution in pbs [ 1.86 mm nah2po4 , 8.41 mm na2hpo4 , 175 mm nacl ] and fixed for an additional 60 - 120 min at room temperature under vacuum .
samples were washed 310 min with pbs containing 0.1% triton - x100 ( pbt ) , and 2x in pbs before mounting in vectashield ( vector labs ) . for immunohistochemistry ,
brains were fixed and washed as described above , followed by overnight incubation on a shaker in 10% normal goat serum ( ngs ) at 4c . for staining using the th antiserum ,
brains were dissected in ice - cold pbs , then fixed in undiluted bouin s solution for 20 minutes .
samples were washed 310 min with pbs , then 210 min with pbt and incubated in 10% ngs overnight as above . the anti - th antiserum was added to a final dilution of 1:300 , together with anti - nc82 antibody ( mouse igg1 ) and anti - gfp antibody ( mouse igg2a ) to a final dilution of 1:200 each and incubated for 3 days . after washing with pbt , alexa594-coupled donkey anti - rat , alexa488 goat anti - mouse igg2a , and alexa649 goat anti - mouse igg1 antibodies were added 1:200 for two days , followed by washing and embedding as described above .. an anti - tyrosine hydroxylase ( th ) antibody raised in rabbit ( ab152 , millipore ) was added to a final dilution of 1:200 and kept in same conditions for another three days . after washing with pbt ,
alexa594-coupled goat anti - rabbit antibody ( a-11037 , invitrogen ) was added 1:200 for one more night , followed by washing and embedding as described before .
imaging was performed on a zeiss lsm 5 pascal confocal microscope and a leica tcs sp5 x. images were processed in amira 5.2 ( mercury systems ) . in some cases ,
debris on the brain surface and/or antennal and gustatory nerves were manually deleted from the relevant confocal sections to permit construction of a clear projection view of the z - stack .
up to 7 day old uas - gcamp3.0;0104-gal4 flies were anaesthetised on ice and waxed to a custom imaging chamber .
the head capsule was opened under 800 l of sugar - free hl3-like saline , and the whole preparation transferred under a slicescope microscope ( scientifica ) .
epifluorescence images were acquired using a pike ccd camera ( allied ) at a rate of 3 images / s at one set gain .
the spontaneous baseline gcamp3.0 response was imaged for 30 s , then either 100 l saline or 100 l mianserin ( 12.5 mm , sigma - aldrich , filtered ) were added to the bath .
after another 30 s , 100 l octopamine ( 50 mm , sigma - aldrich ) or 100 l octopamine ( 50 mm ) + mianserin ( 12 mm , filtered ) were added as before , to reach a final bath concentration of 5 mm octopamine and 2.45 mm mianserin , respectively .
after registration of images ( stackreg plugin ) a standardized region of interest ( roi ) was centered within the area of the mb  lobe tip ( fig .
intensity tables were exported to excel and the f / f was calculated , with an f consisting of the averaged first 24 images .
respective peak intensities within 5 s after saline / octopamine / mianserin application were selected and compared to other groups for significant differences .
total rna from adult fly heads was isolated with trizol ( invitrogen ) and cleaned with rneasy micro kit ( qiagen ) with dnase i treatment .
rna ( 200 ng ) was reverse transcribed using the high capacity cdna reverse transcription kit ( applied biosystems ) and random primers . the cdna was used for quantitative real - time pcr with abi prism 7000 sequence detection system ( applied biosystems ) with standard cycling parameters ( 2 min at 50 c , 10 min at 95 c , and 45 alternate cycles of 15 s at 95 c and 60 s at 60 c ) .
the pcr mixture contained taqman gene expression master mix and the appropriate gene expression assay ( applied biosystems ) .
gapdh ( ab : dm01841185_m1 ) was used as endogenous control for normalization ( ct value ) . the decrease in expression ( ct value )
 dopamine ( da ) is synonymous with reward and motivation in mammals1,2 . 
 however , only recently has dopamine been linked to motivated behavior and rewarding reinforcement in fruit flies3,4 . 
 instead octopamine ( oa ) has historically been considered the signal for reward in insects5 - 7 . here 
 we show using temporal control of neural function in drosophila that only short - term appetitive memory is reinforced by oa . 
 moreover , oa - dependent memory formation requires signaling through da neurons . 
 part of the oa signal requires the -adrenergic like oamb receptor in an identified subset of mushroom body ( mb)-targeted da neurons . 
 oa triggers an increase in intracellular calcium in these da neurons and their direct activation can substitute for sugar to form appetitive memory , even in flies lacking oa . 
 analysis of the -adrenergic like oct2r receptor reveals that oa - dependent reinforcement also requires an interaction with da neurons that control appetitive motivation . 
 these data suggest that sweet taste engages a distributed oa signal that reinforces memory through discrete subsets of mb - targeted da neurons . 
 in addition , they reconcile prior findings with oa and da and suggest that reinforcement systems in flies are more similar to mammals than previously envisaged . 
Methods
Flies
Behavioural analysis
Imaging
None
Real-Time PCR
Supplementary Material