Drosophila Strains We used the following Drosophila strains from the Bloomington Stock Center: elav-Gal4 (stock no. 458), drl-Gal4 (stock no.4669), and UAS-GFP (stock no. 1522). The other Gal4 lines were generously provided by Douglas Armstrong (C547-Gal4, C747-Gal4), Krystyna Keleman (Orb2Δ80), Gerry Rubin (R19H07Gal4), and Troy Zars (MB247). The UAS-dTrpA1 line and MB247-dsRed lines were generously provided by Paul Garrity and Andre Fiala. The following transgenic lines were generated by us: UAS-OrbAEGFP, UAS-Orb2AF5YEGFP, UAS-Δ88Orb2AEGFP, and UAS-Orb2BEGFP. Various genetic combinations were made by standard genetic crosses.

Recombinant Protein Drosophila Orb2A, Orb2B, and Orb2F5Y proteins were expressed in E. coli BL21 cells as C-terminal 6×histidine-tagged proteins and purified using Ni-NTA agarose (QIAGEN) according to the manufacturer's protocol under denaturing conditions and purified recombinant proteins were kept in 6 M GnHCl. For the in vitro assembly ∼400 picomoles of the proteins were diluted or dialysed against a buffer composed of 1 M Urea, 100 mM KCl, 10 mM Na-HEPES (pH 7.6), 1 mM DTT, 0.1 mM CaCl 2 , 1 mM MgCl 2 , and 5% glycerol at 4°C. Dialysis was performed in Slide-A-Lyzer mini Dialysis unit of 7000 MWCO (Pierce).

Antibodies All anti-Orb2 antibodies were raised against recombinant 6×histidine-tagged full-length Orb2A protein. The antibodies 272, 273, and 793 were raised in rabbits (Covance), and antibody 2233 was raised in guinea pigs (Pocono). All antibodies were affinity purified against recombinant Orb2A protein. The antibody 402 was raised in rabbit against a 15 amino acid peptide KHSPSGGASGGGDAS specific to the Orb2B isoform (Sigma). For western analysis, the antibodies were used in following dilutions: Ab793 in 1:500 dilution, Ab273 in 1:1000 dilution, Ab2233 in 1:2000 dilution, and Ab402 in 1:2000 dilution. The rabbit anti-EGFP antibody was used in 1:5000 dilutions. The GFP (ab290), GM130 (ab30637), and grp75 (ab82591) antibodies were obtained from Abcam. All secondary antibodies (Cell Signaling) were used in 1:10,000 dilutions. The following antibodies were obtained from the hybridoma bank (Iowa) and used in the indicated dilution in the western blot anti-CSP (1:20,000), anti-Syntaxin (1:5000), anti-Synapsin1 (1:5000), anti-Dlg (1:5000). The HP1 and DMyc antibodies were kindly provided by the labs of Ali Shilatifard and Julia Zeitlinger in the Stowers institute.

Western Blot and Immunoprecipitation For all biochemical analyses, freshly prepared head extracts were used unless mentioned otherwise. We have found that thawing of head lysates frozen even at −80°C results in loss of Orb2 immunoreactivity. For western blot analysis, fly heads were homogenized (2–4 μl of buffer/head) in a PBS buffer containing 150 mM NaCl, 3 mM MgCl 2 , 0.1 mM CaCl 2 , 5% glycerol, 1% Triton X-100, 1% NP40, and EDTA-free complete protease inhibitors (Roche). Where applicable the extracts were also treated with 50 ng/ml of purified RNaseA (QIAGEN). The total homogenate was rotated at 4°C for 30 min and centrifuged at 10,000 × g for 10 min, and the cleared supernatant was collected. For detection of Orb2 protein, approximately 5–10 head equivalents of extract was analyzed on a 4%–12% gradient SDS-PAGE (Invitrogen) or 8% SDS-PAGE and electroblotted onto PVDF membrane for 16–18 hr at 35 mV in the cold room. The membranes were blocked in 5% non-fat dry milk in TBS-Tween-20 buffer and incubated with the indicated affinity purified antibodies for 12–16 hr at 4°C with constant agitation. The antibody-antigen interaction was visualized by chemiluminescence using HRP-coupled anti-rabbit (for Ab273,793, and 402) or anti-guinea pig (for Ab2233) secondary antibody (Pierce Chemical). For immunoprecipitation, fly heads were homogenized in PBS buffer containing 150 mM NaCl, 3 mM MgCl 2 , 0.1 mM CaCl 2 , 5% glycerol, 1% Triton X-100, 1% NP40, and protease inhibitors. Lysates were clarified two times by centrifugation at 12,000 × g for 10 min at 4°C. For immunoprecipitation, 1.5–2 mg of total protein (corresponding to 100–150 adult fly heads) was incubated with 0.5 to1 μg of the affinity-purified antibodies for 2 hr at 4°C. After 2 hr, the lysates were incubated with protein-A beads (Repligen) for an additional 2 hr. The bead-bound material was collected, washed five times with the homogenization buffer, and subjected to SDS-PAGE and western blot as described above. For immunoprecipitation of Orb2AEGFP, ∼8 mg of total head extracts protein (corresponding to ∼500 adult fly heads) was incubated with either anti-Orb2 antibody 2233 or anti-EGFP chromotek-GFP-Trap (Allele Biotech). The S2 cells were lysed in PBS containing 1% NP40 and protease inhibitors, rotated for 15 min at 4°C and centrifuged at 10,000 × g for 10 min in a tabletop refrigerated centrifuge at 4°C. The clear supernatant was used for western and immunoprecipitation. For the Orb2A and Orb2B interaction experiment, the following lysis buffer was used: PBS + 150 mM NaCl + 0.1%SDS + 1% Triton X-100 + 1% NP40 + 50 ng/ml RNaseA + protease inhibitors. We found that use of nitrocellulose membrane and semi-dry quick transfer methods interferes with detection of the oligomeric Orb2.

Electron Microscopy Recombinant purified Orb2A and Orb2B proteins in 6 M GndHCl-PBS buffer were dialyzed against 1 M Urea buffer for 4 hr. Aliquots were taken and processed for EM by negative staining with Uranyl acetate.

Histology and Imaging For whole-mount preparation of adult brain the cuticle and eyes were removed using forceps in PBS and fixed for 45 min in 4% paraformaldehyde before imaging. Images were acquired using a LSM 5.0 Pascal Confocal Microscope (Carl Zeiss, Germany). High-resolution Z stack images of the fly brain at 512 × 512 pixels (1 μm step) were acquired for the identification of Orb2 puncta. To avoid cross-excitation EGFP and mCherry images were acquired in a multi-track mode.

Image Processing Dickinson et al., 2001 Dickinson, M.E., Bearman, G., Tille, S., Lansford, R., and Fraser, S.E. (2001). Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. Biotechniques 31, 1272, 1274–1276, 1278. Malik et al., 1996 Malik, Z., Dishi, M., and Garini, Y. (1996). Fourier transform multipixel spectroscopy and spectral imaging of protoporphyrin in single melanoma cells. Photochem. Photobiol. 63, 608–614. Wlodarczyk et al., 2008 Wlodarczyk, J., Woehler, A., Kobe, F., Ponimaskin, E., Zeug, A., and Neher, E. (2008). Analysis of FRET signals in the presence of free donors and acceptors. Biophys. J. 94, 986–1000. In order to separate EGFP signal from the bright autofluorescence that is present in live fly brains, it was necessary to use multispectral imaging with linear unmixing (). A GFP reference spectrum was obtained from a brain expressing EGFP from pUAST-Gal4 system. To obtain autofluorescence spectra, a pCasper-Orb2AEGFP expressing brain was analyzed using blind unmixing via the freely available PoissonNMF plug-in for ImageJ (NIH, Bethesda, MD, USA) as described by Neher et al. (). The image was adequately described by two autofluorescence spectra in addition to the EGFP spectrum. These spectra were found to be consistent across several samples and were therefore used for all unmixing experiments without modification. Linear unmixing with the predetermined spectra was performed using a custom written plugin for ImageJ employing standard linear least-squares algorithms. All images were acquired using the Zeiss LSM 710 spectral imaging system (Carl Zeiss, Jena, Germany), with a 40× 1.2 NA C-Apochromat water objective. The 488 nm laser line was used for excitation through an MBS 488 excitation diachronic, and emission was collected from 500 to 660 nm at 9.8 nm increments. Reference spectra are shown in Figure S2 C. With the high NA objective, and high scatter in the brains, only approximately 20 sections could be imaged into the brain before loss of signal. To quantify relative total levels of Orb2 versus Orb2A in live fly brains, 20 z sections with 2 μM spacing were acquired, using a pinhole of 1.8 airy units. Following unmixing, the EGFP signal from the sections was summed. To quantify EGFP intensity in the interior region, the slices that showed this localization were summed and the signal in this area was integrated, taking care to avoid cell bodies above and below this region. For two-color images, DsRed was expressed under the mushroom body-specific promoter MB247 to mark the mushroom body. DsRed expression was sufficiently high to allow excitation also at 488 nm. A reference spectrum for DsRed was acquired at the center of the mushroom body, and this reference spectrum was used throughout the brain. For display of images without unmixing, maximum intensity projections in the wavelength dimension were applied and each pixel was colored approximately according to the visual color of the corresponding maximum wavelength.

Sucrose Gradient Analysis of Orb2AEGFP- and Orb2BEGFP-Overexpressing Flies For the sucrose gradient analysis, fly heads were isolated and immediately crushed in a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 140 mM KCl, 0.5 mM DTT, and 1% Triton X-100. Following centrifugation at 8,000 × g, 1.5 mg of total lysate in a volume of 150 μl was loaded onto a 4 ml linear 10%–40% sucrose (w/v) gradient prepared in a gradient buffer containing 20 mM Tris-HCl (pH 7.5), 140 mM KCl, 5 mM MgCl 2 , and 0.5 mM DTT. The gradient was centrifuged at 55,000 rpm for 5 hr at 4°C in a SW60 rotor and 24 fractions of ∼150 μl were collected. Alternate fractions were loaded onto a 4%–12% SDS-PAGE gradient gel and analyzed by western blot using an Orb2-specific antibody (1:1000 Ab273). Representative molecular weights were determined by fractionating similar gradients loaded with 50 μg of the following standards, BSA (67 kDa), catalase (250 kDa) and thyroglobulin (670 kDa).

Agarose Gel Electrophoresis Halfmann and Lindquist, 2008 Halfmann, R., and Lindquist, S. (2008). Screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis. J. Vis. Exp. Published online July 16, 2008. 10.3791/838. The semi-denaturing agarose gel electrophoresis to analyze the Orb2 oligomers was performed following the protocol of Halfmann et al. (). 4× loading dye (2× TAE, 20% glycerol, 8% SDS) were added to the eluate to a final concentration of 1× and ran in a 1.2% agarose gel in 1× TAE buffer containing 0.1% SDS. To run the eluate under denaturing conditions 4× loading dye containing 0.1 M DTT was used and the sample was boiled for 10 min before running them in the same gel as the nondenatured protein. The gel was transferred overnight using TBS by capillary-transfer method and western blotted with Ab273.

Orb2A Mutant Screening The EGFP-tagged Orb2A was cloned under the constitutively active Actin promoter in pAc5.1 HisV5 vector (Invitrogen) and used as template for PCR. Using limiting amount of nucleotides in the PCR reaction mutations were generated within the first 160 amino acids of Orb2A. The PCR conditions were optimized to generate point mutations. The efficiency of mutagenesis reaction was confirmed by sequencing 93 randomly picked clones, 76 of which had at least one mutation. A library of 1,344 clones was prepared in fourteen 96-well plates. Each clone was transfected in S2 cells using Effectene reagents (QIAGEN) and 16–18 hr after transfection visually screened for clones which produced either completely diffused fluorescence and/or few fluorescence puncta instead of numerous small fluorescence puncta characteristic of wild-type Orb2A protein. The clones which showed diffuse fluorescence were sequenced and out of 72 such clones 59 clones had insertion, deletion, or substitution mutations that resulted in either frame shift or premature stop codon in the N-terminal end. This resulted in the production of low level of N-terminal truncated Orb2AEGFP proteins presumably due to translation initiation from downstream in frame ATG codon and thereby diffused fluorescence. The remaining 13 clones were transfected multiple times and 8 hr, 16 hr, and 48 hr after transfection the fluorescence patterns were analyzed. These 13 clones fell into three main categories: (1) diffused (F5Y), (2) low level of diffused fluorescence and one or two large puncta, and (3) diffused fluorescence as well as fluorescence puncta. The F5Y mutation was reintroduced by site-directed mutagenesis in the wild-type Orb2A, a fresh EGFP-tagged construct was made and retransfected to ensure that the fluorescence pattern we observed during screening was indeed due to the mutation in the Orb2A gene and not due to second site mutation in the vector backbone, in the promoter region or in EGFP.

Genomic Rescue Constructs Bischof et al., 2007 Bischof, J., Maeda, R.K., Hediger, M., Karch, F., and Basler, K. (2007). An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc. Natl. Acad. Sci. USA 104, 3312–3317. Venken et al., 2006 Venken, K.J., He, Y., Hoskins, R.A., and Bellen, H.J. (2006). P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314, 1747–1751. Venken et al., 2008 Venken, K.J., Kasprowicz, J., Kuenen, S., Yan, J., Hassan, B.A., and Verstreken, P. (2008). Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation. Nucleic Acids Res. 36, e114. Bischof et al., 2007 Bischof, J., Maeda, R.K., Hediger, M., Karch, F., and Basler, K. (2007). An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc. Natl. Acad. Sci. USA 104, 3312–3317. Venken et al., 2006 Venken, K.J., He, Y., Hoskins, R.A., and Bellen, H.J. (2006). P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314, 1747–1751. Venken et al., 2008 Venken, K.J., Kasprowicz, J., Kuenen, S., Yan, J., Hassan, B.A., and Verstreken, P. (2008). Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation. Nucleic Acids Res. 36, e114. Venken et al., 2008 Venken, K.J., Kasprowicz, J., Kuenen, S., Yan, J., Hassan, B.A., and Verstreken, P. (2008). Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation. Nucleic Acids Res. 36, e114. F5Y constructs were inserted at the same attP2 site in the 3rd chromosome. The pattB-Orb2ΔA construct was inserted in the attp40 site in the 2nd chromosome and attP2 site in the 3rd chromosome. The tagged genomic rescue constructs were made as described by Bischof and Venken et al. (). 500 bp fragments from the 5′ and 3′ end of the Orb2 locus were cloned into the pattB vector () to generate a capture vector. The linearized capture vector was cotransformed with a PacmanBAC construct () carrying the Orb2 genomic fragment and a 18,761 base pair fragment was captured using recombineering. The resulting untagged pattB-Orb2 construct was subsequently used to generate the EGFP-tagged genomic fragment using recombineering-mediated tagging (). The genomic rescue construct contain 560 base pairs of the of newly assigned Orb2 isoforms Orb2E through H and none of the Orb2I isoform. Therefore the genomic fragment may produce a 1002 nucleotide long Orb2E-H transcript as opposed to the 3747 nucleotides long wild-type Orb2E-H transcript. To introduce the F5Y mutation counter selection BAC modification kit from Genebridges was used. Briefly a Rpls-neo cassette was inserted into the Orb2A-specific exon and replaced with the F5Y mutation using counterselection. To generate Orb2A- and Orb2B-specific deletion in the genomic rescue construct the EGFP-Kanamycin cassette for Orb2A and DsRed-Kanamycin cassette for Orb2B () were introduced downstream of the start ATG codon using recombineering-mediated tagging. To avoid the positional affect on transgene expression the Orb2A and Orb2constructs were inserted at the same attP2 site in the 3chromosome. The pattB-Orb2ΔA construct was inserted in the attp40 site in the 2chromosome and attP2 site in the 3chromosome. The pCasper-Orb2AEGFP construct is comprised of a genomic fragment 1,446 nucleotides 3′of the last Orb2B-E-specific exon and 1,338 bp 5′of the Orb2E-I specific exon and therefore does not contain coding region of any of the Orb2 isoforms except Orb2A. The ∼8.27 Kb genomic fragment was cloned into the SpeI/XhoI site of pCasper4 and EGFP was introduced at the C-terminal end by creating an in frame SgrA1 site.

Hetero-FRET Measurements FRET between EGFP and mCherry labeled proteins was performed using the acceptor photobleaching method. Microscope images were acquired using a Carl Zeiss LSM-510 Confocal microscope (Jena, Germany) with either a C-Apochromat 1.2 NA water objective or a Plan-Neofluar 1.3 NA oil immersion objective with a pinhole size of approximately 2 airy units and a pixel dwell time of approximately 6.4 μs. The red and green signals that were excited with 488 and 561 nm lasers, respectively, were separated with an NFT 565 beam splitter, and collected by photomultiplier tubes (PMT's) through 505–550 and LP 575 filters. Several of the measurements made are plotted as a function of fluorescence intensity (relative concentration). To ensure consistency of relative intensity measurements from day to day and objective to objective we measured the intensity of green and red fluorescent slides (Chroma Technology, VT, USA) and used this value to correct our measurements. Given the large variation in measured intensity among cells used in this study, it is impossible to measure each cell with the same microscope gain and laser power. Therefore the laser power and, to a smaller extent, the microscope gain were adjusted to optimize the signal to fill the available dynamic range for each cell. The relationship between measured intensity and microscope gain and laser power were also corrected for using the same green and red fluorescent slides. Typical laser powers ranged between 0.01% and 0.1%. Apparent FRET efficiencies for each 2 × 2 group of image pixels using the acceptor photobleaching method were calculated as follows: E = 1 − I b e f o r e / I a f t e r . Here I before and I after denote the average fluorescence intensity before and after acceptor photobleaching. Four images were taken before and after photobleaching and averaged before calculating the FRET efficiency. Background intensities obtained from a region near the selected cell were subtracted before averaging. As the majority of measurements made were meant to focus on proteins in puncta, average FRET efficiencies for each cell were calculated by averaging the FRET efficiencies for the top 20% most intense pixels. This analysis was carried out using a custom plugin written for the ImageJ software package. In this way, dim regions with low signal-to-noise were avoided in the analysis. Relative concentrations of mCherry were calculated as described above without correction for the very small contribution of EGFP bleed through. In selected datasets we measured this contribution and deemed it to have little or no effect on the measurement of mCherry relative concentration. A single outlier was removed from the RA + F5Y FRET dataset based on the Grubb's outlier test. P values for FRET, homo-FRET, and FRAP percent recovery were obtained using the one-tailed t test.

Homo-FRET Measurements Grecco et al., 2004 Grecco, H.E., Lidke, K.A., Heintzmann, R., Lidke, D.S., Spagnuolo, C., Martinez, O.E., Jares-Erijman, E.A., and Jovin, T.M. (2004). Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells. Microsc. Res. Tech. 65, 169–179. r = I | | − G I ⊥ / I | | + 2 G I ⊥ . Here r is the anisotropy, I | | is the parallel intensity, I ⊥ is the perpendicular intensity, and G is the correction factor. Homo-FRET measurements between EGFP-labeled proteins were carried out using the fluorescence depolarization method (). In this method, the fluorescence anisotropy is measured. Homo-FRET between GFP molecules within a puncta will result in a decrease in the anisotropy, due to transfer between molecules with slightly offset orientations (i.e., transition dipoles). These measurements were performed on a wide-field Carl Zeiss Axiovert 200 M fluorescence microscope with a Plan-Apochromat 20× 0.8 NA air objective and an AxioCamHR camera. In the excitation path we placed a 450–490 nm band pass filter as well as a vertically oriented polarizer. Identical filter cubes with 510 nm excitation dichroics and polarizers oriented parallel or perpendicular to the excitation polarizer were used for emission polarization discrimination. Finally, a 515–565 nm filter was used for data acquisition. The correction factor for parallel versus perpendicular data acquisition was measured using a solution of fluorescein in 0.1 M NaOH, which has anisotropy of 0.011. Note that this correction factor may not be perfectly quantitative given the difference in polarization between in-focus and out-of-focus light in our set-up, and a dye solution contains a significantly larger percentage of out-of-focus light than a single cell sample. However, we note for this measurement we are only interested in the relative changes in anisotropy, not their absolute values. Once the correction factor has been obtained, the anisotropy is calculated as follows:. Here r is the anisotropy,is the parallel intensity,is the perpendicular intensity, and G is the correction factor. In order to properly analyze the large variation in cell intensities seen in this experiment with sufficient signal to noise yet also avoid saturation, it was necessary to perform both a short and long camera exposure. For data analysis, background was subtracted from both images and pixels close to saturation in the long exposure image were replaced by pixels from the short exposure image multiplied by a factor of 10, essentially producing a single, high dynamic range image. Separate experiments were performed to verify that the intensity observed was indeed a factor of 10 higher in the long exposure image. Once these high dynamic range images were obtained, average anisotropies were calculated for 16 × 16 pixel regions of each image with more than one fourth of their pixels above a background threshold. Only pixels above threshold in both images were included in the average. This subregion size provides approximately 4 subregions per cell and enough spatial resolution to discriminate puncta containing regions from non-puncta containing regions. Compared to our hetero-FRET analysis, this analysis is perhaps more prone to incorporation of diffuse regions, but the simplicity of the method allows for collection of a very large number of cells (>200). This large number of cells provides the needed statistics to observe the small differences in polarization that are characteristic of homo-FRET analysis.

FRAP Measurements FRAP measurements were performed under essentially identical conditions as the hetero-FRET measurements except without the red excitation channel. All images were taken with one second time resolution. Four images were taken before photobleaching of a small region containing a punctate structure. Thereafter, cells were observed for 44 s to assess recovery of both the diffuse pool and individual puncta. Great care was taken to avoid cells in which large cytosolic rearrangements occurred during the recovery as this would invalidate any measurements of percentage recovery of puncta. The averaged recovery curves within the bleached region were fit to a single rising exponential as follows: I ( t ) = A ( 1 − exp [ − t / τ ] ) + B . Here τ is the recovery time constant (inverse rate), A is the recovery amplitude, and B is the background intensity. Note that the half time of recovery is equal to 0.69τ. All of the samples analyzed in this study had relatively similar recovery times—approximately 10 s. The more informative number for our purpose is the fraction mobile, calculated as follows: f m o b i l e = A / ( I p r e f r a p − B ) . Here Iprefrap is the average intensity in the bleach region before bleaching. We typically see around 20% of the recovery intensity coming from dynamics of the diffuse pool. Therefore mobile fractions significantly greater than this are indicative of puncta recovery, i.e., bleached puncta proteins exchanging with the diffuse pool. In these cases, the puncta clearly recovers (see below).

Analysis of FRET Measurements The FRET efficiency is strongly dependent on the distance between fluorophores. In fact, FRET is nonexistent for molecules whose centers are more distant than 10 nm. Fluorescent proteins themselves are approximately 3 nm in diameter, meaning that the maximal FRET efficiency for fluorescent proteins is around 30%. For puncta, the FRET efficiency becomes more complicated because randomly organized oligomers will not necessarily have one EGFP molecule next to one mCherry molecule. In addition, for higher-order oligomers, FRET can occur to multiple acceptors, leading to higher possible FRET than with typical heterodimer scenarios. It is also important to note that FRET is orientation dependent, a factor that could either enhance or diminish the FRET in an organized structure.

Housing of Flies for Behavioral Experiments Flies were maintained using standard fly husbandry methods. For behavioral testing, flies were raised on standard cornmeal food at 25°C and 60% relative humidity on a 12 hr/12 hr light-dark cycle. All flies were collected and placed in a vial with fresh food for 24 hr at 25°C prior to behavioral testing. Before behavioral testing all transgenic lines were backcrossed six generations with white-eyed CantonS flies. The mini white gene is known to influence fly behavior. For that reason all controls and transgenic lines carried one copy of the mini white gene.

Olfactory-Appetitive Conditioning Flies were food deprived for 16 to 20 hr before conditioning in glass vials containing kimwipes paper saturated with water. The wall of the training tube was covered with a Whatman filter paper saturated with 1M sucrose (positive conditioning stimuli, +CS) that was allowed to dry prior to the training session. Another tube was also prepared with a filter paper soaked in water to provide the negative conditioning stimuli (−CS), allowing it to dry before use. Flies starved for 18 hr were introduced into the elevator of a T maze and tested in groups of ∼100. Flies were transferred to the −CS tube and exposed to an odor for 2 min. After 30 s of air stream, the flies were relocated in the elevator and shifted to the +CS tube in the presence of the second odor for 2 min. Memory was tested 2 min, 1 hr, 3 hr, or 24 hr after training. Flies were kept in test tubes with cotton plugs in a humidified chamber when memory tests were less than 3 hr. For the 24 hr test, flies were given standard cornmeal food for 6 to 7 hr after training. They were transferred to test tubes containing a kimwipe soaked with water and starved for 17 hr before testing. For the 48 hr memory test, flies were given standard cornmeal food for 18–24 hr after training and then were starved for 24–30 hr prior to testing. During the memory test, flies were introduced into the elevator and transported to a point where they have to choose between two air streams, one carrying the reward odor and the other with the control odor. Animals were given 2 min to choose between the two odors. Different group of flies were trained in a reciprocal experiment in where the −CS/+CS odor combination were reversed (3-Octanol or 4-Methylcyclohexanol). The performance index (PI) is calculated as the number of flies in the reward odor minus the number of flies in the control odor, divided by the total number of flies in the experiment. A single PI value is the average score of the first and the reciprocal experiment. Test odorants were delivered by bubbling air bottles containing odor dilutions in 50 ml of mineral oil and the air flow was monitored using a flowmeter at 4.5 psi (Allied Healthcare Products, Inc., St. Louis, MO, USA). The odor concentration used for the experiments were: 1.08 × 10−3 for 3-OCT and 1.1 × 10−3 for 4-MCH. The spontaneous response to odors and sucrose were assayed in the T maze. Naive flies were given 2 min to choose between two airstreams, one carrying the test odor (3-Octanol or 4-Methylcyclohexanol) and the other carrying no odor.

Male Courtship Suppression Assay For spaced training, individual males were placed in individual small food tubes (16 × 100 mm culture tubes, VWR) with a mated female for 2 hr. The female was removed and the male was left alone for 30 min. A different mated female was placed in the tube with the male for another 2 hr. The female was removed, and the male again rested for another 30 min. A third mated female was introduced in the tube for 2 hr and removed at the end of the trial. Control males were treated exactly the same way except no mated females were introduced into the tube. Memory test was assayed at 5 min, 4, 15, 24, 36, 48, 60 hr after training. All tests were performed in a 1 cm courtship chamber. Fresh mated females were used for all time points. All memory tests were recorded (for 10 min) and analyzed using a customized software. The courtship index of each male was obtained by manual and/or automatic analysis of the movies by an experimenter blind to the genotype and experimental conditions.

Automated Analysis of Male Courtship We have analyzed the male courtship behavior in an automated fashion to increase the number of flies for each experimental condition and to remove human bias. To accomplish this, we developed a custom software, in which most of the routines procedures are implemented in MATLAB. The source code with instructions is available upon request.