Behavioral experiments were performed in a custom-built, fully automated apparatus (Claridge-Chang et al., 2009) at 32°C unless stated otherwise

(see the Supplemental Experimental Procedures). Data were analyzed in MATLAB 2009b (MathWorks), SigmaPlot 12.5 (Systat Software), and Prism 6 (GraphPad). ePN or iPN projections to the LH were imaged by two-photon laser scanning microscopy (Ng et al., 2002 and Wang et al., 2003). Cuticle and trachea in a window overlying the LH were removed, and the exposed brain was superfused with carbogenated solution (95% O2 and 5% CO2) containing 103 mM NaCl, 3 mM KCl, 5 mM trehalose, 10 mM glucose, 26 mM NaHCO3, 1 mM NaH2PO4, 3 mM CaCl2, 4 mM MgCl2, and 5 mM N-Tris (TES) (pH 7.3). Odors at 10−2 dilution were delivered by switching mass-flow-controlled carrier and stimulus streams (CMOSens performance line, Sensirion) via software-controlled solenoid valves (the Lee Company). AZD5363 in vitro Flow rates at the exit port of the odor tube were 0.5 l per min. Basal plasma membrane fluorescence of ePNs expressing spH was used

to target a suction electrode to the mALT. Spikes were elicited with 1 ms pulses of current (10–30 μA) with a DS3 stimulus isolator (Digitimer). For thermal stimulation of iPNs expressing dTRPA1, the superfusion solution was heated with a closed-loop TC-10 temperature controller (NPI) with a HPT-2 in-line heater (ALA). Temperature shifts from 25°C to 32°C were complete in <1 min. Fixed samples expressing fluorescent proteins and/or stained with

fluorescently labeled antibodies were imaged on a Leica TCS SP5 confocal microscope (see the Supplemental Experimental Procedures). Pfizer Licensed Compound Library We thank Alexei Bygrave and Ruth Brain for generating QUAS-spH flies; Liqun Luo for communicating unpublished results; Bassem Hassan, Kei Ito, Toshi Kitamoto, Tzumin Lee, David Owald, Joachim Urban, Jing Wang, the Bloomington Stock Center, the Vienna Drosophila RNAi Center, and the Kyoto Drosophila Genetic Resource Center for fly strains; and Loren Looger for GCaMP3. This work was supported by grants (to G.M.) from the Wellcome Trust, the Gatsby Charitable Foundation, the Medical Research Council, the National Institutes of Health, and the Oxford Martin 3-mercaptopyruvate sulfurtransferase School. M.P. received postdoctoral fellowships from the European Molecular Biology Organization and the Edmond and Lily Safra Center for Brain Sciences. A.C.L. was a Sir Henry Wellcome Postdoctoral Fellow. M.P. and G.M. conceived and designed the study; M.P. performed and analyzed all experiments; and M.P., A.C.L., and G.M. interpreted the results and wrote the paper. A.C.L. provided fly strains and image analysis scripts. W.H. performed structural imaging. “
“Understanding how nervous systems represent sensory cues, store memories, and support decision making and appropriate action selection is of major interest. Olfactory learning in Drosophila is ideally suited to address these questions.