(2012). GCaMP5 signals were imaged using two-photon microscopy. Adult flies were fixed to a piece of aluminum foil secured to a perfusion chamber (P-1, Harvard Technologies) using dental floss and an Electra Waxer (Almore International). Cuticle, trachea,

and fat bodies obscuring the mushroom body were removed and the exposed brain was superfused with saline (5 mM TES, 103 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 4 mM MgCl2, 26 mM NaHCO3, 1 mM NaH2PO4, 8 mM trehalose, 10 mM glucose [pH 7.3], bubbled with 95% oxygen, 5% carbon dioxide) using a perfusion pump (Watson-Marlow). Fluorescence was excited using 140 fs pulses centered on 910 nm generated by a Ti-sapphire laser (Chameleon Ultra II, Coherent), attenuated by a Pockels cell (Conoptics 302RM). Brains were imaged using Selleck BIBF1120 a Movable Objective Microscope (Sutter) with a Zeiss 20×, 1.0 NA W-Plan-Apochromat objective. Emitted photons were separated

from excitation light by a series of dichromatic mirrors and dielectric and colored glass filters and detected by GaAsP photomultiplier tubes (Hamamatsu Photonics H10770PA-40 SEL). Photomultiplier currents were amplified (Laser Components HCA-4M-500K-C) and passed through a custom-designed integrator circuit to maximize the signal-to-noise ratio. The microscope was controlled through MPScope 2.0 (Nguyen et al., 2006) via a PCI-6110 Lapatinib DAQ board (National Instruments). Odor stimuli were delivered by switching mass-flow-controlled carrier and stimulus streams (CMOSense Performance

Line, Sensirion) via software-controlled solenoid valves (The Lee Company). Flow rates at the exit port of the odor tube were 0.5 l/min. Images were converted to Analyze format and motion corrected by maximizing the pixel-by-pixel correlation between each frame and a reference frame. ΔF/F traces were calculated in ImageJ using manually drawn regions of interest (ROIs) for the background and brain structure of interest. Activity maps were generated in MATLAB from Gaussian-smoothed, background-subtracted images. A baseline fluorescence image was calculated as the average over a 10 s prestimulus interval. Minor z direction movement was ignored by correlating each frame to the baseline fluorescence and discarding it if the correlation fell below a threshold value. This threshold value was manually selected for each brain by noting the constant high correlation value STK38 when the brain was stationary and sudden drops in correlation when the brain moved. For each pixel, the difference between mean intensity during the stimulus and the mean baseline fluorescence (ΔF) was calculated. The ΔF during the presentation of a dummy stimulus (no odor) was subtracted to control for mechanical artifacts from the odor delivery system. If ΔF was less than two times the SD of the intensity of that pixel during the prestimulus interval, that pixel was considered unresponsive. We thank David Owald, Daryl Gohl, Marion Sillies, Tom Clandinin, and Ulrike Heberlein for flies.