Computational protocol: Dendritic Integration of Sensory Evidence in Perceptual Decision-Making

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Protocol publication

[…] Male flies aged 7 days were fixed to a custom mount with soft thermoplastic wax (Agar Scientific). Cuticle, adipose tissue, and trachea were surgically removed in a window large enough to expose the dorsal brain and antennal nerves, and the preparation was superfused with extracellular solution (pH 7.45) containing 5 mM HEPES, 140 mM NaCl, 2 mM KCl, 4.5 mM MgCl2, and 1.5 mM CaCl2. Both antennal nerves were cut as far distally as possible, and the antennae were removed to facilitate access. One nerve was aspirated into a fire-polished suction electrode (40 μm bore diameter) to achieve a 1.5 ± 0.3 MΩ seal. A constant voltage stimulator (Digitimer) was used to deliver trains of 1 ms pulses of 2 V at increasing frequencies for 500 ms, with pauses of 20 s between stimulus trains. The pipette resistance was monitored throughout to guarantee constant stimulation currents.GCaMP6m fluorescence was recorded by two-photon laser scanning microscopy. Excitation light pulses with 140 fs duration and a center wavelength of 910 nm (Chameleon Ultra II, Coherent) were intensity-modulated with the help of a Pockels cell (302RM, Conoptics) and focused by a 20 ×, 1.0 NA water immersion objective (W-Plan-Apochromat, Zeiss) on a Movable Objective Microscope (Sutter Instruments). Emitted photons were separated from excitation light by a series of dichromatic mirrors and dielectric and colored glass filters and detected by a GaAsP photomultiplier tube (H10770PA-40 SEL, Hamamatsu Photonics). Photocurrents were passed through a high-speed amplifier (HCA-4M-500K-C, Laser Components) and a custom-designed integrator circuit to maximize the signal-to-noise ratio (). The microscope was controlled through ScanImage (Vidrio Technologies) via a PCI-6110 DAQ board (National Instruments). Images were acquired at a resolution of 256 × 256 pixels and a frame rate of 5 Hz. All experiments were carried out at room temperature (21–23°C).ΔF/F traces in manually defined regions of interest were calculated in ImageJ by dividing the background-corrected fluorescence by the background-corrected pre-stimulus signal (). To measure the maximal GCaMP6m fluorescence, Ca2+ influx was induced at the end of each experiment by rapidly applying KCl to the bath at a final concentration of 100 mM (). […]

Pipeline specifications

Software tools ScanImage, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Drosophila melanogaster
Chemicals Potassium