Ds qi2 monochrome cmos camera

A detailed description of electrode fabrication methods can be found in SI. The dopamine waveform (−0.4 V to 1.3 V and back to –0.4V) was applied to the carbon-fiber microelectrode (7 μm diameter) every 100 ms at a scan rate of 400 V/s. Prior to the experiment, electrodes were calibrated with 1μM dopamine by using flow cell injection analysis, and this calibration factor was used to convert currents into concentrations. For picospritzing, an empty glass capillary was pulled, the tip was trimmed, and it was filled with 5 mM acetylcholine or 20 μM nicotine for stimulation. A Picospritzer III (Parker Hannfin, Fairfield, NJ) was used to pressure eject acetylcholine or nicotine into the brain tissue. Capillaries were calibrated by picospritzing a droplet in oil and the diameter of the pressure ejected droplet was measured using DS-Qi2 monochrome CMOS camera and NIS-Elements BR imaging software (Nikon Instruments Inc. Melville, NY). The amount of acetylcholine applied was controlled by changing ejected volumes, relying on different pulse durations, and applied pressure was maintained at 10 psi. For 10 pmol stimulation, 2 nL (~78 μm droplet radius, approximately 250 ms pulse, depending on pipette calibration) of 5 mM acetylcholine was applied. The fly brain is 80 nL in volume, so 2 nL is only 1/40 of the volume of the brain.²²

Imaging was performed with a Nikon Ti2-E inverted microscope with DS-Qi2 monochrome CMOS camera controlled with NIS-elements. At least two hemi-sections from each of three arcuate regions were selected for analysis (i.e. rostral, middle, and caudal). Within each region of the arcuate, the total number of GFP cells, and the percentage of GFP cells that contained GR (Experiment 4) or the percentage of GFP cells that contained c-Fos (Experiment 5), were determined in each hemi-section. The mean ± SEM number of GFP cells per hemisection or mean percentage of GFP cells that contained either GR (Experiment 4) or c-Fos (Experiment 5) is reported. Comparisons were then made between treatment groups (within region) with animal as the experimental unit. All cell counting was done by an observer blinded to treatment group using ImageJ software [42 (link)].