In vivo calcium imaging

Intact fly antennae were recorded. The calcium sensor GCaMP1.3 was expressed in the ORNs expressing the odorant receptor Or69a and the odor evoked calcium changes were measured at the receptor neuron dendrites and somata through the intact antennal cuticle. The setup consists of an upright microscope (Olympus BX50WI, Tokyo, Japan) equipped with a 50x air objective (NA = 0.5) and a CCD/monochromator based imaging system (Till Photonics, Gräfelfing, Germany). A monochromator (Polychrome II, TILL Photonics) produced excitation light of 470 nm wavelength that was directed onto the antenna via a 500 nm low-pass filter and a 495 nm dichroic mirror, emission light was filtered through a 505 nm high-pass emission filter.

Images were acquired with a TILL imago CCD camera with a binning of 8x8 on the chip. We varied the exposures time between 180 and 220 ms to adjust for different basal fluorescence values across preparations. Twenty-second films were recorded with an acquisition rate of 4 Hz.

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Odorants were greater than 99% pure or of the highest purity available Aldrich, Taufkirchen, Germany). Pure odorants were diluted in 5 ml mineral oil (Sigma-Aldrich) in 20 ml headspace vials (Schmidlin, Neuheim, Switzerland) to their final concentration ranging from 10^-7 to 10^-2 dilution (v/v). The vials were filled with nitrogen to prevent the odorants from oxidation and sealed and were positioned in a computer-controlled autosampler (CombiPAL, CTC analytics, Zwingen, Switzerland), which was used for odorant delivery to flies and was synchronized with the imaging setup via TTL pulses. A constant air stream (1 ml/s) coming from a synthetic air bottle was guided through a teflon tubing (inner diameter: 1 mm), the tubing exit was placed approximately 5 mm away from the fly’s antennae. We used double pulse protocol for odor stimulation.

During stimulation the constant air stream was interrupted with a computer controlled solenoid valve and the autosampler injected 2 ml of headspace at the speed of 1 ml/s into the tube (2x 1 ml; 6 and 9 s after acquisition onset) with an interstimulus interval of 2 s.

Each stimulus protocol (automated) consisted of 4–6 blocks of 9 or 10 or 12 measurements each with an interstimulus interval of 2 min. Between the blocks the syringe of the autosampler was washed thoroughly (with pentane and afterwards heated to 44°C) for 10 min. Each block started with three control measurements followed by 5 or 6 or 8 odor presentations and ended with a control measurement (room air). For screening 8 different odorants at 10^-2 dilution was tested within a block and for dose responses one odorant was tested with increase in odor concentrations within a block; 5 (10^-6 to 10^-2 dilution) or 6 (10^-7 to 10^-2 dilution) odor presentations for inhibitory and excitatory odorants respectively. After the end of the 4^th block three control measurements were tested again. The control measurements were: (1) a presentation of the solvent - mineral oil, (2) the reference odor isopentanoic acid (IPES) or 2-methybutyl acetate (MBAE) at 10^-2 dilution and (3) room air. The reference odor was used to monitor the fly’s responsive state. An individual fly could show consistent response upto 3 h. Before starting the automated stimulus protocol one or more test measurements with the reference odors at 10^-2 dilution were acquired to optimize focal plane and tubing position, after that automated stimulus protocol explained above was started.

5.3.5 Data analysis

Data analysis was performed with custom made routines written in IDL software (Research Systems, Co, USA) and R (http://www.r-project.org/). Measurements were chosen for further analysis if their flanking control block showed stable response to the reference odor. For response calculation the area showing calcium responses to the first

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reference odor was chosen. For quantification of odor evoked response magnitude (ΔF/F) the average response of 16 frames (4 s; from frame 8–23) before odor stimuli was subtracted from the average response of 20 frames (5 s; from frame 30–50) after 1^st odor stimulus onset (shown in Fig 5.1B). ΔF/F value of mineral oil was subtracted from all the odorants tested and in all plots except for mean traces the data shown were after subtraction of mineral oil response. Significant odor responses were quantified by comparing the mineral oil (solvent) response to non subtracted odor response (ΔF/F value); One way ANOVA and multiple comparisons test (Tukey HSD test performed in R using “agrocolae package”) was performed. Odorants that exhibited statistically significant ΔF/F value were grouped as activating odorants and others were grouped as non activating odorants. Activating odorants were grouped into four categories; best, intermediate, weak and inhibitory ligands based on their ΔF/F value. ΔF/F value greater than “3.5 or 2.8”, “2.5 or 2.4”, “2.1” and lower than “0” was grouped as best, intermediate, weak and inhibitory ligands respectively.

Dose response curves were established for 8 or 10 odorants that elicited excitatory or inhibitory response at 10^-2 dilution respectively. Dose response curves were plotted by averaging the response across the flies tested for one odor at each concentration. A Hill function as shown below was fitted to the concentration series of odorants using the CURVEFIT function in IDL (Pelz et al., 2006; Sachse and Galizia, 2003).

x^h R(x) = Rmax

x^h + EC₅₀^h

Experimentally determined values in the Hill equation are concentration x, the measured response for that concentration R(x) (which entered the algorithm as mean across animals with its variance as weight), and R_max for the overall maximal response to any odor. The estimated values are the Hill coefficient h, which represents a measure of the slope, and the Effective Concentration eliciting a half-maximal (i.e. 50%) response: EC₅₀. Values for EC₅₀ are given as decadic logarithms in this chapter. For example EC₅₀ of –3.5 indicates that a dilution of odor to 10^-3.5 in mineral oil (vol/vol) as used in our apparatus was determined to elicit half-maximal response in the antenna.

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5.4 Results