Minipid

A cotton swab dipped in 99% Methyl Salicylate liquid was placed at the center of the table (Figure 1D, gold star) at approximately 0.5 cm from the surface. Concentration was measured for 30 s at distances from 0.5 cm to 15 cm using a Photoionization Detector (PID) (miniPid, Aurora Scientific). Raw measurements had noise peaks at 60 and 72 Hz, broad spectrum high frequency noise, and substantial baseline drift. To remove the noise peaks, two 2nd order Butterworth IIR notch filters were used. To remove high frequency noise, a (0.2 s backward, 0.02 s forward) moving mean filter was used. To remove baseline drift, a baseline estimation and denoising function was used (Duval, 2018 ). The denoised traces (Figure 1E) show large signal fluctuations and, generally, decreasing mean output with distance. Intermittency was calculated as the mean number of points greater than a threshold of 0.005.

We applied electric shocks (four 1.5 s long 90 V pulses) to the fly's legs by placing the fly on a custom-build copper grid (Figure 2A and Supplementary Figure 4B). We recorded the shock strength received by an individual fly using a bridge circuit (sampling rate: 16 kHz; Figure 3A, Supplementary Figure 4C, and Supplementary Table 2). We used 1-butanol (BUT) and 4-methylcyclohexanol (MCH) diluted in mineral oil (MO; BUT 1:500, MCH 1:1,000) as odorant stimuli, which we presented as 10 s long stimuli with a custom-build stimulator (Szyszka et al., 2011 (link)). We measured the dynamics of the odorant stimuli with a photo ionization detector (miniPID, Aurora Scientific Inc.). Rapid odorant stimulus termination (Supplementary Figure 4A) allowed us to distinguish between responses to the olfactory CS and to the electric shock US.

To estimate odor concentration, we used a mini photo-ionizer detector (miniPID, Aurora Scientific). PID response amplitude depends on the odor identity, its concentration and the distance from the inlet. Each odor elicits a different response that is related to how effectively the measured odor can be ionized. We measured the door PID responses to the odors and their temporal dynamics at different delays. All TOMs elicited similar response dynamics. PID responses varied between these delays and the order of odor presentation, reflecting the between-odor interactions and inter-pipe flow interactions. However, the odor concentration of the odor constituents and the TOMs in three tested delays was highly similar across trials of the same condition. Thus, each TOM was expected to elicit the same odor percept across all trials. Measurements of odor concentration when placing the PID inlet ~5 cm from the odor ports (i.e., the location of the participants’ nose) revealed that the order of odor presentation was preserved, although the odor concentration was more variable across trials when measured at a 5 cm distance (Figure 1—figure supplement 1C).

Odor stimuli were delivered using a custom-made airflow dilution olfactometer with electronic dilution control. All odor stimuli were calibrated using a mini photoionization detector (miniPID, Aurora Scientific, Ontario, Canada) to form square-pulses of 1% concentration (relative to maximum recorded vapor-pressure in air; Figure S1). Odor stimuli used for initial go/no-go training purposes consisted of peppermint oil and almond oil - components that were not present in the odor mixtures later presented in recordings. For stimuli during whole-cell recordings, two were randomly selected from four potential odor mixtures (Figure S1), and for behaving mice randomly assigned to CS+ or CS-. Odor mixtures were comprised of four to six monomolecular odorants selected for their reported ability to activate dorsal glomeruli (Takahashi et al., 2004 (link)), grouped according to similarity of vapor pressure, and added to the mixture in an undiluted quantity inversely proportional to their relative vapor pressures (Figure S1). Odors were presented with a minimum ITI of 10 s. To minimize contamination, a high flow clean air stream was passed through the olfactometer manifolds during this time. Constant air-flow going to the animal was achieved using a final valve, minimizing any tactile component accompanying the odor stimulus.

We used a fast photo-ionization detector (miniPID, Aurora Scientific) to characterize the dynamics of stimulus delivered. Raw data were amplified (gain = 5) and acquired at 15 kHz sampling rate using a custom LabView data acquisition program (Supplementary Fig. 9).

The olfactometer was calibrated using a photo ionization detector (PID; mini PID, Aurora Scientific). The probe was sequentially placed in front of all the odor source outlets under both normal and background odor conditions to verify odor concentration and delivery time. The PID map for odor in the box was generated by placing the probe at X = 1 cm along the breadth and Y = ((15, 33, 45, 57, 69, 81) cm along the length of the box, where Y = 0 marks the entrance of the odor compartments. We were only able to detect the presence and absence of odor at a given position using this technique. Calibration was done using isoamyl acetate as the tracking odor with four repetitions of odor delivery in two sessions each at a given location.