The “activity per second” (Figure 5C)

was calculated for each recording by taking the integral of the activity function and dividing it by the total time in seconds. To pseudocolor an “event” (right panel of Figure 5A), we first performed a Gaussian blur (sigma = 2.0) on all image frames by using ImageJ. We then calculated on a pixel-by-pixel basis an average baseline (Gb and Rb) from five consecutive time points in a trough just prior to the activity event marked with a red arrow in Figure 5B. Then we calculated Galunisertib concentration the normalized GCaMP3.0 and RFP (GN and RN) signal and the resulting activity for the event on a pixel-by-pixel basis by using the calculations shown above. In order to measure synchronization of DAN activity within distinct MB lobes innervations find more and the aimpr, we first computed a normalized cross-correlation (Ryx) function between simultaneously recorded signals as follows:

Ryx(m)=∑t=1N−m+1y(t)x(t+m−1)∑t−1N|x|2∗∑t=1N|y|2,where y and x are the two simultaneous recording activity signals across discrete time t, m is the lag, and N is the total sample length of the recordings. If two signals were synchronized in phase, then Ryx would be maximum with zero lag (m = 1). Therefore, we calculated a zero-lag normalized cross-correlation (Figure 5D) as Normalizedcross-correlation=Ryx(m=1)=∑t=1Ny(t)x(t)∑t−1N|x|2∗∑t=1N|y|2. If two signals are perfectly identical, Ryx(zero lag) = 1. Whole brains were isolated in ice-cold PBS and maintained at 4°C during all steps until mounting them on microscope slides. Brains were fixed in a solution of 4% paraformaldehyde and PBS+T (0.3% Triton X-100 in PBS). After 6 × 10 min washes with PBS+T, the brains were blocked overnight with 5% normal goat serum in PBS+T solution. Brains were then incubated with rabbit anti-GFP (1:200, Molecular Probes) and mouse anti-FasII (1:10, DSHB) primary antibodies overnight. After washing for 6 × 10 min in PBS+T, we incubated the brains overnight in a solution containing

goat anti-rabbit IgG conjugated with Alexa Fluor 488 and goat anti-mouse IgG conjugated with Alexa Fluor 633 (1:1,000, Molecular Probes) secondary antibodies. After an additional washing for 6 × 10 min with PBS+T, we mounted the brains on slides in Vectashield (Vector Laboratories). Images were and collected by using a 10× dry objective and a Leica TCS SP5 II confocal microscope. The step size for z stacks was 1 μm with images collected at 512 × 512 pixel resolution. Excel Stat and Prism were used for statistical analyses. Because PI values obtained from the classical olfactory assay are normally distributed (Tully et al., 1994), we used ANOVAs to make comparisons among different groups. For all comparisons of the effect of temperature across different genotypes, we performed a two-way ANOVA with both temperature and genotype as factors. We followed the two-way ANOVA with a Tukey post hoc comparison among the relevant groups.