To verify that ePN activity vectors and their distances accurately reflect input to the LH, we expressed GCaMP3 (Tian et al., 2009 and Wang et al., 2003) under GH146-GAL4 control and imaged patterns of calcium ISRIB mouse influx into ePN axonal branches in the LH ( Figure 1B). Distances between ePN activity vectors explained more than 50% of the observed variation in the structure

of these activity maps when responses to 21 odor pairs were compared across 13 individuals ( Figure 1C). Behavior was analyzed by tracking individual flies in narrow, 50 mm-long chambers (Claridge-Chang et al., 2009). The left and right halves of each chamber were perfused with independently controlled odor streams whose convergence at the midpoint

defined a ∼5 mm-wide choice zone. Each time a fly entered and left this choice zone, a decision was counted (Figure 2A). Choices in favor of either odor were tallied and combined into a single decision bias score. A bias of 100% indicates that a fly always chose one odor over the other; a bias of 0% signals unbiased or random choices. The measurement period was divided into two 2 min intervals, during which the left-right positions of the odorants were reversed (Figure 2A). We selected odors from the set characterized by Hallem et al. (2004) and Hallem and Carlson (2006) that would create odor pairs spanning the whole range of possible ePN distances (Table S1). Flies made an average of 19.9 ± 8.8 decisions per 4 min measurement OSI-744 nmr period (mean ± SD, n = 10,102 experiments). When the same odor was delivered to both arms of the chamber, choices were unbiased (decision bias = 0.71% ± 3.30%;

mean ± SEM, n = 161 flies) (Figure 2); when different odors were presented, each odor combination elicited a characteristic bias (Table S1), which was expressed in a qualitatively similar fashion by all members of a population (Figures 2B and 2C). Therefore, the lack of a measurable bias in a population is not a consequence of averaging opposing individual preferences. Differences in behavioral bias can arise from two new sources: differences in odor discrimination and differences in odor preference. In our analysis, we conceptually separated the processes of odor discrimination and valuation. In this two-step model of odor choice, the animal must first distinguish the odors in a pair and then decide which (if any) it prefers. If it cannot distinguish the odors, it cannot express a preference. Thus, a measurable preference indicates successful discrimination. The converse is not true: a fly may be able to tell two odors apart but may choose randomly between them if it has no incentive to act on a perceived difference. In other words, our measurements cannot distinguish indiscrimination from indifference. Bearing in mind this limitation, we searched for predictors of behavioral bias across a data set of 51 odor pairs.