(A) Conceptual diagram of memory circuit organization. The parallel axonal fibers of the Kenyon cells represent odor stimuli, modulatory dopaminergic inputs induce plasticity at KC to MBON synapses in distinct compartments (represented by the shaded rectangles) along the length of these axons and MB output neurons from the compartments read out memory. See Aso et al. (2014a) for more details. (B) Left: Design of the optogenetic olfactory arena. See Figure 1—figure supplement 1 and Supplementary file 1 for details. Right: Diagram illustrating odor paths in the arena. Flies are confined in the circular arena at the center (3-mm high and 10-cm diameter). Video 1 illustrates the pattern of airflow. (C) Diagram of the expression pattern of the split-GAL4 line MB320C driving CsChrimson-mVenus (blue) in PPL1-γ1pedc; see Aso et al. (2014a) and www.janelia.org/split-gal4 for primary image data for this and other split-GAL4 lines. (D) Top: Training protocols. For odor delivery, valves were open for 60 s. For training, thirty 1 s pulses of red light (627 nm peak and 34.9 µW/mm2 at the position of the flies) were applied over 60 s starting 5 s after valve opening. Experiments were done reciprocally: In one group of flies, odor A and B were 3-octanol and 4-methylcyclohexanol, respectively, while in a second group of flies, the odors were reversed. All flies went through the same initial training and test protocol, and then the flies, without removal from the arena, went through one of the four diagramed training and test protocols. Bottom: Time course of the performance index (PI) during first test period (from 4–6 min of the experiment; left) and second test period (from 10–12 min of the experiment; right). The PI is defined as [(number of flies in the odor A quadrants) - (number of flies in odor B quadrants)]/(total number of flies). The average PI of reciprocal experiments is shown. The overall PI, which is reported in Figure 1F and Figures 2 and 3 was calculated by averaging the PIs from the final 30 s of each test period (indicated by the black horizontal line on the time axis). In the left panel, thin lines show individual reciprocal experiments and the thick line the mean of all experiments. In the right panel the mean with error bars representing the SEM are shown. Control genotypes did not show any significant memory in the same training protocols: (1) no driver control, pBDPGAL4 in attP2/20xUAS-CsChrimson in attP18 (PI = 0.07, SEM = 0.037, N = 14); and (2) no effector control, MB320C/w1118 (PI = −0.01, SEM = 0.031, N = 10). (E) A single frame of Video 2 showing the position of odor-conditioned flies at the end of the 1-min test period. Lines show trajectory of four flies. Video 2 shows the behavior of flies in the area for the full 1 min test period. (F) Inter-stimulus-interval (ISI) curve. A single training was done for each experiment. The relative timing of a 10 s delivery of odor A and a 10 s period in which three 1 s light pulse were delivered was varied. The diagram on top illustrates the cases of ISI = −10 s, 0 s and +10 s corresponding to the light pulses starting −10 s, 0 s, or +10 s after the initiation of odor delivery, respectively. The data points and error bars show the mean and SEM for MB320C/CsChrimson-mVenus. N = 10–14. Asterisk indicates significance from 0: *p<0.05; **p<0.01; ***p<0.001; n.s., not significant.