Figure 2. Behavioral consequences of optogenetic activation.

(A) The four-armed olfactory arena. Approximately 20 starved female flies were confined in 10 cm diameter and 3 mm high circular area with a hole at the center for air suction. Odor was introduced through four channels at the corners. (B) The protocol for behavioral experiments. Flies were trained by pairing 60 s of odor A with 30 1 s pulses of 627 nm LED light, each separated by 1 s without illumination. A different odor, odor B, was presented without red LED illumination, and then preference between two odors was tested. In the reciprocal experiments, odor B was paired with red light and A was unpaired. The same training was repeated twice more and then a second odor preference test was performed. Finally, six cycles of 10 s 627 nm illumination were applied, spaced by 100 s intervals without odor. Airflow was maintained at 400 mL/min throughout the experiment. (C) Top: The memory scores at the second odor preference test, measured as preference indexes: [(number of flies in the paired odor quadrants)-(number of flies in the unpaired odor quadrants)]/total number of flies during the last 30 s of the 60 s test period. The red asterisks * and ** indicate p<0.05 or p<0.01, respectively: Dunn’s multiple comparison tests compared to empty-split-GAL4 control, following Kruskal-Wallis test. The black * indicates p<0.05 without correction for multiple comparison. N=34 for the empty-split-GAL4 line and N=4–16 for other lines. All the lines were initially tested for four reciprocal experiments; lines with mean preference index above 0.1 or below –0.1 were subjected to additional tests. Cell types, the mushroom body (MB) compartments in which their dendrites lie, their neurotransmitters, the number of synaptic connections they make with dopaminergic (DANs) and octopaminergic (OANs) neurons, and the split-GAL4 driver lines used for the behavioral assays are designated. A summary of connections from all mushroom body output neuron (MBON) subtypes to DANs thought to signal reward or punishment and to OANs is shown in Figure 2—figure supplement 1A. Bottom: Z-scores [(values-mean)/standard deviation] for each parameter: speed, walking speed; angular, absolute of angular change relative to the previous frame at 30 FPS; cos(upwind), cosine of the fly’s orientation toward the upwind direction (i.e. facing away from the center of the arena). ON periods correspond to the first 2 s of the 10 s LED ON periods, whereas OFF periods are the 2 s immediately after the LEDs were turned off. Delta_DistanceFromCenter is change in fly’s mean distance from the center of the arena relative to its position at the onset of LED illumination. Return is a measure of the probability that flies return to the position that they occupied at the end of the LED stimulus. Flies are judged to have returned if they move 10 mm or more from their original position and then return to within 3 mm of the original position within 15 s.

Figure 2—figure supplement 1. Direct connections from mushroom body output neurons (MBONs) to dopaminergic neurons (DANs) and octopaminergic neurons (OANs).

(A) Total number of synaptic connections from each MBON type to DANs and OANs. Based on the valence of memory when activation of DANs is used as unconditioned stimulus in olfactory conditioning (Aso et al., 2012; Aso et al., 2010; Aso and Rubin, 2016; Claridge-Chang et al., 2009; Huetteroth et al., 2015; Ichinose et al., 2015; Lin et al., 2014; Liu et al., 2012; Yamada et al., 2023; Yamagata et al., 2016,Yamagata et al., 2015), we considered PAM01, 02, 04, 05, 06, 07, 08, 09, 10, 11, and 15 as Reward DANs and PAM12, 13, 14, PPL101, 103, and 106 as Punishment DANs. OANs refer to the four types of octopaminergic neurons that innervate the MB: OA-VPM3, OA-VPM4, OA-VUMa2, and OA-VUMa7. (B) Number of connections from MBONs in γ4 and γ5 to DANs in the γ lobe. PPL103, a DAN in the γ2 compartment, has less than five connections with these MBONs and was not included in this table.

Figure 2—figure supplement 2. Activation phenotypes are not compromised by prior optogenetic olfactory conditioning.

Time course of five kinematic parameters shown in Figure 2 for the positive control lines (MB011B and SS49755), negative control (Empty-split-GAL4), and two lines for MBON33 (SS75200 and SS77383) are shown. Lines and dashed lines represent mean of six trial averages for experiments with or without prior optogenetic conditioning sessions as shown in Figure 2B. Lines and shadings are means and SEMs.