Selected images of new split-GAL4 lines.

Panels A and C-H show expression (maximum intensity projections) of the indicated split-GAL4 line in the relevant portion of the brain. In panels A-H, the consensus JFRC2018 unisex brain template is also shown (green). The asterisk in panel H indicates the position of the esophagus. Images showing the full brain, optic lobe and ventral nerve cord of these lines can be found in Figure supplement 1 (for E-H) and Figure supplement 2 (for A-D). Panels B, I and J show images derived from stochastic labeling that reveal the morphology of individual cells. The original confocal stacks from which these images were derived are available for download at https://splitgal4.janelia.org/.

Behavioral consequences of optogenetic activation.

(A) Diagram of 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-second 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 the preference between two odors was tested. In the reciprocal experiments, odor B was paired with red light and odor A was unpaired. The same training was repeated twice more and then a second odor preference test was performed. Finally, six cycles of 10-second 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 a preference index defined as the [(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 in 4 reciprocal experiments; lines with mean preference index above 0.1 or below -0.1 were subjected to additional tests. Cell types, the 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 MBON subtypes to DANs thought to signal reward or punishment and to OANs is shown in Figure supplement 4A. 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 (that is, 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. ΔDistanceFromCenter 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 a fly returns to the position that it 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.

Additional behavioral consequences of optogentic activation.

(A) Time course of mean cos (upwind angle) for flies that express CsChrimson using the designated MBON21 drivers. The trace of empty-split-GAL4 is also shown. Red shading indicates the periods of 627 nm LED illumination. The trajectories from six trials were pooled to calculate a mean for each group of flies. Lines and shadings represent means and SEMs. (B) Mean cos(upwind angle) during the 2 s immediately after the LED was turned off. (C-D) Time course and mean walking speed during the 10 s LED On period. (E-F) The mean cumulative turning angles in 5 movie frames (total elapsed time of 150 ms) for flies expressing CsChrimson in MBON29 and MBON26. (G) The cumulative turning angle during the first 2 s of the LED On period. (A-G) show data from the experiments described in Figure 2. (H) Preference for quadrants with red light. Flies expressing CsChrimson in MBON21, MBON29 or MBON33 were tested with 30 s continuous light (627nm LED) in two quadrants. The test was performed a second time with illumination in opposite quandrants after an 150 s recovery period. (I) Mean preference index to the quadrants with red light during the last 5 s of the two 30 s test periods. Dunn’s multiple comparison tests compared to empty-split-GAL4 control, following Kruskal-Wallis test. *, ** and *** indicate p<0.05, p<0.01 or p<0.001, respectively: N=66 for Empty-split-GAL4 and 8-22 for other lines in (A-G). N=16-26 in (H-I).

Diagrammatic summary of key outputs from selected MBONs.

(A) MBON21 and MBON29 arborize dendrites in the γ4 and γ5 compartments that are innervated by reward representing DANs. MBON21 and MBON29 are cholinergic and preferentially connect with DANs that innervate other compartments to represent punishment, whereas other glutamtergic MBONs from these same compartments preferentially form connections with reward representing DANs going back to the same compartments. In the fly brain, acetylcholine (ACh) is largely excitatory via nicotinic ACh receptors, although the type A muscarinic ACh receptor can mediate an inhibitory effect (18, 19). Glutamate (Glu) can be inhibitory or excitatory depending on the receptors in the postsynaptic cells. Glutamate is known to be inhibitory via the glutamate-gated chrolide channel (GluClα) in the olfactory system (20). All of the ten types of DANs examined with RNA-seq express GluClα and Nmdar2 at high levels whereas expression of Nmdar1 and other glutamate receptors were limited and cell type specific (21). Results in some studies support an excitatory effect of at least a subset of glutamatergic MBONs on DANs (16, 22–24), while electrophysiological recordings indentified inhibitory connection between a glutamatergic MBON and downstream interneurons (15). (B) Diagram showing direct connections between the MB and CX mediated by MBON21, MBON30, MBON09 and MBON04; these MBONs rank first, second, fifth and seventh, respectively, based on the number of direct synaptic connections to the CX; numbers reflect connections between right hemisphere MBONs and right hemisphere FB tangential cells. For circles representing MBONs, the circle diameter represents the fraction of that MBONs direct output that goes to the CX. For the downsteam neurons in the CX, circle diameter represents the fraction of that cell types direct input that comes from MBONs. Arrow width reflects synapse number. See Figure 19 of (3) and Figure 46 of (25) for additional information on the complete set of MB to CX connections.

Maximum intensity projections of the brains and ventral nerve cords of split-GAL4 lines for atypical MBON.

Maximum intensity projections of the brains and ventral nerve cords of split-GAL4 lines for typical MBON.

Summary list of selected split-GAL4 lines for all MBON cell types.

These lines represent what we believe to be the best available split-GAL4 drivers for each cell type. The MB compartments they innervate and neurotransmitters are shown. The neurotransmitters that are shaded in gray were assigned based solely on computational prediction (33). Other transmitters have been confirmed by antibody staining (13) or EASI-FISH (34). The number of cells per hemisphere are shown for each cell type. Available split-GAL4 are listed. Lines listed in boldface are generally of higher quality. Lines whose names are shown in purple font were generated as part of this study. Lines whose names are shown in black font were described in (13). Lines from other studies are shown brown font and their sources were as follows: SS01308, (21); G0239, (35); SS46348, (16); MB618B, SS01127, SS01188, SS01194, SS01298, SS01337 and SS01388, Aso 2021 release: https://splitgal4.janelia.org.

Direct connections from MBONs to DANs and 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 (reviewed in (2)) we considered PAM01, 02, 04, 05, 06, 07, 08, 09, 10, 11 and 15 to be Reward DANs and PAM12, 13, 14, PPL101, 103 and 106 to be 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.