Supplemental Information. Memory-Relevant Mushroom Body. Output Synapses Are Cholinergic

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1 Neuron, Volume 89 Supplemental Information Memory-Relevant Mushroom Body Output Synapses Are Cholinergic Oliver Barnstedt, David Owald, Johannes Felsenberg, Ruth Brain, John-Paul Moszynski, Clifford B. Talbot, Paola N. Perrat, and Scott Waddell

2 OK107-GAL4 + + ChATRNAi OK107-GAL4 ChATRNAi αchat SMP MB αvt 2 + VTRNAi OK107-GAL4 VTRNAi

3 Figure S1, related to Figure 1A and B. ChAT and VT immunostaining labels Kenyon cells. Single confocal sections through resentative sample of brains of UAS- ChAT RNAi ; OK107-GAL4 and UAS-VT RNAi ; OK107-GAL4 flies and their genetic controls, shown at the level of the MB γ lobe neurons. αchat labeling is visibly lower in comparable images from brains of UAS-ChAT RNAi ; OK107-GAL4 flies demonstrating that the signal in control flies resents intrinsic labeling of KCs. Each lane sents both channels of single confocal sections at the level of the γ lobe from three to four resentative brains per genotype. All flies were raised and processed for immunostaining in parallel before being imaged using the same confocal settings. Scale bars 20 µm. White dashed rectangles denote areas taken for quantification. See Movie S1 for comparison of a whole stack. Similar levels of staining and RNAi-reduction were observed for the other MB lobes (not shown). 3

4 A Immediate Appetitive Memory 0.4 B Immediate Aversive Memory Performance Index Performance Index C 0 OK107 / + + / VT RNAi OK107 / VT RNAi!"#$%&'(%)%(%!%&+,- -0.8!"#$%&'(%)%(%!%&.,/ OK107 / + + / GAD RNAi OK107 / GAD RNAi + / VGAT RNAi OK107 / VGAT RNAi + / Vglut RNAi OK107 / Vglut RNAi "67!&8&3!&8&9 42:;3&<&2:27 42:1=&<&2:2>?@A4!"#$ BC@D!&8&3!&8&12 42:1;&<&2:2> 42:1E&<&2:2F?@A4%#!"$ BC@D!&8&9!&8&12 42:7=&<&2:29 42:1F&<&2:2>.0123&G&!"#$ &'#(!&8&3!&8&12 42:E7&<&2:29 42:EE&<&2:2F.0123&G&%#!"$ BC@D!&8&9!&8&12 42:7E&<&2:29 42:1=&<&2:2F Figure S2, related to Figure 1C. RNAi directed towards but not GABAergic and glutamatergic transmission compromises olfactory memory. (A) KC-driven UAS- VT RNAi impairs immediate appetitive memory (n = 10-12, asterisk denotes p < 5; oneway ANOVA, Tukey s HSD post-hoc test). (B) KC-driven UAS-GAD RNAi, UAS-VGAT RNAi (both required for GABAergic signaling), and UAS-VGlut RNAi (required for glutamatergic signaling) did not significantly impact immediate aversive memory performance, as compared to the relevant genetic control flies (n = 4-7, p > 5; one-way ANOVA, Tukey s HSD post-hoc test). Error bars in (A) and (B) resent SEM. (C) Naïve OCT and MCH avoidance performance of all fly strains tested for aversive behavioral memory in Figure 1C. Displayed are mean performance index values +/- SEM. No statistical differences were evident between the groups (n = 7-10, all p > 5, one-way ANOVA, Tukey s HSD post-hoc test) other than OK107; UAS-ChAT RNAi and +; UAS-ChAT RNAi flies which exhibit defective MCH avoidance compared to OK107; + (n = 7, p < 5, one-way ANOVA, Tukey s HSD post-hoc test). MCH concentration was 1:500. 4

5 5 ms A 7 ms 15 ms B 50 ms R21D02-GAL4 > UAS-GCaMP6f C 1% ΔF/F0 0. A Sad GCaMP6f ΔF x104 ΔF 1% ΔF/F0 20 msec D s 5% i 100 μm s 20% i s 200% i s i 100 μm onto M4/6 imaged from M4/6 R21D02-GAL4 > UAS-GCaMP6f 100 μm onto M4/6 imaged from M4/6 R21D02-GAL4 > UAS-GCaMP6f % ΔF/F sec 10 mm onto calyx imaged from M4/6 R21D02-GAL4 > UAS-GCaMP6f Mean Peak ΔF/F0 Mean Peak ΔF/F0 0.8 Mean Peak ΔF/F0 E 10 mm onto calyx imaged from calyx 247-LexA > lexaop-gcamp6f 0.4 normal Mg mm Mg2+ F 247::GCaMP5 1mM R21D02 > GCaMP5 1mM normal Mg2+ df/f0 normal Mg mm Mg mm Mg2+ 247::GCaMP5 Vehicle 247::GCaMP5 Nic 100 um R21D02 > GCaMP5 Nic 100 um 5 normal Mg mm Mg2+

6 Figure S3, related to Figure 2. Only local dendritic application of evokes calcium transients in M4/6 neurons. 100 µm was applied to the M4/6 dendritic region in R21D02-GAL4; UAS-GCaMP6f brains. (A) The breadth of the evoked calcium response in M4/6 dendrites depends on the sad of the applied. Upper panels follow the sad of Texas Red that was co-applied with 100 μm. The ejection dose was successively increased from a 5 ms to 50 ms application. Lower panels depict ΔF (mean of responses 1.5 s after application mean 1 s before application). Each pixel is color-coded. (B and C) elicits calcium responses in M4/6 neurons only when locally applied to the dendritic region. (B) 100 μm was ejected at different positions within the protocerebrum, while the sad of the solution was monitored with Texas Red. Sad outlines are marked with colored dashed ellipses. (C) Mean trace of three application trials, each trace relates to the corresponding application outline shown in (B). Only delivery close to M4/6 dendrites (the orange and bright blue traces) evoked calcium responses. A magnified view of the individual traces underlying the bright blue response reveals a ms response onset. The experiments in A-C contained 1 µm tetradotoxin (TTX). (D) The injection pipette was moved to successively more distant locations away from the dendrite, while calcium responses were recorded using widefield imaging. Each row depicts a different brain. Dashed white line resents the midline of the brain for orientation purposes; s - superior, i - inferior. Colored dashed ellipses resent sad of, monitored by Texas Red; colors of calcium traces correspond to the matching dashed colored ellipses. Scale bars in A, B and D 20 µm. (E) 100 μm still evokes M4/6 responses under conditions of high magnesium in AHLS. Addition of 10 mm Mg 2+ did not affect responses evoked in M4/6 neurons when 100 μm was applied to the M4/6 neuron dendrites (first panel; n = 6, p > 5, independent samples t-test). Calcium responses in M4/6 also show similar kinetics with and without additional 10 mm Mg 2+ (second panel). Mean traces are shown with SEM in shade. Calcium responses in M4/6 neurons were significantly decreased at 10 mm Mg 2+ when they were stimulated via 10 mm applied in the calyx (third panel; n = 3-4, p < 5, independent samples t-test). Additional 10 mm Mg 2+ did not decrease KC responses evoked by applying 10 mm to the MB calyx (fourth panel; n = 2, p > 5, independent samples t-test). Error bars resent SEM. (F) KCs do not respond to cholinergic stimulation in the MB lobes. Application of 1 mm or 100 μm nicotine to the MB lobes does not elicit calcium responses in KCs (upper row), while both agonists evoke strong calcium responses in M4/6 neurons when applied in the same location (bottom panels). Displayed are mean traces with the respective SEM as shade, each color resenting an individual brain. Note that the reduced signal observed in the upper panels likely results from tissue displacement caused by ssure-evoked release of or vehicle. 6

7 A MBON-β 2mp (M4β ) B MBON-γ5β 2a (M6) C MBON-β 2mp (M4β ) MBON-β 2mp_bi MBON-γ5β 2a (M6) MBON-β 2mp (M4β ) 20 μm D MBON-β 2mp (M4β ) MBON-β2β 2a (M4β) E MBON-α 3ap (V2α ) MBON-α 3m (V2α ) Figure S4, related to Figure 3. evokes responses in multiple classes of MBONs. Each panel shows the anatomy of the relevant MBON and below the corresponding GCaMP6f measured physiological responses elicited by 1 mm acetylcholine application. Data resent individual -evoked traces from one brain per genotype. (A) MB002B labels MBON-βʹ2mp (M4β ). (B), MB011B labels MBON-γ5βʹ2a (M6), MBON-βʹ2mp (M4β ) and MBON-βʹ2mp_bi.(C), MB210B labels MBON-γ5βʹ2a (M6) and MBON-βʹ2mp (M4β ). (D) R93F01-GAL4 labels MBON-βʹ2mp (M4β ) and MBON-γ5βʹ2a (M6). (E) MB027B labels MBON-αʹ3ap (V2α ) and MBON-αʹ3m (V2α ). Initial increase in traces of (D) is an artifact of the LED trigger. 7

8 A 100 μm Acetylcholine 250 μm Mecamylamine B post 10 sec post Mecamylamine Peak 0.1 post C 100 μm 100 μm D Acetylcholine Mecamylamine E G 100 μm Acetylcholine Vehicle 10 sec 10 sec post Mecamylamine post Vehicle Peak F Peak post 100 μm 100 μm Acetylcholine Hexamethonium post H Peak 0.4 I 100 μm Acetylcholine 10 μm MLA 20% 10 sec post Hexamethonium J post Peak % 10 sec post MLA K 100 μm Mecamylamine 30 min washout L ns Norm. Peak Δ F/F % 5 sec Meca wash M 250 μm Mecamylamine 30 min washout N ns Norm. Peak Δ F/F % df/f0 5 sec Meca wash 8

9 Figure S5, related to Figure 4. M4/6 neuron calcium responses can be reversibly blocked by nicotinic receptor antagonists. (A-J) Local evoked calcium responses in M4/6 dendrites are blocked by the non-selective nicotinic receptor antagonists mecamylamine, hexamethonium, and MLA. (A-B) responses vanish within 100 s of applying 250 µm mecamylamine to the recording chamber. (A) Sample trace and (B) quantification, n = 5 brains, average of four trials, asterisk denotes p < 5, paired samples t-test. (C-D) responses also decrease within 100 s of applying 100 µm mecamylamine to the recording chamber. (C) Sample trace and (D) quantification, n = 3 brains, average of four trials, asterisk denotes p < 5, paired samples t-test. (E-F) Application of a vehicle control has no effect on the -evoked responses. (E) Sample trace and (F) quantification, n = 5 brains, average of four trials, p > 5, paired samples t-test. (G-H) responses vanish within 100 s of applying 100 µm hexamethonium to the recording chamber. (G) Sample trace and (H) quantification, n = 3 brains, average of four trials, asterisk denotes p < 5, paired samples t-test. (I-J) responses vanish within 100 s of applying 10 µm MLA to the recording chamber. (I) Sample trace and (J) quantification, n = 3 brains, average of four trials, asterisk denotes p < 5, paired samples t-test. (K-N) Optogenetic stimulation of KCs triggers M4/6 neuron calcium responses that are significantly reduced by application of mecamylamine, and partially recovered after washout. (K) Sample trace and (L) quantification of applying 100 µm mecamylamine, n = 2 brains, average of four trials, asterisk denotes p < 5, paired samples t-test. Genotype: lexaop-cschrimson / UAS- GCaMP6f; R21D02-GAL4 / 247-LexA::VP16. (M) Sample trace and (N) quantification of applying 250 µm mecamylamine, n = 2 brains, average of four trials, asterisk denotes p < 5, paired samples t-test. Genotype: R15B01-LexA, lexaop-gcamp6f / UAS-CsChrimson; R13F02-GAL4 / +. was normalized to each experiment s average initial four responses. 9

10 Movie S1, related to Figure 1A. Kenyon cells are ChAT- and VT-immunopositive. ChAT and VT immunofluorescence in the mushroom body lobes are greatly reduced by OK107-GAL4 directed exssion of UAS-ChAT RNAi or UAS-VT RNAi respectively (middle panels). Movie shows a resentative sample stack at 0.5 µm/frame. All flies were raised and processed for immunostaining in parallel before being imaged using the same confocal settings. Scale bar 50 µm. Movie S2, related to Figure 4A and D. Mecamylamine abolishes Kenyon cell-driven M4/6 responses. Optogenetic KC activation via 247-LexA exssed lexaop-cschrimson and red light illumination leads to robust calcium responses in M4/6 axons. R21D02-GAL4; UAS-GCaMP6f explant brains monitored with two-photon imaging. Frame rate: 5.92 Hz. Two resentative samples before and after addition of the nicotinic receptor antagonist mecamylamine (250 µm) or vehicle, are shown. The data in these movies is also sented as static views in Figures 4 G-L. White horizontal stripes that appear at regular intervals are artifacts from LED stimulation. 10