Sparse, decorrelated odor coding in the mushroom body enhances learned odor discrimination

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1 Sprse, decorrelted odor coding in the mushroom ody enhnces lerned odor discrimintion Andrew C. Lin, Alexei Bygrve, Alix de Clignon, Tzumin Lee, nd Gero Miesenöck Supplementry Mteril R64C08-GAL4 (γ) R35B12-GAL4 (α β ) Supplementry Figure 1. GAL4 driver lines leling α β nd γ Kenyon cells. (,) Mximum intensity z-projections of confocl imge stcks. () γ Kenyon cells re leled in R64C08-GAL4/UAS-CD8::GFP flies. () α β Kenyon cells re leled in R35B12-GAL4/UAS-CD8::GFP flies. Scle rs, 50 µm. Representtive imges selected from t lest 3 dissected rins. Nture Neuroscience: doi: /nn.3660

() Anterior view of GFP expression in NP2631-GAL4, GH146-Flp/UAS-CD8::GFP; tup-frt-gal80-frt/+ fly with oth APL neurons leled. Scle rs, 50 µm.

2 Supplementry Figure 2. Intersectionl strtegy llows specific leling of APL neurons with insertions of tup-frt-gal80-frt on chromosome II nd III. () Anterior view of GFP expression in NP2631-GAL4, GH146-Flp/tuP-FRT-GAL80-FRT, UAS-CD8::GFP; UAS-shi ts1 /+ fly with oth APL neurons leled. () Anterior view of GFP expression in NP2631-GAL4, GH146-Flp/UAS-CD8::GFP; tup-frt-gal80-frt/+ fly with oth APL neurons leled. Scle rs, 50 µm. Representtive imges selected from 10 () nd 5 () confocl stcks nd nerly 700 rins dissected (see Fig. 6). Nture Neuroscience: doi: /nn.3660

3 Mnipultion: c d Control dtrpa1 leled leled TeTx leled e Control f g h i dtrpa1 leled shi ts1 leled TeTx leled Mximum F/F Mximum F/F TeTx: Mximum F/F t / mximum F/F t Loe imged: α α α α β β α β F/F 20% 5 s Inctive Unleled Inctive Unleled Inctive Inctive Inctive β α β β Loe imged TeTx-inctive TeTx control dtrpa1 leled leled γ γ γ Supplementry Figure 3. APL inhiits Kenyon cells in ll loes. (-d) Impct of different APL mnipultions (rows) on odor-evoked C 2+ influx in different mushroom ody loes (columns). Blck rs indicte 5 s pulses of ethyl cette. () In control hemispheres of flies where APL ws unleled, odor-evoked C 2+ influx in Kenyon cells declined slightly t. () In hemispheres of dtrpa1 flies where APL ws leled, odor-evoked C 2+ influx in Kenyon cells ws lmost completely olished t. (c) In hemispheres of flies where APL ws leled, odor-evoked C 2+ influx in Kenyon cells incresed gretly t. (d) In APL-leled hemispheres of TeTx flies (red), odor-evoked C 2+ influx in Kenyon cells ws much higher thn in APL-leled hemispheres of TeTx-inctive flies (lue). (e-h) Br grphs summrizing mximum F/F from (-d) nd including dt fter recovery to fter heting (e-g) nd hemispheres from TeTx flies where APL ws unleled (green rs, h). n, left to right, given s numer of rin hemispheres [numer of flies]: (e) n=24 [17] (α nd α ); 7 [6] (β, β, γ) (f) n=9 [5], 8 [5]. (g) n=30 [21] (α nd α ); 15 [10] (β, β, γ). (h) n=9 [7], 10 [8], 7 [6] (α nd α ); 10 [6], 7 [6] (β, β, γ). (i) Rtios of odor-evoked C 2+ influx t vs., corresponding to pnels e-h. Error rs show s.e.m. P < 5, P < 1, P < 01, repeted-mesures ANOVA with Geisser-Greenhouse correction nd Holm-Sidk multiple comprisons test (e,f α, g α, β, β, γ), Friedmn test with Dunn s multiple comprisons test (f α, g α), one-wy ANOVA with Tukey-Krmer post hoc test (h α, α ), unpired Welch t-test (h,i β, β, γ) or Mnn-Whitney U test (i α, α ). See Supplementry Tle 1 for full genotypes. Nture Neuroscience: doi: /nn.3660

m247-lexa> GAL80 no GAL80 150% Ethyl utyrte F/F Sprseness Isomyl cette Inter-odor correltions 0% r c Correltion 0.3 0.

Kenyon cell expression of TeTx is required for the effect of OK107>TeTx on sprse coding.

Color-coded correltion mtrices s in Fig. 5,. Scle rs, 10 µm. n given s numer of hemispheres [numer of flies].

4 m247-lexa> GAL80 no GAL80 150% Ethyl utyrte F/F Sprseness Isomyl cette Inter-odor correltions 0% r c Correltion m247-lexa, lexaop-gcmp3 OK107-GAL4, UAS-TeTx tup-gal80 ts 0 m247-lexa> GAL80 no GAL80 Supplementry Figure 4. Kenyon cell expression of TeTx is required for the effect of OK107>TeTx on sprse coding. () Activity mps of odor responses in OK107>TeTx, m247-lexa>gcmp3,gal80 flies (left, n = 7 [5]) nd OK107>TeTx, m247-lexa>gcmp3 (right, n = 6 [6]). Color-coded correltion mtrices s in Fig. 5,. Scle rs, 10 µm. n given s numer of hemispheres [numer of flies]. (, c) Removing Kenyon cell expression with m247-lexa>gal80 increses the popultion sprseness () nd decreses the inter-odor correltions (c) of ctivity mps of odor responses in flies crrying OK107>TeTx. P < 5, unpired Welch t-test. See Supplementry Tle 1 for full genotypes. Nture Neuroscience: doi: /nn.3660

5 α loe α loe δ-dl IA:EB 1:4 IA:EB 4:1 δ-dl IA:EB 1:4 IA:EB 4:1 unleled leled F/F 40% 5 s unleled leled Mximum F/F Supplementry Figure 5. Blocking APL synptic output ffects Kenyon cell responses to rod odors more thn Kenyon cell responses to nrrow odors. F/F trces nd mximum F/F dt corresponding to the rtios in Fig. 6. Shding on trces indictes s.e.m. Blck rs indicte 5-s pulses of δ-dl, IA:EB 1:4, or IA:EB 4:1 s leled. P < 5, P < 1, P < 01, repeted-mesures ANOVA with Geisser-Greenhouse correction nd Holm-Sidk multiple comprisons test. n = 12 [8] ( leled), n = 6-7 [5] ( unleled). n given s numer of hemispheres [numer of flies]. Nture Neuroscience: doi: /nn.3660

6 % time spent in CS % time spent in CS ºC OCT vs. MCH Neither Left Right Both 21 ºC 21 ºC Dissimilr shi ts1 in APL: Similr Supplementry Figure 6. Lerned odor discrimintion of flies with only one APL neuron leled nd with OCT vs. MCH. () Lerned odor discrimintion of 3-octnol (OCT) vs. 4-methylcyclohexnol (MCH). Protocol ws s in Fig. 7 except the CS+ ws OCT nd the CS ws MCH. n, left to right, given s numer of flies [numer of experiments]: 29 [6], 14 [6], 19 [6], 41 [6], 25 [6], 18 [6], 16 [6], 32 [6]. Kruskl-Wllis ANOVA revels no significnt differences etween the 8 groups (P = 9). 2-wy ANOVA revels min effect of temperture (P < 5) ut no significnt interction etween genotype nd temperture cross flies with neither, left, right, or oth APL neurons leled (P = 2) or compring flies with neither or oth APL neurons leled (P = 0.82). () Dt s in Fig. 7c, dding single-apl-leled flies. CS+ ws IA:EB 4:1 nd CS ws δ-dl ( dissimilr ) or IA:EB 1:4 ( similr ). At, flies with only the left or right APL leled y shi ts1 re somewht impired on lerned discrimintion of similr odors compred to flies with neither APL leled, ut the difference is not sttisticlly significnt. n, left to right, given s numer of flies [numer of experiments]: 23 [6], 28 [6], 19 [6], 26 [6], 16 [7], 32 [7], 34 [7], 44 [7], 18 [8], 30 [8], 22 [8], 55 [8], 32 [9], 33 [9], 37 [9], 51 [9]. P < 5, P < 1, P < 01, Kruskl-Wllis ANOVA with Holm-Bonferroni correction for post hoc tests, testing only pirs of dt points with one vrile chnged (tsk, temperture, nd APL leling). P < 5, 3-wy ANOVA for interction of tsk, temperture, nd APL leling. P < 1, 2-wy ANOVA for interction of genotype nd temperture for discrimintion of similr odors. P < 5, 2-wy ANOVA for interction of tsk nd APL leling t. P < 5, 2-wy ANOVA for interction of tsk nd temperture for flies with oth APL neurons or the left APL neuron leled. Other 2-wy ANOVAs did not revel ny significnt interctions. Error rs show s.e.m. Nture Neuroscience: doi: /nn.3660

F/F, α loe 0.8 0.4 # # /+ c 0 Sprseness 0.95 0.90 GH146> NP2631> d 0.3 Correltion 0.

tup-frt-gal80-frt UAS-mCherry UAS-shi ts1 () UAS-TeTx-inctive UAS-TeTx UAS- APL leled

RNAi knockdown of GABA iosynthesis in the APL neuron cuses only prtil effects on Kenyon cell

[7], 10 [8], 21 [19], 22 [19], 10, 11, 6, 6. n given s numer of hemispheres [numer of flies].

(d) Men inter-odor correltions etween cell ody responses for the pnel of odors used in Fig. 5.

Scle rs, 10 µm. Color grids represent pirwise correltions s in Fig.

7 Mximum F/F, α loe 1.5 # # e unleled Ethyl utyrte Isomyl cette Inter-odor correltions unleled Mximum F/F, α loe # # /+ c 0 Sprseness GH146> NP2631> d 0.3 Correltion 0.1 NP2631>, mcherry shi ts1 TeTx rod drivers 0 F/F (%) r NP2631-GAL4 GH146-GAL4 GH146-Flp tup-frt-gal80-frt UAS-mCherry UAS-shi ts1 () UAS-TeTx-inctive UAS-TeTx UAS- APL leled Supplementry Figure 7. RNAi knockdown of GABA iosynthesis in the APL neuron cuses only prtil effects on Kenyon cell odor responses. (-d) See grid t ottom for full genotypes. () α loe responses to ethyl cette. () α loe responses to ethyl cette. (,) n, left to right, given s numer of rin hemispheres [numer of flies]: 25 [17], 30 [21], 9 [7], 10 [8], 21 [19], 22 [19], 10, 11, 6, 6. n given s numer of hemispheres [numer of flies]. (c) Men popultion sprseness of cell ody responses to the pnel of odors used in Fig. 5. (d) Men inter-odor correltions etween cell ody responses for the pnel of odors used in Fig. 5. (c,d) n, left to right, given s numer of rin hemispheres [numer of flies]: 7 [7], 8 [7], 9 [7], 9 [7], 8 [8], 10 [8], 10, 11, 5, 5. (e) Smple ctivity mps of cell ody responses nlyzed in pnels c nd d. Compre to Fig. 5,. Scle rs, 10 µm. Color grids represent pirwise correltions s in Fig. 5: 1) ethyl cette, 2) 3-octnol, 3) utyl cette, 4) isomyl cette, 5) ethyl utyrte, 6) 2-pentnol, 7) 4-methylcyclohexnol. P < 5, P < 1, P < 01 significnt difference etween colored rs nd the relevnt controls (gry rs), y unpired Welch t-test for nd nd y Kruskl-Wllis ANOVA nd Dunn s multiple comprisons test for GH146 nd NP2631 driving. P < 5 significnt difference etween effect of nd effect of y 2-wy ANOVA. P < 5 significnt difference etween effect of mnipultion mrked with nd effect of TeTx y 2-wy ANOVA. # P < 5 significnt difference etween effect of mnipultion mrked with # nd effect of y 2-wy ANOVA. Error rs show s.e.m. Nture Neuroscience: doi: /nn.3660

8 Sprseness (MCH) Sprseness (OCT) c Correltion (MCH vs. OCT) NP2631-GAL4 GH146-GAL4 GH146-Flp tup-frt-gal80-frt UAS-mCherry UAS-shi ts1 () UAS- APL leled shi ts1 rod drivers Supplementry Figure 8. RNAi knockdown of GABA iosynthesis in the APL neuron does not ffect sprseness or correltion of Kenyon cell responses to 4-methylcyclohexnol or 3-octnol. (-c) See grid t ottom for full genotypes. () Popultion sprseness of cell ody responses to 4-methylcyclohexnol (MCH). Sttisticl comprisons, left to right: Mnn-Whitney U test (P = 94), unpired Welch t-test (P = 0), one-wy ANOVA (P = 0.48). () Popultion sprseness of cell ody responses to 3-octnol (OCT). Sttisticl comprisons: Mnn-Whitney U test (P = 54), unpired Welch t-test (P = 0.49), Kruskl-Wllis ANOVA (P = 0.41). (c) Correltion etween cell ody responses to MCH nd OCT. Sttisticl comprisons: unpired Welch t-test (P = 0.10), Mnn-Whitney U test (P = 0.75), Kruskl-Wllis ANOVA (P = 0.34). n, left to right, given s numer of rin hemispheres [numer of flies]: 7 [7], 8 [7], 8 [8], 10 [8], 10, 11, 5, 5. Error rs show s.e.m. Nture Neuroscience: doi: /nn.3660

9 Supplementry Tle 1. Summry of revited genotypes Figure Shorthnd Full genotype 1, 2, 5 m247-lexa>gcmp3 lexaop-gcmp3/+; m247-lexa/+ 1, 2, 5 m247-lexa>gcmp3,shi ts1 lexaop-gcmp3/lexaop-shi ts1 ; m247-lexa/+ 1c, 5 OK107>TeTx tup-gal80 ts, UAS-TeTx/+; UAS-GCMP3/+; OK107-GAL4/+ tup-gal80 ts, UAS-TeTx-inctive/+; UAS-GCMP3/+; OK107-1c, 5 OK107>TeTx-inctive GAL4/+ OK107>TeTx, m247- tup-gal80 ts, UAS-TeTx/+; m247-lexa, lexaop-gcmp3/+; 1c, S4 LexA>GCMP3 OK107-GAL4/+ 1c, S4 NP225>GCMP3, m247-1d LexA>shi ts1 NP225-GAL4/lexAop-shits1 ; UAS-GCMP3/m247-LexA 1d NP225>GCMP3, shi ts1 /+ NP225-GAL4/lexAop-shi ts1 ; UAS-GCMP3/+ 1e m247-lexa m247-lexa/lexaop-cd2::rfp 1f OK107-GAL4, GAL80 ts tup-gal80ts, UAS-TeTx-inctive/+; UAS-GCMP3/+; OK107- GAL4/+ 1g m247-lexa>gcmp3, 2 c739>shi ts1 c739-gal4/+; m247-lexa, lexaop-gcmp3/uas-shits1 m247-lexa>gcmp3, 2 R35B12>shi ts1 UAS-shits1 /+; m247-lexa, lexaop-gcmp3/r35b12-gal4 m247-lexa>gcmp3, 2 R64C08>shi ts1 UAS-shits1 /+; m247-lexa, lexaop-gcmp3/r64c08-gal4 3 GH146>spH, m247-3 GH146-GAL4/UAS-spH; m247-lexa/lexaop-dtrpa1 LexA>dTRPA1 3c GH146>GCMP3, dtrpa1/+ GH146-GAL4/UAS-GCMP3, MB-dsRed; lexaop-dtrpa1/+ 3d GH146>spH, dtrpa1/+ GH146-GAL4/UAS-spH; lexaop-dtrpa1/+ 3e 3e 4, 7 NP2631-GAL4, GH146-Flp/tuP-FRT-GAL80-FRT,UAS-,GFP CD8::GFP; UAS-shi ts1 /+ ts1 NP2631-GAL4, GH146-Flp/UAS-mCherry; m247-lexa, lexaop- 4-8, S7 shi GCMP3, tup-frt-gal80-frt/uas-shi ts1 NP2631-GAL4, GH146-Flp/UAS-mCherry; m247-lexa, lexaop- 4, 5 dtrpa1 GCMP3, tup-frt-gal80-frt/uas-dtrpa1 NP2631-GAL4, GH146-Flp/tuP-GAL80 ts, UAS-TeTx; m247-4, 5, S7 TeTx LexA, lexaop-gcmp3/tup-frt-gal80-frt NP2631-GAL4, GH146-Flp/tuP-GAL80 ts, UAS-TeTx-inctive; 4, 5, S7 TeTx-inctive m247-lexa, lexaop-gcmp3/tup-frt-gal80-frt RNAi NP2631-GAL4, GH146-Flp/UAS-mCherry; m247-lexa, lexaop- 8, S7 GAD GCMP3, tup-frt-gal80-frt/uas- 8, S7 /+ +; m247-lexa, lexaop-gcmp3/uas- 8, S7 GH146> GH146-GAL4/+; m247-lexa, lexaop-gcmp3/uas- 8, S7 NP2631> NP2631-GAL4/+; m247-lexa, lexaop-gcmp3/uas- OK107>TeTx, m247- OK107-GAL4, GAL80 ts, m247- GH146>GCMP3, m247- GH146>GCMP3, m247- tup-gal80 ts, UAS-TeTx/+; m247-lexa, lexaoptup-gal80, UAS-TeTx-inctive/+; UAS-GCMP3/m247- GH146-GAL4/UAS-GCMP3, MB-dsRed; m247-lexa/lexaopdtrpa1 GH146-GAL4, lexaop-shi ts1 /UAS-GCMP3, MB-dsRed; m247- LexA>GCMP3,GAL80 LexA>Gl80 LexA>dTRPA1 LexA>shi ts1 GCMP3/m247-LexA, lexaop-gal80; OK107-GAL4/+ LexA, lexaop-gal80; OK107-GAL4/+ LexA/+ GH146>GCMP3, shi ts1 /+ GH146-GAL4, lexaop-shi ts1 /UAS-GCMP3, MB-dsRed; + 8, S7 NP2631>, mcherry NP2631-GAL4/UAS-mCherry; m247-lexa, lexaop- GCMP3/UAS- Nture Neuroscience: doi: /nn.3660

Report. Social Facilitation of Long-Lasting Memory Retrieval in Drosophila

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