Activation of SS04185 inhibits rolling evoked by activation of Basin neurons

(A) Ethograms of Basin activation (top panel) and co-activation of SS04185 and Basins (bottom panel). Each row represents an individual larva. Pink, blue, green, orange, and purple lines represent bouts of rolling, turning, crawling, backward crawling, and hunching. The red bar and dashed lines indicate the time window during which neural activation was present. Genotypes: 20xUAS-IVS-CsChrimson::mVenus/+;+; R72F11-Gal4/+ (top); 20xUAS-IVS- CsChrimson::mVenus/+; R54B01-Gal4.AD/+; R46E07-Gal4.DBD/ R72F11-Gal4 (bottom). Genotypes in (B, D-G) are the same as those mentioned here.

(B) Time series of rolling probabilities of larvae during co-activation of SS04185 and Basins (red) and activation of Basins alone (black), as well as crawling speed of larvae during activation of Basins alone (green). Shaded areas represent 95% confidential intervals for rolling probabilities. The red bar and dashed lines denote the optogenetic stimulation window.

(C) Rolling probabilities of larvae with activation of different neurons. Error bars represent the 95% confidence interval. Genotypes from left to right: 1) 20xUAS-IVS- CsChrimson::mVenus/+;;, 2) 20xUAS-IVS-CsChrimson::mVenus/+; R54B01-Gal4.AD/+; R46E07-Gal4.DBD/+, 3) 20xUAS-IVS-CsChrimson::mVenus/+;; R72F11-Gal4/+, 4) 20xUAS-IVS-CsChrimson::mVenus/+; R54B01-Gal4.AD/+; R46E07-Gal4.DBD/ R72F11-Gal4. n = 120, 118, 231, 155 from left to right. Statistics: Chi-square test, χ2 = 0, p > 0.05 for the first two groups; χ2 = 83.85, p < 0.001 for the last two groups; and χ2 = 365.51, p < 0.001 for the comparison between the first two groups and the last two groups.

(D) Cumulative plot of rolling duration. Statistics: Mann-Whitney test, p < 0.001, n = 652, 120.

(E) A violin plot showing start of first rolling bout for each larva during stimulation. Statistics: Mann-Whitney test, p = 0.027, n = 225, 89.

(F) A violin plot displaying end of first rolling bout for each larva during stimulation. Statistics: Mann-Whitney test, p < 0.001, n = 225, 89.

(G) A violin plot presenting start of first crawling bout for each larva during stimulation. Statistics: Mann-Whitney test, p < 0.001, n = 214, 70.

**p < 0.01, ***p < 0.001.

SS04185-DN, but not SS04185-MB, inhibits rolling when co-activated with Basins

(A) Morphology of SS04185 neurons. GFP, grey (left), green (right); nc82, magenta. Anterior, up; dorsal view; scale bar, 100 µm. Genotype: 10xUAS-IVS-mry::GFP/+; R54B01-Gal4.AD/+; R46E07-Gal4.DBD/+.

(B) Kenyon cells are less labeled in SS04185 with MB>Kipper Zipper. CsChrimson expression in Kenyon cells of SS04185 in Control and SS04185 with Killer Zipper in mushroom body (MB). GFP, grey (left), green (right); nc82, magenta. Anterior, up; dorsal view; scale bar, 20 µm. Genotype: 20xUAS-IVS-CsChrimson::mVenus/+; R54B01-Gal4.AD/+; R46E07-Gal4.DBD/+ (Control); 20xUAS-IVS-CsChrimson::mVenus/+; R13F02-LexA,LexAop-KZip+/R54B01- Gal4.AD; R72F11-Gal4/R46E07-Gal4.DBD (MB>KZip+).

(C) Rolling probabilities of larvae with activation of SS04185 reduce the expression of CsChrimson in mushroom body (MB) neurons. Error bars, 95% confidence interval. n = 78, 55, 100 from left to right. Statistics: Chi-square test, χ2 = 2.32, p > 0.05 for the two groups with SS04185 expression; χ2 = 37.50, p < 0.001 for the comparison between the two groups on the left; χ2 = 70.45, p < 0.001 for the comparison between the groups with MB>KZip+ expression which reduce expression of CsChrimson in MB. Genotypes: 20xUAS-IVS- CsChrimson::mVenus/+; R13F02-LexA,LexAop-KZip+/+; R72F11-Gal4/+ (black); 20xUAS-IVS- CsChrimson::mVenusR54B01-Gal4.AD/+; R46E07-Gal4.DBD/R72F11-Gal4 (dark red); 20xUAS-IVS-CsChrimson::mVenus/+; R13F02-LexA,LexAop-KZip+/R54B01-Gal4.AD; R72F11- Gal4/R46E07-Gal4.DBD (red). Genotypes in (D-E) are the same as mentioned here.

(D) Cumulative plot of rolling duration. Statistics: Kruskal-Wallis test: H = 8.28, p = 0.016; Bonferroni-corrected Mann-Whitney test, p > 0.05 for all pairwise post-hoc tests, n = 103, 20, 27 from left to right.

(E) A violin plot of start of first crawling bout for each larva during stimulation. Statistics: Kruskal-Wallis test: H = 15.02, p < 0.001; Bonferroni-corrected Mann-Whitney test, p > 0.05 for the two groups with SS04185 expression; p < 0.001 for the comparison between the group without SS04185 expression and the groups with full SS04185 expression, n = 65, 20, 7 from left to right.

(F) The probabilities of larval rolling during first 5 s of stimulation. Error bars, 95% confidence interval. n = 101, 126. Statistics: Chi-square test, χ2 = 4.27, p = 0.039. Genotype: 13xLexAop2-IVS-CsChrimson::tdTomato/w+, hs-FLP; R54B01-Gal4.AD/72F11-LexA; 20XUAS-(FRT.stop)- CsChrimson::mVenus/R46E07-Gal4.DBD. Genotypes in (G-H) are the same as mentioned here.

(G) Cumulative plot of rolling duration. Statistics: Mann-Whitney test, p < 0.001, n = 350, 473.

(H) A violin plot of start of first crawling bout for each larva during stimulation. Statistics: Mann-Whitney test, p < 0.001, n = 97, 120.

**p < 0.01, ***p < 0.001.

SS04185-DN is identical to SeIN128

(A) TEM neuron reconstruction of SeIN128 neurons. Left panel: anterior, up; dorsal view. Right panel: anterior, up; dorsal, right; lateral view. Red dots, presynaptic sites. Cyan dots, postsynaptic sites.

(B) A transverse section of larval CNS from EM reconstruction data. SeIN128 (green), Basins (blue), and A00c (orange) are located in ventromedial tract (VM). mdIV, red; magenta, neural tracts. DM, dorsomedial tract; VM, ventromedial tract. Dorsal, up; anterior view; scale bar, 1 µm.

(C) Cartoon generated based on transverse section of SeIN128, Basin-1 to Basin-4, A00c, and mdIV from EM neuron reconstruction data and (D). Nerve tracts are shown in magenta. Dorsal, up; posterior view. DM, dorsomedial tract; VM, ventromedial tract; CI, central-intermediate tract; CL, central-lateral tract; DL, dorsolateral tract; VL, ventrolateral tract. SeIN128, green; Basin-1 to Basin-4, blue; A00c, orange; mdIV, red.

(D) SS04185-expressing neurons co-stained with N-Cadherin. A cell body of SS04185- Descending neuron located in ventral part of the subesophageal zone (SEZ). SS04185, Green; N- Cadherin, magenta. Anterior, up; left, dorsal view; right, longitudinal section; scale bar, 100 µm. Genotype: 10xUAS-IVS-mry::GFP/+; R54B01-Gal4.AD/+; R46E07-Gal4.DBD/+. SS04185,

Green; Cadherin, magenta. Anterior, left, dorsal, up; lateral view; scale bar, 100 µm.

(A) (E) Transverse section of SS04185-DN co-stained with Fas2. SS04185-DN located at ventromedial tract (VM). SS04185, Green; Fas2, magenta. Dorsal, up; posterior view; scale bar, 20 µm. DM, dorsomedial tract; VM, ventromedial tract; CI, central-intermediate tract; CL, central-lateral tract; DL, dorsolateral tract; VL, ventrolateral tract. Genotype: 10xUAS-IVS- mry::GFP/+; R54B01-Gal4.AD/+; R46E07-Gal4.DBD/+.

(F, H, J) SS04185-DN co-localized with Basins or A00C neuron tract but not MdIV. SS04185, Green; Basins (F), A00c (H) or mdIV (J), magenta;. Genotype: w; R54B01-Gal4.AD/R72F11- LexA(F) 71A10-LexA(H) or ppk1.9-LexA(J); R46E07-Gal4.DBD/13xLexAop2-IVS-CsChrimson::tdTomato,20xUAS-IVS-GCaMP6s. Top panel: anterior, up; dorsal view; scale bar, 10 µm. Bottom panel: dorsal, up; posterior view; scale bar, 5 µm.

(G, I, K) SeIN128, Basin-2, A00c or mdIV morphologies from the TEM neural reconstruction. Anterior, up; dorsal view. SS04185, green; Basin-2, blue; A00C, orange; mdIV, red.

SeIN128 receives input from Basin and A00c neurons

(A) Summary of the connectivity between SeIN128 and the escape circuit. SeIN128 receives inputs from Basin-2 and A00c and provide feedback to Basin-2 and A00c. Synapse number shown next to connection arrows, where line width is proportional to synapse number. All connections in the ventral nerve cord are shown except unilateral synapses, <5 synapses, between neurons. Each polygon represents a pair of the indicated neuron and segment (segment number is shown under the neuron name). SeIN128, green; Basin-2, blue; A00c, orange; mdIV, red.

(B, C) SeIN128 is functionally downstream of Basins (B) or A00c (C). Calcium transients, ΔF/F0 traces of GCaMP6s in SeIN128 axons (black line, mean) during 610-nm optogenetic activation of Basins at various intensities. Vertical gray line represents optogenetic activation. Genotype: w; R72F11-LexA (B) or R71A10-LexA (C) /R54B01-Gal4.AD; 13xLexAop2-IVS—CsChrimson::tdTomato, 20xUAS-IVS-GCaMP6s/R46E07-Gal4.DBD.

(A) (D) A00c responses are faster and stronger than SeIN128 responses during activation of Basins. Calcium transients (black line, mean; gray line, single recording) represented by ΔF/F0 in A00c by of 610-nm optogenetic activation of Basins at various intensities. Genotype: w; R72F11- LexA/+; 13xLexAop2-IVS-CsChrimson::tdTomato, 20xUAS-IVS-GCaMP6s/R71A10-Gal4.

For (B) to (D), irradiances from left to right are 0.04, 0.1, 0.3, 0.5, and 1.4 µW/mm2. For each irradiance (n = 6), individual traces are shown with gray lines whereas the average of individuals is shown in black. The shaded gray area indicates the period of optogenetic activation (0 to 1 s).

(A) (E) The timing of the peak ΔF/F0correlated with the identity of the neurons but not the peak ΔF/F0 value. SeIN128 neurons are shown as orange dots, whereas A00c is shown as a green dot.

SeIN128 is GABAergic and negatively controls rolling

(A) Immunostaining of SeIN128 cell body (green) and GABAergic neuron (magenta). Genotype: 10xUAS-IVS-mryGFP; R54B01-Gal4.AD/13xLexAop-dsRed; R46E07-Gal4.DBD/Trojan-GAD-T2A-LexA. White triangles indicate locations of SeIN128 cell bodies. Anterior, up; dorsal view; scale bar, 10 µm.

(B) Time series of rolling probabilities of larvae with Basin activation (black), or VGAT RNAi in SS04185 and Basin activation (red). The red bar and dashed lines display the window of optogenetic stimulation eliciting larval escape responses. Shaded areas show 95% confidential intervals of rolling probabilities. Genotypes: 13xLexAop2-IVS-CsChrimson::mVenus; R72F11- LexA/+; HMS02355/+ (black); 13xLexAop2-IVS-CsChrimson::mVenus; R72F11-LexA/R54B01- Gal4.AD; HMS02355/R46E07-Gal4.DBD (red). Genotypes in (C, D) are the same as mentioned here.

(C) Binned larval rolling probabilities during first 5 s of stimulation in (A). Error bars, 95% confidence interval. n = 110, 73. Statistics: Chi-square test, χ2 = 9.34, p < 0.001.

(D) Cumulative plot of rolling duration. Statistics: Mann-Whitney test, p = 0.015, n = 55, 73.

**p < 0.01, ***p < 0.001.

Inhibition of SeIN128 prolongs rolling and delays initiation of crawling

(A) Time series of rolling probabilities of larvae with Basin activation (black), or SS04185 inhibition and Basin activation (red). Shaded regions show 95% confidential intervals of rolling probabilities. Genotypes: 13xLexAop2-IVS-CsChrimson::mVenus; R72F11-LexA/+; UAS- TeTxLC.tnt/+ (black); 13xLexAop2-IVS-CsChrimson::mVenus; R72F11-LexA/R54B01-Gal4.AD; UAS-TeTxLC.tnt/R46E07-Gal4.DBD (red). Genotypes in (B-F) are the same as mentioned here.

(B) Rolling probabilities during first 5 s of stimulation in (A). Error bars, 95% confidence interval. n = 241, 164. Statistics: Chi-square test, χ2 = 44.02, p < 0.001.

(C) A violin plot of total time spent rolling for each individual larva during stimulation. Statistics: Mann-Whitney test, p < 0.001, n = 221, 258.

(D) Cumulative plot of rolling duration. Statistics: Mann-Whitney test, p < 0.001, n = 160, 154.

(E) A violin plot of end of first rolling bout for each larva during stimulation. Statistics: Mann-Whitney test, p < 0.001, n = 160, 154.

(F) A violin plot of start of first crawling bout for each larva during stimulation. Statistics: Mann-Whitney test, p < 0.001, n = 65, 105.

**p < 0.01, ***p < 0.001.

SeIN128 sends feedback inhibition to Basins

(A) Rolling probabilities for larvae with GABAR-RNAi in their Basin neurons. From left to right, the genotypes are 20xUAS-IVS-CsChrimson::mVenus/+;; R72F11-Gal4/+ (black), 20xUAS—IVS-CsChrimson::mVenus/+;; R72F11-Gal4/UAS-HMC03388 (dark green), 20xUAS- IVS-CsChrimson::mVenus/+;; R72F11-Gal4/UAS-JF02989 (light green), 20xUAS-IVS- CsChrimson::mVenus/+;; R72F11-Gal4/UAS-HMC02975 (yellow), and 20xUAS-IVS- CsChrimson::mVenus/+;; R72F11-Gal4/UAS-HMC03643 (orange). Genotypes in (B) are the same as mentioned here. N = 320, 205, 159, 183, 182 from left to right. Statistics: Chi-square test, Bonferroni correction. GABA-B-R11 group: χ2 = 8.76, p = 0.012. GABA-B-R12 group: χ2 = 24.70, p < 0.001. GABA-B-R2 group: χ2 = 25.77, p < 0.001. GABA-A-R group: χ2 = 16.29, p < 0.001.

(B) Cumulative plot of rolling duration. Statistics: Kruskal-Wallis test: H = 69.52, p < 0.001; Bonferroni-corrected Mann-Whitney test, p < 0.001 for GABA-B-R12, GABA-B-R2, and GABA-A-R RNAi groups, n = 520, 488, 387, 582, 306 from left to right.

(C) Summary of peak ΔF/F0 in Basin axons with or without SeIN128 activation under various irradiances. Control groups shown in black are without SeIN128 activation while experimental groups shown in red are with SeIN128 activation. Statistics: Mann-Whitney test, p > 0.05 for irradiances of 0.04, 0.1, 1.4 µW/mm2; p = 0.016 for irradiance of 0.3 µW/mm2; p = 0.032 for irradiance of 0.5 µW/mm2. Genotype: 20xUAS-Syn21-opGCaMP6s, 10xUAS-Syn21- CsChrimson88::tdTomato/+; CyO/+;R72F11-Gal4/TM6 (black); 20xUAS-Syn21- opGCaMP6s,10xUAS-Syn21-CsChrimson88::tdTomato/+;CyO/R54B01-Gal4.AD;R72F11- Gal4/R46E07-Gal4.DBD (red).

**p < 0.01, ***p < 0.001.

SeIN128 inhibits rolling elicited by both Basin-2 and Basin-4 activation

(A) Binned larval rolling probabilities during the first 5 s of stimulation. Error bars, 95% confidence interval. n = 81, 119. Statistics: Chi-square test, χ2 = 35.51, p < 0.001. Genotypes: 20xUAS-IVS-CsChrimson::mVenus/+; R72F11-Gal4.AD/+; R38H09-Gal4.DBD/+ (black); 20xUAS-IVS-CsChrimson::mVenus/+; R72F11-Gal4.AD/R54B01-Gal4.AD; R38H09-Gal4.DBD/R46E07-Gal4.DBD (red). Genotypes in (B-D) are the same as mentioned here.

(B) Cumulative plot of rolling duration. Statistics: Mann-Whitney test, p = 0.0034, n = 206, 83.

(C) A violin plot of end of first rolling bout for each larva during stimulation. Statistics: Mann-Whitney test, p = 0.0047, n = 57, 38.

(D) A violin plot of start of first crawling bout for each larva during stimulation. Statistics: Mann-Whitney test, p = 0.045, n = 107, 38.

(E) Binned larval rolling probabilities during first 5 s of stimulation. Error bars, 95% confidence interval. n = 192, 213. Statistics: Chi-square test, χ2 = 64.81, p < 0.001. Genotypes: 20xUAS-IVS- CsChrimson::mVenus/+; R72F11-Gal4.AD/+; R57F07-Gal4.DBD/+ (black); 20xUAS-IVS- CsChrimson::mVenus/+; R72F11-Gal4.AD/R54B01-Gal4.AD; R57F07-Gal4.DBD/R46E07-Gal4.DBD (red). Genotypes in (F-H) are the same as mentioned here.

(F) Cumulative plot of rolling duration. Statistics: Mann-Whitney test, p = 0.032, n = 231, 71.

(G) A violin plot of end of first rolling bout for each larva during stimulation. Statistics: Mann-Whitney test, p = 0.0047, n = 129, 61.

(H) A violin plot of start of first crawling bout for each larva during stimulation. Statistics: Mann-Whitney test, p < 0.001, n = 159, 71.

**p < 0.01, ***p < 0.001.

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