Supplemental Information. Characterization of Proprioceptive System. Dynamics in Behaving Drosophila Larvae. Using High-Speed Volumetric Microscopy

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1 urrent Biology, Volume 29 Supplemental Information haracterization of roprioceptive System Dynamics in Behaving Drosophila Larvae Using High-Speed Volumetric Microscopy Rebecca D. Vaadia, Wenze Li, Venkatakaushik Voleti, diti Singhania, Elizabeth M.. Hillman, and Wesley B. Grueber

2 , raw signals inter-cell distance.5 F/F GF control, raw signals GF control, green/red ratios GF GF- ratio inter-cell distance B inter-cell distance B F/F, green/red ratios.5 - ratio inter-cell distance.8 N.S. *** GF GF / ratio Resting p>.1 ontraction 6f / ratio Resting p<.1 ontraction.5 D.5 neurons tracked in GF control Figure S1. Ratiometrically measured calcium dynamics properly control for motion artifacts, related to Figure 2. () hange in fluorescence from baseline ( F/F ) in 6f (green) and (red) during crawling in vpda neuron somas. Segment contraction is depicted with inter-cell distance (dashed lines). ( ) hange in ratio of 6f to fluorescence (, blue). Increases can be seen during segment contraction. (B) hange in fluorescence from baseline ( F/F ) in GF (green) and (red) during crawling in vpda neuron somas. Segment contraction is depicted with inter-cell distance (dashed lines). (B ) hange in ratio of GF to fluorescence (, blue). No increase is associated with segment contraction. () omparison of GF- and 6f-tdtomato ratios between resting and contraction phases (see methods). For GF- analysis, n= 2 animals, 7 cells, 14 events, for 6f-tdtomato analysis, n=3 animals, 22 cells, 26 events. Note that there is no difference between GF- ratios in the resting versus contraction phases, while there is a significant increase in 6f- ratios during contraction (p<.1, as measured by two-tailed t-test). (D) SE imaging of 41-Gal4, 2XUS md8::gf, US-D4- animals during forward crawling, Ventral side. Imaging shows vpda neurons. GF channel is shown. osterior is to the left. Images are shown on a square root grayscale to reduce dynamic range for visualization of both cell bodies and dendrites. Scale bar=1µm.

3 Inter-cell dorsal class I neurons 41-Gal4, 2XUS-IVS-6f (x2), US-D4- D dmd1 dbd vpda calcium signal calcium signal dmd1calcium signal dbd calcium signal calcium signal nterior inter-cell distance a t = s t =.5 s t = 1. s t = 1.5 s t = 2. s all dorsal proprioceptors 1D5-Gal4, 2XUS-IVS-6f (x2), US-D4- B dbd dmd1 E.8.8 & dmd B B neurons 1129-Gal4, 2XUS-IVS-6f (x2), US-D4- t= s t=.7 s t=1.1 s t=1.4 s t=2 s dbd tdtomato c tdtomato t= s t=.5 s t=1 s t=1.5 s t=2 s Figure S2. Examples of SE imaging of dynamics, related to Figure 3. osterior is to the left for all images. For (-), images show representative SE MIs over a 35-9 µm depth range from a 16-2 deep volume (to exclude

4 gut autofluorescence, square root grayscale). Dashed box indicates neurons examined in time lapse sequences below, shown for both and channels. (- ) SE imaging of 41-Gal4, 2XUS-IVS-6f (x2), US-D4- larva. See Video S5, first section. rrowheads indicate increases in dendritic 6f, arrows indicate increases in axon bundle (containing both and axons). Note dendrites are active before. (B-B ) SE imaging of GMR1D5-Gal4, 2XUS-IVS-6f (x2), US-D4- larva. See Video S5, second section. Orange arrowhead marks dbd cell body, pink arrowhead marks dmd1 cell body. (- ) SE imaging of 1129-Gal4, 2XUS-IVS-6f (x2), US-D4- larva. See Video S5, third section. (D) Schematic of larval proprioceptive system. (E) Examples of single cell calcium activity dynamics during forward crawling. The calcium response is plotted in solid lines (quantified as ). The distance between the measured neuron and the posterior neuron ( distance) is plotted in black dashed lines. The distance between the measured neuron and the anterior neuron (anterior intercell distance) is also plotted in brown dashed lines on the plot, since this is a better proxy for dendrite folding.

5 t=4.5s 111 µm 116 µm B over glass t=6s 88 µm 116 µm Glass slide Standard ompressed t=8s 82 µm 116 µm Dendrite length alcium signal t=1s 96 µm 114 µm 1.5 t=11.5s 114 µm 113 µm % change in dendrite length Figure S3. Sensory activity does not occur in the absence of dendritic folding, related to Figure 3. () Time lapse of SE imaging of dorsal class I neurons labeled with 41-Gal4, 2XUS-IVS-6f (x2), US-D4-, in a compressed preparation, which prevents dendritic folding in (see (B) and methods). TdTomato channel is shown to depict dendrite dynamics. Larva is 3 rd instar. osterior is to the left. (MI) over a 5µm depth range from a 16µm deep volume. ( ) Tracing of time lapse data shown in (), posterior cells. is blue and is green. Dotted lines and shaded areas represent extent of arbor in a relaxed segment. Measurements represent dendrite length (µm), a measure of dendrite folding. rrows denote frames with dendrite folding. Note that dendrites fold, but not. (B) Schematic of compressed preparation. () alcium responses (, solid lines) and % change in dendrite length (dotted lines) in a compressed preparation of (blue) and (green) during segment contraction. ctivity correlates with dendrite folding. Scale bar=1µm.