Direction Selectivity in Drosophila Proprioceptors Requires the Mechanosensory Channel Tmc

Abstract

Drosophila Transmembrane channel-like (Tmc) is a protein that functions in larval proprioception. The closely related TMC1 protein is required for mammalian hearing and is a pore-forming subunit of the hair cell mechanotransduction channel. In hair cells, TMC1 is gated by small deflections of microvilli that produce tension on extracellular tip-links that connect adjacent villi. How Tmc might be gated in larval proprioceptors, which are neurons having a morphology that is completely distinct from hair cells, is unknown. Here, we have used high-speed confocal microscopy both to measure displacements of proprioceptive sensory dendrites during larval movement and to optically measure neural activity of the moving proprioceptors. Unexpectedly, the pattern of dendrite deformation for distinct neurons was unique and differed depending on the direction of locomotion: ddaE neuron dendrites were strongly curved by forward locomotion, while the dendrites of ddaD were more strongly deformed by backward locomotion. Furthermore, GCaMP6f calcium signals recorded in the proprioceptive neurons during locomotion indicated tuning to the direction of movement. ddaE showed strong activation during forward locomotion, while ddaD showed responses that were strongest during backward locomotion. Peripheral proprioceptive neurons in animals mutant for Tmc showed a near-complete loss of movement related calcium signals. As the strength of the responses of wild-type animals was correlated with dendrite curvature, we propose that Tmc channels may be activated by membrane curvature in dendrites that are exposed to strain. Our findings begin to explain how distinct cellular systems rely on a common molecular pathway for mechanosensory responses.

Keywords: Drosophila melanogaster; Tmc; behavior; mechanotransduction; microscopy; proprioception; sensory.

Figure 1.. Dendrite Deformation and GCaMP6f Activation Patterns in Larval Forward Locomotion

(A) Dendrite morphology of class I da neurons ddaE and ddaD visualized by expression of mCD8::GFP driven by2-21-GAL4. (B) Dendrite deformation pattern of ddaE and ddaD during forward locomotion. The left panel shows individual time points of a maximum-intensity projection from a volumetric time series. The right panel depicts the model of dendritic architecture reconstituted by a computer vision framework for neurite tracing from the volume shown in the left panel. ddaE and ddaD are colored in magenta and green, respectively. (C) GCaMP6f activation pattern of ddaE and ddaD during forward locomotion. The images at the nearly similar phase of segmental contraction cycle shown in the left panel of (B) are selected to show the activation of GCaMP6f. ddaE and ddaD cell bodies are marked with magenta and white circles, respectively. Arrows point to activated GCaMP6f in ddaE dendrites. Maximum-intensity projections of confocal z time series are shown in (A) and (C). Stage ofsegmental contraction cycle (Θ) and corresponding time stamp (s) are shown in (B) and (C). All images are shown as dorsal side up and anterior on the right. Scale bar, 40 μm. See also Figures S1 and S2 and Videos S1, S2, S3, and S5.

Figure 2.. Dendrite Deformation and GCaMP6f Activation Patterns in Larval Backward Locomotion

(A) Dendrite morphology of class I da neurons ddaE and ddaD visualized by expression of mCD8::GFP driven by 2-21-GAL4. (B) Dendrite deformation pattern of ddaE and ddaD during backward locomotion. The left panel shows individual time points of a maximum-intensity projection from a volumetric time series. The right panel depicts the model of dendritic architecture reconstituted by a computer vision framework for neurite tracing from the volume shown in the left panel. ddaE and ddaD are colored in magenta and green, respectively. (C) GCaMP6f activation pattern of ddaE and ddaD in backward locomotion. Representative images at the nearly similar phase of segmental contraction cycle shown in the left panel of(B) are selected to show the activation of GCaMP6f. ddaE and ddaD cell bodies are marked with magenta and white circles, respectively. Arrows point to activated GCaMP6f in ddaD dendrites. Maximum-intensity projections of confocal z time series are shown in (A) and (C). Stage of segmental contraction cycle (Θ) and corresponding time stamp (s) are shown in (B) and (C). All images are show as dorsal side up and anterior on the right. Scale bar, 40 μm. See also Figures S1 and S2 and Videos S1, S2, S3, and S5.

Figure 3.. Preferential Activation of Class I da Neurons ddaE and ddaD during Larval Forward and Backward Locomotion

(A) Comparison of peak ΔF/F in ddaE and ddaD during forward and backward locomotion. ***p < 0.001, Student’s t test. n = 54 (ddaE) and n = 51(ddaD) in forward movements; n = 36 (ddaE) and n = 38 (ddaD) in backward movements. Error bars indicate the SEM. (B) Trace of GCaMP6f ΔF/F for ddaE and ddaD neuron of the same segment during three consecutive waves of backward and three waves of forward locomotion. The bottom frame shows the speed of the ddaE neuron movement throughout the time series. (C and D) Comparison of mean ΔF/F for ddaE and ddaD in the segmental contraction cycle during forward (C; n = 25 for both ddaE and ddaD) and backward (D; n = 17 for ddaE and n = 21 for ddaD) locomotion. (E and F) Comparison of mean ΔF/F in ddaE (E) and ddaD (F) in forward and backward locomotion in a segmental contraction cycle. These two panels used re-grouped data from (C) and (D). (C-F) Position along the radial axis represents ΔF/F (percent change). The phase angle of the segmental contraction cycle depicts the distance between neurons of adjacent segments, which is decreasing from 0–180 degrees and increasing from 180–360 degrees. Darker colored line indicates mean ΔF/F, and the lighter colored shading denotes the SEM. n indicates the number of neurons examined. Neuronal activities were recorded from nine animals for forward and 11 animals for backward locomotion. See also Figure S2 and Videos S4 and S5.

Figure 4.. Relationship between Neuron Activity and Locomotion in Freely Moving Larvae

Representative traces from simultaneous recordings of ddaE and ddaD during larval locomotion obtained using a two-photon tracking microscope. (A) Activity (top) and instantaneous speed (bottom) during a 400 s period completed with backward and forward locomotion bouts. Shading indicates behavioral state (magenta, forward crawling; green, backward crawling; white, not crawling). Activity (red, ddaE; blue, ddaD) is measured as a ratio of GCaMP6f fluorescence to mCherry fluorescence divided by the baseline ratio. (B) Activity and velocity aligned to time within a bout, averaged across many bouts. A bout is defined to be a period of rapid movement. Velocity is defined as the movement of the neuron relative to the position of the tail. Forward bouts (magenta) have positive velocity (away from the tail), and backward bouts (green) have negative velocity. The time axis is aligned so that the fastest movement is at t = 0. No measurements of activity were used to detect bouts or align the time axis. Shaded regions represent mean ± SEM. The left panels show ddaE, and the right panels show ddaD (n = 182 forward bouts, both neurons; n = 224 backward bouts, both neurons). (C) Activation of dorsal neurons during body bends. The box-and-whisker plot shows the median, 25^th and 75^th percentiles, range of data, and outliers for activity at moment of maximum body bend, as determined by visual inspection of raw behavioral video. When a group is composed of fewer than ten data points, all points are shown. Ipsi indicates a bend toward the neuron (body wall is convex at location of neuron); contra indicates a bend away from the neuron (body wall is concave at location of neuron). Groupings are as follows: all, all sampled body bends of the given type; forward, all sampled body bends in which the larva was crawling forward before and after the bend; backward, all sampled body bends in which the larva was crawling backward before and after the bend; and simultaneous recording, subset of bends measured when ddaD and ddaE were tracked simultaneously. ***p < 0.001 rejects the hypothesis that both groups are drawn from the same random normal distribution, using a two-sample t test. See also Figure S4, Table S1, and Video S6.

Figure 5.. Comparison of Dendrite Curvature and GCaMP6f Activation during Larval Locomotion

(A and B) Dendrite curvature of ddaE and ddaD neurons plotted versus phase of the segmental contraction cycle. Position along the radial axis represents absolute value of curvature. The phase angle of the segmental contraction cycle depicts the distance between adjacent neurons, which is decreasing from 0–180 degrees and increasing from 180–360 degrees. Solid lines represent the mean ofcurvature (n = 21 from seven animals for forward locomotion; n = 10 from six animals for backward locomotion), and colored shading represents the SEM. Overall, the curvature of ddaE is larger than that of ddaD during forward locomotion (A) and vice versa during backward locomotion (B). (C and D) Normalized dendrite curvature and GCaMP6f signals plotted on the same polar coordinates show that dendrite curvature precedes the rise in GCaMP signal and decreasing of curvature anticipates the decline of GCaMP6f signals for ddaE during forward locomotion (C) and ddaD during backward locomotion (D). Solid lines represent the mean of normalized curvature or GCaMP6f (curvature: n = 11 from four animals and n = 8 from four animals forforward and backward locomotion, respectively; GCaMP6f: n = 12 from five animals and n = 9 from five animals for forward and backward locomotion, respectively). Colored shading represents the SEM. n indicates the number of neurons examined. See also Figures S2 and S3 and Video S8.

Figure 6.. Representative Still Images from GCaMP6f Recordings on Tmc Mutant and Tmc Rescue Larvae

Maximum-intensity projections of confocal z time series are shown in (A)–(D). In all panels, the ddaE cell soma is encircled in magenta, and ddaD is encircled in white. Each panel indicates the value for theta and the time stamp for the relevant image. (A and B) Still images of a larva undergoing forward locomotion; note that there is increased fluorescence intensity in ddaE soma, and the dendrites light up in ddaE rescue larva compared with Tmc¹ mutant. (C and D) Still images of a larva undergoing backward locomotion; note that there is increased fluorescence intensity in ddaD soma, and the dendrites light up in ddaD rescue larva compared with Tmc¹ mutant. White arrows point to the activated dendrites. In (A) and (C), the mutant genotype is w;UAS-GCaMP6f; 2-21-GAL4 Tmc¹. In (B) and (D), the rescue genotype is w;UAS-GCaMP6f UAS-Tmc; 2-21-GAL4 Tmc¹. Scale bar, 40 μm. See also Figure S2 and Video S7.

Figure 7.. Tmc Is Required for Idiothetic Activation of ddaE and ddaD in Larval Forward and Backward Locomotion

(A) Comparison of peak ΔF/F (percentage) in ddaE and ddaD neurons in Tmc wild-type control (n = 54 for ddaE and n = 51 for ddaD), mutant (n = 36 for ddaE and n = 43 for ddaD), and rescue (n = 19 for both ddaE and ddaD) larvae in larval forward locomotion. (B) Comparison of peak ΔF/F (%) in ddaE and ddaD in Tmc wild-type control (n = 36 for ddaE and n = 38 for ddaD), mutant (n = 35), and rescue (n = 20) larvae in larval backward locomotion. (C) Comparison of mean ΔF/F (%) in ddaE of Tmc wild-type control (n = 25), mutant (n = 17), and rescue (n = 16) larvae in larval forward locomotion during the segmental contraction cycle. (D) Comparison of mean ΔF/F (%) in ddaD of Tmc wild-type control (n = 25), mutant (n = 17), and rescue (n = 15) larvae in larval forward locomotion in the segmental contraction cycle. (E) Comparison of mean ΔF/F (%) in ddaE of Tmc wild-type control (n = 17), mutant (n = 26), and rescue (n = 18) larvae in larval backward locomotion in a muscle contraction cycle. (F) Comparison of mean ΔF/F (%) in ddaD of Tmc wild-type control (n = 21), mutant (n = 26), and rescue (n = 18) larvae in larval backward locomotion in a muscle contraction cycle. Genotypes are as follows: Tmc wild-type control is w; UAS-GCaMP6f;2-21- GAL4. Tmc mutantis w; UAS-GCaMP6f;2-21-GAL4 Tmc¹. Tmc rescue is w;UAS-GCaMP6f UAS-Tmc; 2-21-GAL4 Tmc¹. ** p < 0.01, ***p < 0.001, Student’s t test. Error bars in (A) and (B) indicate the SEM. The position of the radial axis in (C)-(F) represents DF/F (percentage). Solid colored lines represent the mean values, and colored shading denotes the SEM. n indicates the number of neurons examined. Neuronal activities in forward movements were recorded from nine, ten, and seven animals for Tmc wild-type control, Tmc mutant, and Tmc rescue, respectively. Neuronal activities in backward movements were recorded from eleven, eleven, and nine animals for Tmc wildtype control, Tmc mutant, and Tmc rescue, respectively. See also Figures S2 and S5 and Video S7