Drosophila mechanical nociceptors preferentially sense localized poking

  1. Zhen Liu
  2. Meng-Hua Wu
  3. Qi-Xuan Wang
  4. Shao-Zhen Lin
  5. Xi-Qiao Feng
  6. Bo Li
  7. Xin Liang  Is a corresponding author
  1. School of Life Sciences, Tsinghua University, China
  2. Institute of Biomechanics and Medical Engineering, Department of Engineering Mechanics, Tsinghua University, China

Abstract

Mechanical nociception is an evolutionarily conserved sensory process required for the survival of living organisms. Previous studies have revealed much about the neural circuits and sensory molecules in mechanical nociception, but the cellular mechanisms adopted by nociceptors in force detection remain elusive. To address this issue, we study the mechanosensation of a fly larval nociceptor (class IV da neurons, c4da) using a customized mechanical device. We find that c4da are sensitive to mN-scale forces and make uniform responses to the forces applied at different dendritic regions. Moreover, c4da showed a greater sensitivity to localized forces, consistent with them being able to detect the poking of sharp objects, such as wasp ovipositor. Further analysis reveals that high morphological complexity, mechanosensitivity to lateral tension and possibly also active signal propagation in dendrites contribute to the sensory features of c4da. In particular, we discover that Piezo and Ppk1/Ppk26, two key mechanosensory molecules, make differential but additive contributions to the mechanosensitivity of c4da. In all, our results provide updates into understanding how c4da process mechanical signals at the cellular level and reveal the contributions of key molecules.

Editor's evaluation

Liu et al. present a fascinating study that significantly advances fundamental knowledge about the molecular and cellular pathways underlying mechanical nociception. The use of a combination of fine biophysics and neurogenetics provides unprecedented insight into mechanosensory functions in an intact tissue environment of the Drosophila larva. The results of this work have strong implications for our understanding of the sensation of acute pain.

https://doi.org/10.7554/eLife.76574.sa0

eLife digest

Being able to sense harm is essential for survival. Animals have to be able to tell the difference between a gentle touch and a dangerous pressure. They do this using nerve cells called mechanical nociceptors which switch on when the body feels a potentially painful pressure, such as a sharp object poking the skin. Once activated, the nerves send outputs to other parts of the central nervous system which coordinate the motions needed to escape the source of the pain.

One popular model to understand harm-sensing is the larvae of fruit flies which automatically roll back and forth when they sense the pointy sting of a wasp. This process is initiated by sensory nerve cells called class IV dendritic arborization neurons (or c4da for short) which sit under the fly’s skin. However, it is still not fully understood how these mechanical nociceptors detect the poking forces of the wasp’s tail.

To investigate, Liu, Wu et al. built a device that could poke sections of fly larvae under a microscope so they could see how different types of pressure affected the activity and shape of c4da cells. This revealed that c4da nerves were most sensitive to sharp objects that illicit a more localized force, which may explain why these cells are so good at responding to wasp attacks.

Further analysis showed that this sensitivity was due to the high number of branches, or dendrites, protruding from the body of c4da nerves. Liu, Wu et al. discovered that the dendrites were coated in a touch-sensitive protein that can sense and amplify both squashing and pulling, resulting in a signal that activates c4da nerves to send outputs to other parts of the central nervous system. This mechanism increases the likelihood that a c4da cell will detect a mechanical pressure even if it is far away from the body of the nerve.

These findings shed light on how sensory cells like c4da are optimized to carry out specific roles. This could be important for understanding other nerve systems which sense mechanical pressure, such as those involved in touch or auditory processes. However, further work is needed to see whether the molecules and mechanism identified by Liu, Wu et al. are also present in humans.

Introduction

Mechanosensation is a physiological process that transduces mechanical stimuli into neural signals (Chalfie, 2009). It underlies the perception of gentle touch, sound, acceleration and noxious force. To cope with manifold environmental forces, mechanoreceptor cells are diversified (Lumpkin et al., 2010; Zimmerman et al., 2014). Among different types of mechanoreceptor cells, those activated by noxious forces, that is mechanical nociceptors, are of particular importance because they are one of the sensory organs that are essential for the survival of living organisms (Lumpkin et al., 2010; Tracey, 2017).

Much effort has been made to understand mechanical nociception in several model organisms. In mammals, free nerve endings of nociceptive neurons penetrate the keratinocyte layer of skin and serve as the primary nociceptors. The axons of these neurons output to neural circuits in the spinal cord, which then transmit pain signals to local interneurons or up to the brain to initiate neural reflexes (Tracey, 2017). At the molecular level, transient receptor potential (TRP) channels, Piezo and other channels are found to be involved in mechanical nociception (Kwan et al., 2006; Tracey, 2017; Murthy et al., 2018). In invertebrates (such as worms and flies), mechanical nociception has also been extensively studied (Chatzigeorgiou et al., 2010; Tracey, 2017). A widely used model is fly larval mechanical nociception mediated by class IV dendritic arborization neurons (c4da). C4da are polymodal nociceptors that can be activated by light, thermal and mechanical stimuli (Hwang et al., 2007; Xiang et al., 2010; Kim et al., 2012; Terada et al., 2016). The axon of c4da synapses to several targets in the ventral nerve cord, which then output signals to trigger the rolling behavior, a stereotyped locomotion in escaping from nociceptive stimuli (Grueber et al., 2007; Ohyama et al., 2015; Gerhard et al., 2017; Hu et al., 2017; Kaneko et al., 2017; Takagi et al., 2017; Yoshino et al., 2017; Burgos et al., 2018). Furthermore, two parallel molecular pathways in c4da, one mediated by Ppk1/Ppk26 and the other by Piezo, are proposed to be responsible for the behavioral responses in mechanical but not thermal nociception (Kim et al., 2012; Zhong et al., 2010; Gorczyca et al., 2014; Guo et al., 2014).

An intriguing question is whether c4da neurons are optimized at the cellular level for its function as mechanical nociceptor? A recent study shows that the nociceptive response of c4da to thermal stimuli depends on the dendritic calcium influx through two TRPA channels and the L-type voltage-gated calcium channel, suggesting the presence of neuronal processing of heat-induced responses (Terada et al., 2016). In a more general sense, it is also intriguing how mechanoreceptor cells are optimized for their specific stimuli. An interesting study reports unique neuronal mechanisms in the mechanosensory neurons in tactile-foraging birds (Schneider et al., 2014), also demonstrating the presence of cellular mechanisms that facilitate the response to specific stimuli. So far, how c4da process mechanical inputs in nociception at the neuronal level remains unclear.

To address this issue, we build a ‘mechanical-optical’ recording system that is able to measure the in vivo sensory response of c4da to controlled forces. We find that c4da are sensitive to mN-scale forces and use the entire dendritic territory (including the non-neuronal part of the epidermis) as the force-receptive field. In particular, c4da show greater responses to smaller probes, suggesting their sensory preference to localized forces. Further analysis reveals the cellular mechanisms that facilitate the sensory features of c4da and the contributions of key molecules. In all, these findings update the current model for the c4da-mediated mechanical nociception and provide novel insights into how mechano-nociceptor cells process force signals at the cellular level.

Results

Mechanical recording and the ‘sphere-surface’ contact model

We set out by building a mechanical device that was able to exert and measure compressive forces onto fly larval fillets (Figure 1A). This device contained three parts: a piezo stack actuator, a metal beam coupled with a strain gauge and a glass force probe (Figure 1A and Figure 1—figure supplement 1). The whole device was mounted on the working stage of an inverted spinning-disk confocal microscope (Figure 1A and Figure 1—figure supplement 1). To record the force-evoked response of sensory neurons, freshly prepared larval fillet was spread and mounted on a polydimethylsiloxane (PDMS) pad (thickness: 1 mm) with the exterior surface of the cuticle accessible to the force probe and the interior surface visible to the confocal microscope (Figure 1A). When the force probe, driven by the piezo actuator, delivered compressive forces onto the larval fillet, the strain gauge converted the deflection of the metal beam into an electrical signal. The electrical signal could be translated into a force signal using the calibration curve (Figure 1B–C, see Materials and methods). By making spherical probes in different diameters (Figure 1D), we can study how mechanoreceptor cells respond to the probes of different sizes. In the meantime, we also monitored the position of force probes, dendritic morphology (membrane marker) and neuronal response (GCaMP6s) (Figure 1E).

Figure 1 with 1 supplement see all
The 'mechanical-optical' recording system.

(A) The cartoon schematics for the ‘mechanical-optical’ recording system (left) and the contact model between a spherical probe and the larval fillet (right). Vin was the driving voltage of the piezo actuator. Vout was the readout voltage of the strain gauge. (B) The force calibration curve of the strain gauge. The data points were mean values from three measurements. (C) Representative traces for the input (driving voltage of the piezo actuator, upper panel) and the mechanical output (stimulating force, lower panel) of the recording system. (D) The scanning electron microscopy images of glass force probes of different sizes. Scale bar, 100 μm. (E) Left panel: a representative image of c4da (membrane, red channel), Middle panel: a bright field image of larval fillet. Right panel: two representative images showing the GCaMP6s signals in c4da at resting (left) and exciting (right) conditions. Yellow arrowhead: soma. White arrowhead: force probe. Scale bar, 100 μm. Genotype: uas-cd4-tdTom; ppk-gal4/20×uas-ivs-gcamp6s. (F) The mechanical schematics of the 'sphere-surface' model. Note that the contact interface had a spherical crown shape. The definitions of all model parameters were described in Materials and methods. (G) The cartoons schematics showing the relationship among indentation depth of the force probe (d) deflection of the beam (B) and stepping distance of the piezo (D). (H) The plots of D (red) and B (blue) versus the driving voltage of the piezo (Vin). (I) The comparison between the calculated (red and blue) and experimentally measured (black) contact forces. In our calculations, the maximal (4 MPa, red) and minimal (1.6 MPa, blue) values of the elastic modules of PDMS were from the literatures (Johnston et al., 2014; Vlassov et al., 2018). Data were presented as mean ± std (n=9 assays).

The contact mechanics between the force probe and fillet can be approximated using a classic ‘sphere-surface’ contact model (Johnson, 1985), which would allow us to separate the effects of different parameters, such as pressure and contact area. Note that to guarantee the accuracy of the contact model, the indentation depth (d) needs to be smaller than the radius of the spherical probe (r) (Figure 1F). This prerequisite is acceptable for our measurements because our pilot experiments showed that when the step distance (D) of the piezo actuator was greater than r, the force probe often penetrated the cuticle and caused tissue damage. The small deformation constraint would naturally minimize tissue damage (Figure 1—figure supplement 1) and allow us to focus on mechanical nociception (i.e. no major interference from chemo-nociception due to tissue damage). To confirm if the modeling approximation is valid, we calculated contact force (fmodel) from d (Equations 1 and 2, see Materials and methods), which could be obtained from the measured values of D and beam deflection (B) (Equation 3, see Materials and methods). fmodel was then compared to the experimentally measured forces (fexp) (Figure 1G–I). As shown in Figure 1I, the relationship between fexp and d fell in the range of model calculations, demonstrating that the model approximation is valid. Note that different values for the elastic modulus of PDMS (EPDMS) (Johnston et al., 2014; Vlassov et al., 2018) were used to calculate the upper and lower bounds of contact forces (Figure 1I).

C4da are sensitive to mN-scale forces and more sensitive to small probes

Using the customized mechanical device, we applied poking forces onto c4da at the positions of about 100 μm away from the soma (i.e. the ‘proximal’ region, denoted as p in the plots) (Figure 2A and Video 1). By simultaneously monitoring the position of the probes and neuronal morphology, we ensured that the probe made direct compression onto the dendrites. Force titration experiments using a 60 μm probe showed a half-activation force (f50) of ~3 mN and a full-activation force (f90) of ~4 mN, while those using a 30 μm probe showed a f50 of ~0.7 mN and a f90 of ~1.5 mN (Figure 2B–C). This observation suggests that the mechanosensitivity of c4da is dependent on probe size. To explore the role of the entire dendritic field in force detection, we compressed the dendrites of c4da at the positions of about 200 μm away from the soma (i.e. the ‘distal’ region, denoted as d in the figures) (Figure 2D). When the saturation force (4 mN) of the 60 μm probe was used, the responses of c4da were not significantly different from those to the proximal stimuli (Figure 2E). In the case of using a 30 μm probe and applying the saturation force (1.5 mN), the responses of c4da were to some extent weaker than those to the proximal stimuli, but most of the neurons still made clear responses (Figure 2E). Therefore, our results showed that c4da could respond to the forces applied onto any part of their dendritic arbors.

Figure 2 with 3 supplements see all
The mechanosensory features of c4da.

(A) A representative image of c4da. The forces were applied at about 100 μm from the soma, i.e. along the green dashed circle. The representative force application points were marked using the filled circles (Green: 30 μm probe. Red: 60 μm probe). Genotype: uas-cd4-tdtom; ppk-gal4. (B) Representative responses of c4da (ΔF/F0, i.e. the change in calcium signal in the soma, unless otherwise stated hereinafter) to mN-scale forces delivered using the 30 μm (green) and 60 μm (red) probes. The black arrowhead indicated the defocused period of the soma caused by the stimulating force (2 s). Genotype: ppk-gal4/+; ppk-cd4-tdtom/20×uas-ivs-gcamp6s. (C) The force-response (ΔF/F0) plots of c4da (n=12 cells). The dashed lines were Boltzmann fitting. (D) The schematic showing the force application points (filled circles, green: 30 μm probe, red: 60 μm probe) of different stimuli. The dashed concentric circles were 100 and 200 μm in radius, respectively. (p) proximal dendrite. (d) distal dendrite. d-off: the ‘dendrite-off’ region. (E) The responses of c4da (ΔF/F0) to the proximal and distal stimuli using the 30 (1.5 mN) and 60 μm (4 mN) probes. Mann Whitney U test was used. *: p<0.05. ns: no significance. (F) The schematic showing the stimuli (filled circles, green: 30 μm probe, red: 60 μm probe, orange: 100 μm probe, blue: 200 μm probe) delivered using the probes of different sizes. The dashed circle was 200 μm in radius. (G) The responses of c4da (ΔF/F0, n=10 cells) to the forces applied on the distal dendrites using the probes of different sizes. (H) The plots of the responses of c4da (the same dataset as panel (G)) to distal stimuli versus central pressure (P0) (left panel) and contact areas (Ac) (right panel), respectively. Also see Figure 2—figure supplement 2 for the plots of the responses versus the pressures at other positions (Px μm). (I) The cartoon schematics of the modified behavior assay for mechanical nociception. VFF: Von Frey fiber, FP: force probe. (J) The behavioral responses of wild-type larvae to the mechanical poking stimuli using the probes of different sizes. The experiments were performed three times and the total numbers of larvae used for each type of probe were as following: 30 μm probe (n=92), 60 μm probe (n=67), 100 μm probe (n=69), 200 μm probe (n=70). One-way ANOVA test was used. **: p<0.01. ns: no significance. In panels (A), (D) and (F), scale bar: 100 μm. Asterisk: the soma. In panels (C), (G) and (H), data were presented as mean ± sem. In panel (E) and (J), data were presented as mean ± std and the numbers of cells were indicated below the scattered data points.

Video 1
The representative response of a c4da cell to the 4 mN force stimulus.

The asterisk indicated the force application point and the white arrowhead indicated the soma.

To further test the validity of this assay, we performed the same assay on c1da (class I da neuron) and c3da (class III da neuron). C1da showed no responses to compressive forces (Figure 2—figure supplement 1), consistent with their function being a proprioceptor rather than a tactile receptor (Hughes and Thomas, 2007; Guo et al., 2016; He et al., 2019). C3da showed a much smaller threshold of activation, suggesting a higher sensitivity in detecting tactile forces (using both 30 and 60 μm probes). This is consistent with their known function as a larval gentle touch receptor (Figure 2—figure supplement 1; Tsubouchi et al., 2012; Yan et al., 2013).

The observation of different responses of c4da to the 30 and 60 μm probes suggests that c4da are more sensitive to smaller probes. To test this idea, we stimulated c4da using probes of different diameters (30, 60, 100, and 200 μm) at the distal regions (Figure 2F). We found that c4da made stronger responses (ΔF/F0) to smaller probes at the same force (Figure 2G). In addition, the slope of the force-ΔF/F0 curve increased as the probe became smaller (Figure 2G), reflecting an enhanced mechanosensitivity to the change in stimulating force. To further understand this result, we calculated central pressure (P0) and contact area (Ac) based on the ‘sphere-surface’ model (Equations 4–7, see Materials and methods), and then plotted the responses against these two parameters separately (Figure 2H). In the P0F/F0 plots (left panel in Figure 2H), we found that increasing contact area decreased the threshold of cell activation but had only a minor impact on the slopes of the curves, suggesting that c4da have a robust sensitivity to the change in pressure. Similar observations were obtained when the pressures at other positions (Px μm) were plotted against the responses (ΔF/F0) (Figure 2—figure supplement 2). In the AcF/F0 plots (right panel in Figure 2H), when the contact area was the same, higher pressure led to stronger responses (Figure 2H), also consistent with c4da being a pressure sensor. The comparison of the ΔP (the change of pressure) and ΔAc (the change of contact area) of different probes showed that for a given change of forces, the smaller probes caused a smaller ΔAc but a greater ΔP in comparison to the larger probes (Figure 2H), explaining the higher sensitivity of c4da to the smaller probes.

To further understand how the sensory preference of c4da to localized poking contributes to the behavioral response of larvae in nociception, we performed the behavior assay of mechanical nociception using a modified Von Frey fiber. To test the effect of different probe sizes, we attached the glass probes (shorter than 1 mm) of different diameters (from 30 to 200 μm) to the end of a Von Frey fiber (Figure 2I). Using these modified fibers, we were able to deliver poking forces of 15–20 mN to fly larvae. Note that mean value and variation of the stimulating forces were independent on the probe size (Figure 2—figure supplement 3). The defensive rolling behaviors were recorded and scored to reflect the sensory response of c4da. We found that fly larvae showed stronger behavioral responses to smaller probes (Figure 2J), in agreement with our observations on the cellular level.

In summary, our results demonstrate that c4da are sensitive to mN-scale compressive forces applied onto their dendrites. In particular, c4da are more sensitive to smaller probes, consistent with c4da acting as a larval nociceptor to sense mechanical poking or sting from sharp objects, for example the wasp ovipositor (Hwang et al., 2007; Robertson et al., 2013; Cerkvenik et al., 2017).

Morphological complexity likely contributes to the sensory features of c4da

Having characterized the mechanosensory responses of c4da, we wondered what could be the cellular mechanisms underlying the sensory preference of c4da to small probes. The key thing is to ensure that an adequate number of mechanosensory molecules can be activated upon a localized force. Intuitively, a densely distributed dendritic arbor would be helpful. We tested this hypothesis by studying the sensory responses of a knock-down mutant of cut (cti), a key transcription factor underlying the high morphological complexity of c4da (Grueber et al., 2003; Jinushi-Nakao et al., 2007).

C4da-cti neurons showed a sparser dendritic morphology than c4da-wt (Figure 3A). Modified Sholl analysis showed that the dendritic density of c4da-wt was uniform across almost the entire dendritic field, while that of c4da-cti was similar to c4da-wt in the proximal region but rapidly decreased towards the distal region (Figure 3B). In comparison to c4da-wt or c4da-scrambled RNAi, c4da-cti showed a similar response to the stimuli on the proximal dendrites, but a significantly weaker response to those on the distal dendrites (Figure 3C). We noted that the overall responses of c4da-cti to different probes were generally reduced and in particular, the responses to the 30 μm probe were reduced to the greatest extent (Figure 3D). Consistent with the cellular observations, the defensive rolling behaviors of c4da-cti in response to the mechanical poking of small probes (30 and 60 μm) were largely weakened in comparison to those of c4da-wt, while those to large probes (e.g. 100 μm) showed nearly no change (Figure 3E). Based on these observations, we conclude that the sensory preference of c4da to small probes was lost in c4da-cti.

Figure 3 with 1 supplement see all
The contribution of dendritic morphology to the sensory features of c4da.

(A) The schematic showing the force application points (filled circles, green: 30 μm probe, red: 60 μm probe, orange: 100 μm probe) on c4da-cti. The dashed concentric circles were 100 and 200 μm in radius, respectively. (p) proximal dendrite. (d) distal dendrite. d-off: the ’dendrite-off’ region. Asterisk: the soma. Scale bar, 100 μm. Genotype: ppk-gal4/+, ppk-cd4-tdtom/uas-cti. (B) Modified Sholl analysis on the morphology of c4da-wt and c4da-cti. Note that there was a broad region in c4da-wt (red bar) in which the dendritic density was nearly constant. The shadow areas represented standard deviations. n=5 cells for each genotype. The black arrowhead indicated the regions of proximal dendrites. (C) The responses of c4da-ctiF/F0) to the force stimuli (4 mN) applied onto the proximal and distal dendrites using a 60 μm probe. The numbers of cells were indicated below the scattered data points. Mann Whitney U test was used. *: p<0.05. ns: no significance. cti: ppk-gal4/20×uas-ivs-gcamp6s, ppk-cd4-tdtom/uas-cti. scrambledi: ppk-gal4/20×uas-ivs-gcamp6s, ppk-cd4-tdtom/uas-scrambledi. (D) The responses of c4da-ctiF/F0) to the forces applied on the distal dendrites using the probes of different sizes. Data were presented as mean ± sem (n=10 cells). (E) The behavioral responses of cti larvae to mechanical poking using the probes of different sizes. wt: w1118. ppk-gal4: ppk-gal4; +/+. uas-cti: +/+; uas-cti. cti: ppk-gal4/+; uas-cti/+. ppk-gal4 larvae: 30 μm probe (n=63 larvae from three experiments), 60 μm probe (n=60 larvae from three experiments) and 100 μm probe (n=68 larvae from three experiments). uas-cti larvae: 30 μm probe (n=68 larvae from three experiments), 60 μm probe (n=72 larvae from three experiments) and 100 μm probe (n=63 larvae from three experiments). cti larvae: 30 μm probe (n=97 larvae from four experiments), 60 μm probe (n=91 larvae from four experiments) and 100 μm probe (n=91 larvae from four experiments). One-way ANOVA test was used. **: p<0.01. ns: no significance. In panels (C), (D) and (E), the corresponding data from c4da-wt were provided for comparison. In panel (C) and (E), data were presented as mean ± std.

Because Cut is a transcription factor, the reduction of its expression level might lead to other changes in addition to the disrupted morphology. To explore the effects of these potential factors, we performed several control experiments. First, we checked the expression level and subcellular localization of two mechanosensory molecules, that is Piezo and Ppk1/Ppk26, in c4da-cti. They showed similar expression and localization as in c4da-wt (Figure 3—figure supplement 1). Second, the changes in dendritic cytoskeleton might indirectly affect mechanosensation by altering the mechanical homeostasis of the dendrites, so we checked the dendritic signals of F-actin and microtubules. No significant difference was found (Figure 3—figure supplement 1). These two observations suggest that although the reduction in the expression level of Cut decreases the number of dendritic branches, the expression and localization of mechanosensory molecules and cytoskeletal elements are nearly unchanged in the existing dendrites of c4da-cti. Third, we also performed the same set of mechanical recording experiments on c3da-wt, in which there is no additional manipulation at the genetic level, but the dendritic density differs from that of c4da-wt in the way as in the case of c4da-cti (Figure 2—figure supplement 1). The responses of c3da-wt to the distal stimuli were significantly weaker than those to the proximal stimuli (Figure 2—figure supplement 1). In addition, no preference to small probes was found in c3da-wt (Figure 2—figure supplement 1). These sensory features were markedly different from those in c4da-wt, but similar to those in c4da-cti. In all, our observations are consistent with the dendritic morphology making a direct contribution in supporting the sensory features of c4da. Note that the potential contributions of other unknown factors cannot be absolutely excluded (see Discussion).

Mechanosensitivity to lateral tension expands the force-receptive field

An unexpected finding in studying c4da-cti, primarily due to the sparse dendritic morphology, was that when the force probe was not directly placed on a dendrite but on an adjacent region without any dendrite (i.e. the ‘dendrite-off’ mode, denoted as ‘d-off’ in the plots), the response of c4da was nearly unchanged, as if the force was directly applied on the dendrite (Figure 3C). We wondered if this reflects impalpable difference due to the overall reduction in the sensory response of c4da-cti or that the dendrites of c4da have an expanded force-receptive field. To verify this, we performed the same experiments on c4da-wt (d-off in Figure 2D). The responses of c4da-wt to the d-off stimuli were also unchanged from those to the distal stimuli (Figure 4A). Further experiments on c4da-wt showed that the responses kept nearly constant until the force was applied at least 40–60 μm away from a dendrite (Figure 4A). Based on these results, we conclude that the dendrites of c4da have an expanded force-receptive field.

Figure 4 with 1 supplement see all
The mechanosensitivity of c4da to lateral tension.

(A) The responses of c4da (ΔF/F0) to the d-off stimuli (4 mN, 60 μm probe) applied at different positions.The numbers of cells were indicated below the scattered data points. Mann Whitney U test was used. **: p<0.01. *: p<0.05. ns: no significance. Genotype: ppk-gal4/+; ppk-cd4-tdtom/20×uas-ivs-gcamp6s. (B) Left panel: representative heat maps showing the 2D distributions of pressure perpendicular to the cuticle surface (PP) and tension parallel with the cuticle surface (TL) of the 30 μm (upper) and 60 (lower) μm probes. Scale bar, 30 μm. Right panels: representative line profiles (normalized) showing the distribution of PP and TL versus the distance to the center of the force probe. Indentation depth: 20 μm. (C) The representative color map for the distance to the nearest dendrite, in which the distance was color-coded as indicated. Random positions were chosen (the red crosses) as the force application points in our simulations. Scale bar, 100 μm. (D–E) The activation probability in three conditions: (1) only sensitive to PP; (2) only sensitive to TL; (3) sensitive to both PP and TL. The dendritic coverage threshold (Cd) was the minimal length of activated dendrites that could excite neuronal responses. For each condition, 100 random positions and two sizes of probes (panel D) 30 μm; (panel E) 60 μm were used in our simulations for each cell (n=5 cells). In panel (A), (D) and (E), data were presented as mean ± std.

The expansion of force-receptive field suggests that in addition to the sensitivity to the pressure perpendicular to cuticle surface (Pp), c4da are also sensitive to lateral tension (parallel to cuticle surface, TL). By simulating tissue mechanics of the larval fillet (Figure 4—figure supplement 1, also see Materials and methods), we calculated the distribution of Pp and TL as a result of the poking stimuli from the 30 and 60 μm probes (indentation depth: 20 μm). In both cases, we noted that as the distance to the center of the force probe increased, Pp showed a monotonic decrease but TL peaked at around 20–25 μm away from the probe center (Figure 4B). Therefore, modeling analysis predicts that if c4da were only sensitive to ΔPp, their sensory response to the d-off stimuli should decrease. However, if c4da were also sensitive to ΔTL, the increase in TL would compensate for the reduction in Pp. In this scenario, the effective force-receptive field is expanded.

Morphological analysis of c4da showed that almost all positions in the dendritic territory were within 20 μm distance from a dendrite (Figure 4C). To examine how the sensitivity to TL promotes the likelihood of cell activation, we overlaid the 2D profiles of Pp and TL to the dendrites of c4da at random positions (Figure 4C) and calculated the probability of cell activation. Note that the calculations were made based on two assumptions. First, we assumed 10% of the peak value of Pp or TL as the activation threshold of a dendritic segment. Second, we assumed a dendritic coverage threshold (Cd), i.e. the minimal amount of excited dendritic segments that would lead to neuronal excitation. To explore the parameter space, we tested three values for Cd, i.e. 0.1% (20 μm) (low threshold), 1% (200 μm) (intermediate threshold) and 2% (400 μm) (high threshold) of the total dendritic length (mean ± std: 19560±1667 μm, n=5 cells). We considered three scenarios in our simulations, in which the cell is sensitive to either Pp or TL or both. Simulation results showed that with the lower threshold assumption (e.g. Cd = 20 μm), the cells could be activated in all three conditions. However, if the threshold was of intermediate (e.g. Cd = 200 μm) or high (e.g. Cd = 400 μm) level, lateral mechanosensitivity could largely enhance the probability of neuronal excitation (Figure 4D–E), consistent with our hypothesis. Therefore, our analysis suggests that the sensitivity to lateral tension enhances the sensitivity of c4da in force detection, especially when dendritic coverage is small.

Piezo and Ppk1/Ppk26 differentially contribute to the mechanosensitivity of c4da

We then wondered what is the molecular basis underlying the mechanosensitivity of c4da, especially the sensitivity to lateral tension? Previous behavior assays suggest that Ppk1/Ppk26 and Piezo mediate two parallel mechanosensory pathways in c4da (Zhong et al., 2010; Kim et al., 2012; Gorczyca et al., 2014; Guo et al., 2014; Mauthner et al., 2014). This remains to be confirmed at the cellular level. Moreover, it is also unclear how Ppk1/Ppk26 and Piezo contribute to the mechanosensitivity of c4da, respectively.

We first confirmed that the dendritic morphologies of c4da-piezoKO, c4da-ppk261 (a null mutant) and c4da-piezoKO, ppk1Δ5 (a double null mutant) were not affected (Figure 5A–B), excluding the potential effect of morphological changes. We then measured the force-evoked responses of these mutants using two types of probes (30 and 60 μm) and at different dendritic regions. In all conditions, the responses of c4da-ppk261 were significantly reduced and the sensory preference to small probes was completely lost (Figure 5C–D). In contrast, the response of c4da-piezoKO was changed in a different way. C4da-piezoKO showed a mildly reduced response to the large probe (60 μm) but a significantly lower response to the small probe (30 μm). This phenotype was more prominent for the distal stimuli than for the proximal stimuli (Figure 5C–D). As a result, the preference to small probes was only attenuated for the proximal stimuli but completely lost for the distal stimuli (Figure 5C–D). Based on these results, we conclude that Ppk1/Ppk26 contributes to the overall mechanosensitivity of c4da and Piezo is particularly important for detecting localized forces. Furthermore, c4da-piezoKO, ppk1Δ5 (the double mutant) showed almost no response to any force stimuli (Figure 5E), suggesting that the contributions of Ppk1/Ppk26 and Piezo are additive. Finally, the responses of c4da-piezoKO and c4da-ppk261 to the d-off stimuli were comparable with those to the distal stimuli, showing that both mutants are still able to sense lateral tension (Figure 5F). However, their responses to the d-off stimuli were significantly weaker than that of c4da-wt (Figure 5F), suggesting that Ppk1/Ppk26 and Piezo both make contributions to the lateral mechanosensitivity of c4da.

The differential contributions of Piezo and Ppk1/Ppk26 to the mechanosensitivity of c4da.

(A) Representative images of c4da-piezoKO (piezoKO; ppk-cd4-tdtom/+), c4da-ppk261 (uas-cd4-tdtom/ppk-gal4; ppk261), c4da-piezoKO, ppkΔ5 (piezoKO, ppkΔ5; ppk-cd4-tdtom/+). Scale bar: 100 μm. (B) Modified Sholl analysis on the morphologies of c4da-wt (n=5), c4da-piezoKO (n=3), c4da-ppk261 (n=3) and c4da-piezoKO, ppkΔ5 (n=3). The shadow areas represented standard deviations. (C–D) The responses of c4da-piezoKO and c4da-ppk261F/F0) to the proximal (C) and distal (D) stimuli delivered using the probes of two different sizes. Solid lines: 30 μm probe. Dashed lines: 60 μm probe. piezoKO: piezoKO; ppk-gal4/20×uas-ivs-gcamp6s (n=12 cells). ppk261: ppk-gal4/20×uas-ivs-gcamp6s; ppk261 (n=12 cells). (E) The responses of c4da-piezoKO, ppkΔ5F/F0) to the proximal and distal stimuli delivered using a 60 μm probe. Solid lines: proximal stimuli. Dashed lines: distal stimuli. Genotype: piezoKO, ppk1Δ5: piezoKO, ppk1Δ5; ppk-gal4/20×uas-ivs-gcamp6s (n=8 cells). (F) The responses of c4da-piezoKO and c4da-ppk261F/F0) to the d-off stimuli (4 mN) delivered using a 60 μm probe. The numbers of cells were indicated below the scattered data points. Mann Whitney U test was used. **: p<0.01. ***: p<0.001. ns: no significance. (G) The behavioral responses of the c4da-piezoKO, c4da-rescue, c4da-ppk261 and c4da-piezoKO, ppkΔ5 larvae to mechanical poking using the probes of different sizes. wt: w1118. piezoKO: piezoKO; +/+. rescue: piezoKO;uas-gfp-piezo/ppk-gal4. ppk261: +/+; ppk261. piezoKO, ppk1Δ5: piezoKO, ppk1Δ5; +/+. piezoKO larvae: 30 μm probe (n=70 larvae from three experiments), 60 μm probe (n=63 larvae from three experiments). rescue larvae: 30 μm probe (n=50 larvae from three experiments), 60 μm probe (n=47 larvae from three experiments). ppk261 larvae: 30 μm probe (n=72 larvae from three experiments), 60 μm probe (n=93 larvae from three experiments). piezoKO, ppk1Δ5 larvae: 30 μm probe (n=61 larvae from three experiments), 60 μm probe (n=60 larvae from three experiments). One-way ANOVA test was used for the comparison among different genotypes. ***: p<0.001. ns: no significance. In panels (C), (D) and (E) data were presented as mean ± sem. In panels (F) and (G), data were presented as mean ± std. In panels (C), (D), (E), (F) and (G), the data from c4da-wt were provided for comparison.

To further verify the physiological contributions of Ppk1/Ppk26 and Piezo in mechanical nociception, we performed the behavior assay of mechanical nociception on these mutants using the 30 and 60 μm probes. C4da-piezoKO showed a largely reduced response to the 30 μm probe but an almost unchanged response to the 60 μm probe. Because the expression of Piezo is not restricted to c4da, we performed tissue-specific rescue experiment by driving the expression of Piezo using a c4da-specific driver (ppk-gal4). In the rescue strain, the behavioral defect of c4da-piezoKO in mechanical nociception could be rescued, suggesting that the behavior phenotype could be largely accounted for by the loss of Piezo in c4da (Figure 5G). Meanwhile, c4da-ppk261 showed very weak responses to the probes of both sizes and the double mutant (i.e. c4da-piezoKO, ppk1Δ5) showed almost no responses to all mechanical stimuli (Figure 5G). In all, these behavioral observations are consistent with the idea that Ppk1/Ppk26 and Piezo make differential but additive contributions in the c4da-mediated mechanical nociception.

Ca-α1D contributes to the dendritic signal propagation in c4da

Finally, we addressed if the cellular mechanosensitivity of c4da relies on active signal propagation in the dendrites. The observation of the unchanged responses to the distal and proximal stimuli using the 60 μm probe (Figure 2E) supports the idea of active signal propagation in the dendrites. Meanwhile, the lower response to the distal stimuli than that to the proximal stimuli using the 30 μm probe in both c4da-wt (Figure 2E) and c4da-piezoKO (Figure 5C–D) suggests that the dendritic signal propagation mechanism is not all-or-none but graded, possibly dependent on the amounts of dendritic segments being activated. This hypothesis predicts that when the dendritic signal propagation, if any, is weakened, the cellular response of c4da to smaller probes would be affected to a greater extent. We then tested this hypothesis.

Previous studies showed that Ca-α1D, a voltage-gated calcium channel (VGCC), contributes to the somatic and dendritic calcium transient evoked by heat or laser stimulation of c4da (Terada et al., 2016; Basak et al., 2021), suggesting its function in facilitating dendritic signal propagation. Therefore, we first examined the roles of VGCCs in the force-evoked responses. When c4da-wt were stimulated by a 60 μm probe, the calcium increase was not only observed in the soma but also in the dendrites on the homolateral (p1 position: between the soma and the force probe; Figure 6A) and contralateral (p2 position: opposite side of the force probe; Figure 6A) sides of the stimulating forces. These observations demonstrate the occurrence of dendritic signal propagation. The calcium responses in both homolateral and contralateral dendrites can be suppressed by nimodipine (5 μM) (Figure 6B and Figure 6—figure supplement 1), an antagonist of VGCCs, suggesting that VGCCs contribute to the observed force-evoked dendritic calcium increases.

Figure 6 with 2 supplements see all
Active signal propagation in the dendrites of c4da.

(A) Representative images and curves showing the responses (ΔF/F0) of the soma (red arrowhead), the homolateral dendrite (position p1: yellow arrowhead) and contralateral dendrite (position p2: white arrowhead) of c4da-wt, nimodipine-treated c4da-wt, c4da-Ca-α1Di and Ca-α1DX10/AR66 to the stimuli (4 mN, 60 μm probe). The asterisk indicated the force application point. Scale bar: 50 μm. C4da-wt: ppk-gal4/+; ppk-cd4-tdtom/20×uas-ivs-gcamp6s. Ca-α1Di: ppk-gal4/20×uas-ivs-gcamp6s; ppk-cd4-tdtom/ uas-Ca-α1Di. Ca-α1DX10/AR66: Ca-α1DX10/ Ca-α1DAR66; ppk-gal4/20×uas-ivs-gcamp6s. (B) Statistical quantification of calcium increases in the homolateral dendrites in response to the stimuli (4 mN, 60 μm probe). The genotypes were the same as those shown in panel (A). Mann Whitney U test was used for the comparison between c4da-wt and the mutants. Kruskal Wallis test was used for the comparison among the groups with drug treatment. ***: p<0.001. **: p<0.01. ns: no significance. (C) and (D) The responses (ΔF/F0) of nimodipine treated c4da-wt to the proximal and distal stimuli from the probes of different sizes. (C): 60 μm probe stimuli. (D) : 30 μm probe stimuli. Kruskal Wallis test was used. ***: p<0.001. **: p<0.01. *: p<0.05. ns: no significance. (E) and (F) The responses (ΔF/F0) of c4da-wt (n=10 cells) treated with 2.5 μM nimodipine to the proximal (E) and distal (F) stimuli delivered using the probes of two different sizes. Solid lines: 30 μm probe. Dashed lines: 60 μm probe. (G) The responses (ΔF/F0) of c4da-Ca-α1Di and c4da-Ca-α1DX10/AR66 to the stimuli applied at different dendritic regions (proximal and distal) and delivered using the probes of two different sizes (60 μm: 4 mN, 30 μm: 1.5 mN). (H) The behavioral responses of the c4da-Ca-α1Di larvae to mechanical poking using the probes of two different sizes. wt: w1118. ppk-gal4: ppk-gal4; +/+. uas-Ca-α1Di: +/+; uas-Ca-α1Di. Ca-α1Di: ppk-gal4/+; uas-Ca-α1Di /+. Ca-α1DX10/AR66: Ca-α1DX10/ Ca-α1DAR66; +/+. uas-Ca-α1Di larvae: 30 μm probe (n=61 larvae from three experiments), 60 μm probe (n=61 larvae from three experiments). Ca-α1Di larvae: 30 μm probe (n=56 larvae from three experiments), 60 μm probe (n=60 larvae from three experiments). Ca-α1DX10/AR66 larvae: 30 μm probe (n=43 larvae from three experiments), 60 μm probe (n=47 larvae from three experiments). One-way ANOVA test was used for the comparison among different genotypes. ***: p<0.001. ns: no significance. In panels (B), (C), (D), (G) and (H), data were presented as mean ± std. In panels (B), (C), (D) and (G), the numbers of cells were indicated below the scattered data points. In panels (E) and (F), data were presented as mean ± sem. In panels (C), (D), (E), (F), (G) and (H), the corresponding data from c4da-wt were provided for comparison.

Drug titration experiments showed that in the condition of using a 60 μm probe, 5 μM nimodipine, the full inhibition concentration in the literatures (Scriabine and van den Kerckhoff, 1988; Terada et al., 2016), significantly suppressed the responses of c4da to both proximal and distal stimuli, while 2.5 μM nimodipine showed nearly no effect on the response to the proximal stimuli but a moderate suppression on that to the distal stimuli (Figure 6C). We interpreted this observation as when the VGCCs in the dendrites of c4da were partially inhibited, the responses to the distal stimuli were affected to a greater extent, suggesting that the dendritic signal propagation is more important in sensing distal stimuli. When a 30 μm probe was used, 2.5 μM nimodipine showed an inhibitory effect on the response to the proximal stimuli and an even stronger effect on that to the distal stimuli (Figure 6D). Further measurements on the responses of c4da to the full range of stimulating forces in the presence of 2.5 μM nimodipine showed that the drug largely suppressed the responses to the 30 μm probe but had only a mild effect on those to the 60 μm probe (Figure 6E–F), suggesting the loss of the sensory preference of c4da to small probes (Figure 6E–F). These observations demonstrate that the loss of VGCCs reduces the sensory preference to small probes, possibly by disrupting the dendritic signal propagation.

Inspired by the previous studies (Eberl et al., 1998; Terada et al., 2016), we then performed further experiments on the genomic (Ca-α1DX10/AR66) and knockdown (Ca-α1Di) mutants of Ca-α1D. Ca-α1DX10/AR66 has been genetically and functionally characterized in previous studies (Eberl et al., 1998; Terada et al., 2016). The Ca-α1Di mutant has been used for functional studies in a previous report (Terada et al., 2016) and we here also demonstrated that the expression of Ca-α1Di could significantly reduce the expression level of Ca-α1D (Figure 6—figure supplement 2). We noted that in comparison to c4da-wt, the calcium responses observed in the dendrites were largely reduced in these mutants (Figure 6B and Figure 6—figure supplement 1). In addition, the calcium responses in the soma of these mutants were also significantly reduced, in particular for the responses to the distal stimuli from the small probe (Figure 6G). Using the behavior assay, we found that the Ca-α1Di and Ca-α1DX10/AR66 larvae showed largely weakened defensive behaviors in response to the mechanical poking, especially to those from the smaller probes (Figure 6H), which accords with our cellular observations. In all, our genetic and functional analysis suggests that Ca-α1D, as a subunit of VGCC, contributes to the sensory preference of c4da, likely by facilitating dendritic signal propagation. Further expression analysis using the existing sequencing datasets (Li et al., 2022) showed that Ca-α1D had a broad expression in various types of neurons, including those expressing ppk1/ppk26 (e.g. c4da) (Figure 6—figure supplement 2). However, despite much effort, all of our further attempts to visualize the subcellular localization of Ca-α1D at both endogenous and over-expression conditions were so far not successful, possibly due to the low expression level as shown in the analysis on the sequencing datasets or other unknown technique reasons, for example that the tagged-Ca-α1D may not be stable (Figure 6—figure supplement 2). Therefore, the cell biological features of Ca-α1D in c4da are still required to be characterized in future studies (see Discussion).

Discussion

In the present study, we show that c4da are sensitive to mN-scale forces and could respond to the forces applied at different dendritic regions. In particular, c4da appear to be more sensitive to small probes, given that the applied forces are the same. We reveal three cellular mechanisms that could facilitate the sensory features of c4da (Figure 7). First, the high dendritic complexity ensures dense and uniform distribution of mechanosensory molecules. Second, the mechanosensitivity to lateral tension, which depends on both Ppk1/Ppk26 and Piezo, expands the force-receptive field of the dendrites. Third, the active signal propagation in the dendrites, possibly mediated by a voltage-gated calcium channel subunit Ca-α1D, promotes the overall sensitivity of c4da and has a more prominent effect on the sensory preference to small probes. We now discuss the potential implications of our findings in understanding the mechanosensory and nociceptive functions of c4da.

The cellular mechanisms that facilitate the mechanosensory features of c4da.

Left panel: c4da showed a greater sensitivity to localized poking forces and made uniform responses to the forces applied at different dendritic regions (p) proximal stimuli. (d) distal stimuli. Right panel: the key cellular mechanisms that facilitate the sensory features of c4da and the important contributing molecules.

Mechanical considerations on the differences between the semi-intact fillet preparation and intact larvae

In our assay, the semi-intact fillet preparation facilitates the coupling of mechanical stimulation to the recording of neuronal activities. However, it should be noted that the mechanics of our semi-intact fillet preparation is different from that of the intact animal, which is mainly reflected in two aspects. First, the fillet used in the present study is a tissue composite of cuticle, epidermis, neuron, extracellular matrix and muscle. In intact animals, this tissue composite is stretched and the tissular tension contributes to the mechanical homeostasis of c4da. In our semi-intact preparation, the tissue composite is also stretched when mounted on the PDMS pad. However, the resulting tissular tension is unlikely to be the same as that in the intact animals. This may change the mechanosensitivity of c4da. In addition, the change in the mechanical properties of tissue would alter the deformation of the fillet caused by the same force, which could also affect apparent force-dependent mechanosensitivity of c4da. Second, intact larvae are filled with the hemolymph but the fillet in our preparation is laid on a PDMS pad. The mechanical rigidity provided by the internal liquid pressure (from the hemolymph) and the muscle layer in the intact animals is possibly lower than that of the PDMS pad (Kohane et al., 2003). Therefore, the same force may cause different tissue deformations and in turn excite the neurons to a different level. These differences may explain the apparent discrepancy between the lower force used in the present work (<5 mN) in comparison to the higher forces used in the behavioral assays in the previous reports (Hwang et al., 2007; Zhong et al., 2010; Kim et al., 2012; Lopez-Bellido et al., 2019). Although these mechanical differences may affect the range of forces required to excite c4da in different studies, we do not think that they would significantly affect the way of how c4da encode the spatial parameters of stimulating forces because the overall dendritic morphology and the expression of key sensory molecules are not affected.

Implications for the mechano-nociceptive functions of c4da

C4da were first implicated as mechanoreceptor cells using the behavior assays (Zhong et al., 2010; Kim et al., 2012; Gorczyca et al., 2014; Guo et al., 2014; Mauthner et al., 2014). Cellular responses to force stimuli were observed on isolated ppk1-positive cells and whole mount larval fillet preparations (Kim et al., 2012; Tsubouchi et al., 2012; Yan et al., 2013; Guo et al., 2014; Walcott et al., 2018). Here, we provide quantitative characterizations on the responses of c4da to controlled forces. Our results add to the current model of understanding how c4da act as a mechanical nociceptor in the following two aspects.

First, at the cellular level, we find that c4da are more sensitive to smaller probes. This provides a cellular basis to understand the physiological function of c4da in sensing mechanical poking from sharp objects, for example the ovipositor of wasp (Hwang et al., 2007). From mechanical perspective, the geometry of the force probes (spherical in the present study) determines that for a given change of force, smaller probes caused a smaller ΔAc but a greater ΔP in comparison to larger probes. Because c4da are pressure sensors, the greater ΔP would elicit a greater change in the neuronal response. Meanwhile, c4da need to ensure that the ΔP over a small contact interface could be detected, integrated and amplified in order to excite neuronal responses. For this purpose, c4da develop several optimization mechanisms at the cellular level (Figure 7). First, complex dendritic morphology allows small probes to have a high dendritic coverage. Second, lateral mechanosensitivity expands the force-receptive field of dendrites, thereby acting as a mechanical amplifier. Third, active signal propagation, as an intracellular electrochemical amplifier, could compensate for signal attenuation in the dendrites. Altogether, these mechanisms optimize the mechanosensitivity of c4da by increasing the probability of activating the mechanosensory pathways that are required to excite neuronal responses.

An recent study reported that c4da can be excited by focal laser ablation through a noncontact pathway, namely that when the laser stimuli were not directly applied onto the dendrites, the neuronal responses could only be observed in axon but not in dendrites (Basak et al., 2021). This pathway is thought be mediated by extracellular molecules released from damaged cells and implies that at least in some signaling processes of c4da, the axon has a lower threshold of activation than the dendrites (Basak et al., 2021). In the present work, we did not observe noncontact responses, including those using the d-off stimuli. We think that there are mainly two reasons for this difference. First, in our experiments, the small deformation constraint minimizes tissue damage (Figure 1—figure supplement 1). Second, in our semi-intact preparation, the tissue was bathed in the insect hemolymph-like buffer, so the extracellular signaling molecules, if any, might be rapidly diluted and are unlikely to reach the effective working concentration. Therefore, we argue that mechanical stimulations used in our study primarily stimulate the contact pathway, in which the dendrites are activated in the first place. This is also supported by two additional observations. First, we note that the force-evoked neuronal responses are dependent on Ppk1/Ppk26 and Piezo channels. These channels have clear dendritic localizations and are expected to contribute to the dendritic responses. Second, although the dendritic localization of Ca-α1D is still required to be clarified, the dependence of dendritic responses on VGCCs (Figure 6B) suggests that an active signal propagation mechanism is involved in the force-evoked dendritic responses. Comparatively speaking, we think that the previous study provides novel insights into how c4da respond to local tissue damage (Basak et al., 2021), while our study advances the model of how c4da detect forces as a mechanoreceptor cell. These two studies, in a complementary way, promote our understanding of the function and working mechanism of c4da as a nociceptor.

Second, at the molecular level, we find that Piezo and Ppk1/Ppk26 make differential contributions to the mechanosensitivity of c4da. Previous behavior assays suggest that Ppk1/Ppk26 and Piezo in c4da mediate two parallel pathways in mechanical nociception (Kim et al., 2012; Gorczyca et al., 2014; Guo et al., 2014; Mauthner et al., 2014). This raises the question of why c4da need two sets of mechanosensory pathways. Furthermore, patch clamp recordings showed that the force-evoked electrical response of isolated ppk-positive cells is entirely dependent on Piezo (Kim et al., 2012), which contradicts the result from the behavior assay. Therefore, it is necessary to explore how Ppk1/Ppk26 and Piezo contribute to the in-vivo mechanosensitivity of c4da. Here, we discover that Piezo is particularly important for c4da to detect localized forces but plays a relatively minor role in sensing large probes. In contrast, the loss of Ppk1/Ppk26 reduces the responses of c4da to the entire range of stimulating forces used in our experiments, suggesting the contribution of Ppk1/Ppk26 to the overall mechanosensitivity. In addition, the lack of responses in the double knockout mutant strain confirms that the contributions of Ppk1/Ppk26 and Piezo are additive, as suggested by the previous behavior assays (Kim et al., 2012). Therefore, our findings add to the current model by showing the differential roles of Ppk1/Ppk26 and Piezo in mechanosensation, and in particular, Piezo seems to act as a functional module to specifically enhance the mechanosensory features of c4da. Furthermore, we also show that Ppk1/Ppk26 and Piezo both contribute to the sensitivity of c4da to lateral tension. Consistent with this idea, members in the Piezo and the DEG/ENaC channel families have been proposed to be gated by the changes in lateral membrane tension (Cueva et al., 2007; Guo and MacKinnon, 2017; Liang and Howard, 2018; Lin et al., 2019). However, it remains unclear how Piezo could facilitate the greater sensitivity to localized forces. We think that the underlying molecular mechanisms may reside in the gating property of Piezo, such as gating sensitivity, conductance and the property of the gating force.

Contribution of dendritic morphology to the function of c4da

Based on the observations in the cti strain (Figure 3), we argue that the morphological complexity is a key factor in supporting the sensory features of c4da. Because Cut is a transcription factor that contributes to the morphological determination of c4da, it is formally possible that the altered mechanosensory responses observed in the cti mutant is due to other downstream changes. While this possibility cannot be absolutely excluded, we think that the contribution of dendritic morphology is likely valid based on several lines of evidence and thoughts. First, the responses of c4da-cti to the stimuli on the proximal dendritic region, where the dendritic density is similar to that of c4da-wt, is comparable to those of c4da-wt, suggesting that the essential mechanosensory elements are fairly normal in the existing dendrites of c4da-cti (Figure 3C). Second, the expression and localization of mechanosensory molecules (Piezo and Ppk1/Ppk26) and cytoskeletal elements (F-actin and microtubule) in c4da-cti are comparable to those in c4da-wt (Figure 3—figure supplement 1), although it is possible that the fine organization of these molecular components could still be different in c4da-cti. Third, at least in terms of how a cell responds to different probe sizes and to the stimuli at different dendritic regions, c4da-cti are similar to c3da-wt, whose morphological pattern is markedly different from c4da-wt but close to c4da-cti (Figure 2—figure supplement 1). Assuming that the spatial parameters of forces are primarily encoded at the cellular level, the similarity between c3da-wt and c4da-cti would reflect the contribution of dendritic morphology. Therefore, our observations are consistent with the idea that the dendritic complexity is a contributing parameter underlying the force-encoding mechanism of c4da.

Materials and methods

Fly stocks

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All flies were maintained on standard medium at 23–25℃ in 12:12 light-dark cycles. 20×uas-ivs-gcamp6s (BL42746, BL42749), ppk-gal4 (BL32078, BL32079), uas-cd4-tdTom (BL35837, BL35841), ppk-cd4-tdtom (BL35845), ppk-cd4-tdgfp (BL35842, BL35843) uas-lifeact-rfp (BL58715), Ca-α1DX10 (BL25141) and Mi{ET1}Ca-α1DMB06807 (BL25527) were from Bloomington Drosophila Stock Center (BDSC). Gal4-19-12 was from the Jan Lab (UCSF). ppk26-gfp was from the Tracey Lab (Indiana University). Uas-mcherry-jupiter was from the Han Lab (Cornell University). Tmc-gal4, gfp-piezo, piezoKO, uas-gfp-piezo, ppk261 and piezoKO, ppkΔ5 and were from Wei Zhang (Tsinghua University). tub-gal4 was from Bing Zhou (Tsinghua University). cti (THU1309), Ca-α1Di (THU0766) and the control strains were from Tsinghua Fly Center. Ca-α1DAR66 was from Daniel Eberl (University of Iowa).

Larval fillet preparation

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Third instar larvae were dissected on a 35 mm dish coated with a PDMS pad (thickness: 1 mm) in the insect hemolymph-like (HL) buffer (Stewart et al., 1994). The cuticle, epidermis and muscle tissue were kept as intact as possible. The fillet was mounted on the PDMS pad using 8–10 insect pins (Austerlitz Insect Pins, Czech) and kept as stretched. HL buffer: 103 mM NaCl (10019318 Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), 3 mM KCl (P9541 Sigma-Aldrich, USA), 5 mM TES (T5691 Sigma-Aldrich, USA), 8 mM trehalose (T1067 Sigma-Aldrich, USA), 10 mM glucose (G7528 Sigma-Aldrich, USA), 5 mM sucrose (V900116 Sigma-Aldrich, USA), 26 mM NaHCO3 (A500873 Sangon Biotech, Co., Lt., Shanghai, China), 1 mM NaH2PO4 (S8282 Sigma-Aldrich, USA), 2 mM CaCl2 (Xilong Scientific Co., Ltd., Shantou, China), and 4 mM MgCl2 (M8266 Sigma-Aldrich, USA). The PDMS pad was made using the Sylgard 184 kit (Dow, UAS).

Mechanical device setup and calibration

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The mechanical device consisted of a piezo actuator (PZT 150/7/60 vs 12, SuZhou Micro Automation Technology Co., Ltd., Suzhou, China), a strain gauge (customized in Nanjing Bio-inspired-tech Co., Ltd., Nanjing, China) and a force probe. The stepping distance (D) of the piezo actuator and the deflection of the metal beam (B) were measured using a digital camera with a pixel size of 1.1 μm (AO-3M630, AOSVI optical instrument Co., Ltd. Shenzhen, China). The voltage readout of the strain gauge after the addition of pure water with a minimal step of 100 μL (i.e. 0.98 mN) was recorded using a data acquisition card (NBIT-DSU-2404A, Nanjing Bio-inspired-tech Co., Ltd., Nanjing, China). The force probes were made from capillary glass tubes using an electrode puller (PC-10, Narishige, Japan) and then polished using a microforge (MF-830, Narishige, Japan). Finally, the assembled mechanical device was mounted on the working stage of the spinning-disk confocal microscope (Andor, UK).

Confocal microscopy

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The calcium fluorescent signals were recorded using an Andor inverted spinning disk confocal system (Andor, UK) equipped with an inverted microscope (Olympus, IX73), an iXon 897 EMCCD and a long working-distance 20×objective (UCPlanFL N, N.A. 0.70, working distance: 2.1 mm) (Olympus, Japan).

The images of Piezo-GFP, Ppk26-GFP, mCherry-Jupiter and Lifeact-RFP were acquired using a Zeiss 780 confocal microscope equipped with a 63×objective (Zeiss Plan-Apochromat, 1.4 N.A., Germany) and GaAsP detectors (Zeiss, Germany).

Scanning electron microscopy

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The scanning electron microscopy (SEM) pictures of the glass probes were imaged using FEI Quanta 200 (Thermofisher, USA) with 15-kV voltages and 500×magnification in the high vacuum mode.

Modified behavior assay for mechanical nociception

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The Von Frey fiber (6lb. Omniflex Line, Zebco) was mounted onto a holder. The free fiber was 20 mm long. The customized glass probe was fixed at the free end of the fiber using ergo 5400 (Kisling, Switzerland). The poking force was measured using an electronic precision balance (XPR204S, Mettler Toledo, USA) and was found to be independent on probe size (Figure 2—figure supplement 3). The behavior assays were carried out as the previously described (Hwang et al., 2007; Zhong et al., 2010). Briefly, we used the modified fiber to poke the dorsal side of larval middle segments and recorded their behavioral responses. If the larva showed a typical rolling behavior (rolling over 360°) upon each mechanical stimulation, it was considered as making an effective response. The percentage of responding animals was then calculated. Statistical analysis was performed using one-way ANOVA using Matlab (MathWorks, USA).

Drug treatment

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Nimodipine (N149 Sigma-Aldrich, USA) was dissolved in DMSO (276855 Sigma-Aldrich, USA). The incubation time for all drugs was 20 min.

The ‘sphere-surface’ contact mechanics model

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Based on the classical ‘sphere-surface’ contact model (Johnson, 1985), we had

(1) f=43Er12d32
(2) E*EPDMS1ν2

where f was the total force, r was the radius of the sphere, EPDMS was the elastic modulus of PDMS, E* was the effective modulus, ν was the Poisson’s ratio of PDMS and d was the indentation depth. d can be calculated as

(3) d=DB

where D was the stepping displacement generated by the piezo stack actuator and B was the bending deflection of the metal beam (Figure 1A). In this model, the pressure at the center of contact area (P0) can be calculated as

(4) P0=3f2πa2

where a was the radius of the contact area and could be calculated as

(5) a=rd

The area of contact surface (Ac) can be calculated as

(6) Ac=2πr(rr2rd)

The pressures at a given point to the soma can be calculated as

(7) Pxμm=P0(1x2a2)12

where x (μm) was the distance to the center of contact area.

Finite element analysis

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To theoretically investigate the stress field in the cuticle caused by indentation, finite element simulations were performed using the commercial software ABAQUS 6.14.1. (Dassault Systèmes, France). Due to symmetry of the load and the geometry, an axisymmetric mechanical model was established (Figure 4—figure supplement 1). The model includes the spherical probe and the underlying composite that consists of two layers of materials: (1) i.e. the cuticle layer (thickness 10 μm, elastic modulus Ecuticle = 1–10 MPa, Poisson’s ratio νcuticle=0.45); (2) the underlying PDMS substrate (thickness 1000 μm, elastic modulus EPDMS = 2.6 MPa, Poisson’s ratio νPDMS=0.45). Because the muscle layer (elastic modulus Emuscle = 10 kPa) was much more compliant than cuticle and PDMS (Kot et al., 2012), it is expected to make little contribution to the force distribution. Therefore, we omitted the muscle layer in our finite element model for simplicity. The probe was treated as a rigid object, while the cuticle layer and PDMS substrate were assumed to be linearly elastic. Displacement load in the vertical direction was applied to the probe. The lower surface of the cuticle layer and the upper surface of the PDMS substrate were assumed to be tightly coupled. Fixed boundary conditions were applied to the bottom surface of the PDMS substrate. The compressive pressure (perpendicular to cuticle surface) and lateral (parallel to cuticle surface) tension were both calculated. The simulation of random positioned stimuli was performed using Matlab.

Image analysis

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GCaMP6s signal was measured using Fiji (Schindelin et al., 2012). The calibration bars and scale bars were generated in Fiji. Sholl analysis on neuronal morphology was carried out using Fiji (Ferreira et al., 2014). The density of intersections was calculated as the number of intersections divided by the circumference of corresponding circle in the Sholl analysis (Figure 3, Figure 5 and Figure 2—figure supplement 1).

The single-cell transcriptomic atlas analysis

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We used the FlyCellAtlas (Li et al., 2022) single-cell RNA-sequencing datasets to explore the expression profiles of Ca-a1D. Specifically, we downloaded the stringent analysis result of the Body 10×Genomic sample from FlyCellAtlas website. We visualized the expression pattern with the use of Python packages scanpy (version 1.9.1) (Wolf et al., 2018) and anndata (version 0.8.0) (Virshup et al., 2021). We first explored the expression pattern of Ca-a1D in the whole dataset. To further check if the ppk-specific neurons also express Ca-a1D, we plotted the expression of both genes in the neuron clusters.

Conventional data plotting and statistical analysis

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Data plotting and statistical analysis were performed using Origin (OriginLab, USA). The heat maps of the PP and TL were generated using Matlab. Statistical analysis was performed using Origin.

Generation of transgenic flies for Ca-α1D expression

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To construct mCherry tagged Ca-α1D expressed under its own promoter, the coding sequence of Ca-α1D-RK transcript was fused with mCherry tag and its promoter sequence (a 3 kb sequence upstream of the Ca-α1D-RK coding region). The regulation sequence at 3’ end, which is ~2.3 kb downstream of the Ca-α1D-RK coding region, was fused at the 3’ end. The entire construct of ‘promoter-mCherry-Ca-α1D –3’ regulation sequence’ was then cloned into pJFRC81 (Addgene 36432) between the HindIII and FseI sites. The plasmid was then injected into the attp40 strain to generate the promoter fusion mCherry-Ca-α1D transgenic line.

To construct uas-mcherry-Ca-α1D, the coding sequence of mcherry-Ca-α1D-RK was cloned into pJFRC81 between the NotI and XbaI sites. The plasmid was then injected into the attp2 strain to generate the uas-mcherry-Ca-α1D transgenic fly.

Immunostaining

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Immunostaining followed the procedure described previously (Hsu et al., 2020). 3rd larvae were filleted in PBS and fixed in 4% PFA in PBS. The primary antibody used was: chicken anti-Dmca1D (1:100, shared by John Y. Kuwada). Secondary antibody used was: Donkey anti-chicken Alexa Fluor 488 (1:500, SIGMA, 18C0824).

RT-PCR

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The total RNA was extracted from whole larva using the RNeasy Mini Kit (QIAGEN, 74104). The cDNA was synthesized using SuperScript III Reverse Transcriptase (RT) (Invitrogen, 18080051). All polymerase chain reactions were performed using the Phusion DNA polymerase (New England Biolabs, Ipswich, MA). Rp49 was used as the reference gene. Rp49 forward primer: 5’-TACAGGCCCAAGATCGTGAA-3’. Rp49 reverse primer: 5’-TCTCCTTGCGCTTCTTGGA-3’. Ca-α1D forward primer: 5’-CATTGCAAACATTCCGGAAACC-3’. Ca-α1D reverse primer: 5’-CAATTGGGAGAGTGCAGTACT-3’. The gel image was taken using the Tanon image system (SHTN 1600, China). The intensity of DNA bands on the gel images was analyzed using Fiji.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. The source data for all plots have been provided as Excel files.

The following previously published data sets were used
    1. Li H
    (2021) ArrayExpress
    ID E-MTAB-10628. Fly Cell Atlas: single-cell transcriptomes of the entire adult Drosophila - Smartseq2 dataset.
    1. Maxime DW
    2. Li H
    3. Janssens J
    (2021) ArrayExpress
    ID E-MTAB-10519. The Fly Cell Atlas: single-cell transcriptomes of the entire adult Drosophila - 10x dataset.

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Decision letter

  1. Matthieu Louis
    Reviewing Editor; University of California, Santa Barbara, United States
  2. Claude Desplan
    Senior Editor; New York University, United States
  3. Martin Göpfert
    Reviewer

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Drosophila Mechanical Nociceptors Preferentially Sense Localized Poking" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Claude Desplan as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Martin Göpfert (Reviewer #3).

The reviewers have discussed at length their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. The reviewers were generally very impressed by the quality of your study and the novelty of your results. Nonetheless, a series of significant technical concerns were raised. We are confident that you will be able to address these concerns, and ask that you do so in a revised manuscript. Although the proposed revisions will entail additional work and new data, we encourage you to build on existing results. Specially:

1. Please check if dendrite/epidermal breakage occurs with the used probes. Ruling out such breakages is important to evaluate the data and its interpretation both at the level of the calcium signals and behavior. You should use ppk-tdtomato or ppk-mCD8GFP for this to make sure dendrites are bright enough and visible. Also, magnification higher than 20X is probably needed.

2. Please use the existing set of recordings to evaluate the possibility of dendritic calcium signals. The idea is to analyze the spatial distribution of dendritic calcium responses to the pokes. If no or very weak dendritic calcium signals are found, this observation should be stated in the manuscript. If significant dendritic calcium signals are observed, you should determine the dependence of these signals on Ca-alpha1D.

3. Please validate the RNAi knockdown and mutant results, particularly for Ca-alpha1D and piezo. For the RNAi lines used, qPCR data on the knockdown-efficiency should be added. If the qPCR do not work, you should consider using piezo-GFP as a readout for assessing the knockdown efficiency. For Ca-alpha1D, you should check the relevant mutants that could be used to provide better genetic evidence in support of a role for Ca-alpha1D. Data on protein localization is presented for the mechanosensitive channels, but not for Ca-alpha1D. Knowing the distribution of the Ca-alpha1D subunit is critical to the interpretation of the rest of your data. To do this, we recommend you use an antibody against Ca-alpha1D that was generated in I-Uen Hsu et al., PNAS 2020, PMID:33168737. Confirming expression, localization and RNAi knockdown should be feasible if the antibody proves to be specific enough. If the antibody's specificity is insufficient, you should check whether Ca-alpha1D is expressed in the cell using e.g. Mi{ET1}Ca-α1D[MB06807] that is available at Bloomington. For mutant alleles of Ca-alpha1D, combinations of available reagents were used in a previous study cited by the author (Terada et al. ELife 2016): Ca-α1DX7/AR66 and Ca-α1DX10/AR66. While we realize these extra experiments represent substantial work, their results will be important to clarify the role of Ca-alpha1D and piezo to support several or your statements. In case the experiments listed above are unsuccessful, we ask you to tone down the interpretation of your results, which would be unfortunate.

4. Please check/redo the calculations related the elastic modulus (PDMS vs. larval cuticle) and clarify the differences in the mechanical properties of the open filet preparation vs. the intact animal. In particular, transferring the elastic modulus of muscles characterized in humans to flies does not seem to be legitimate. The modeling should be repeated with a realistic value for the elastic modulus of the larva, along with an improved discussion of how the force distributions would compare to those in an intact larva. In the discussion, you should address the apparent discrepancy between the force used in your work (<5 mN) compared to the 5-10x higher forces typically used in previous publications studying rolling responses in live larvae.

5. Please provide a minimal set of controls for behavioral data. This should include the driver control for the RNAi, the driver alone, and the driver plus RNAi. You have conducted these controls for the experiments related to cut but you are missing driver alone for Ca-alpha1D.

6. Please address the points raised by the reviewers regarding the interpretation of the results, the statistics and grammatical errors. Among the missing references, it would be important to incorporate into the discussion the following paper from the Howard lab: Focal laser stimulation of fly nociceptors activates distinct axonal and dendritic Ca 2+ signals by Basak, Sutradhar and Howard (PMID: 34175294, DOI: 10.1016/j.bpj.2021.06.001). This paper shows that axonal activation of the c4da neurons can happen even in the absence of any dendritic calcium signals. This argues that any active properties of the dendrites only occur with very strong activation. In the methodology, you should provide details about the creation of the piezo-GFP line (along with better imaging of its distribution in the dendrites). You should also add missing information about the regions of interest in which calcium responses were measured.

Reviewer #1 (Recommendations for the authors):

Liu et al. present findings that significantly extend the understanding of molecular and cellular pathways of mechanical nociception in Drosophila larvae. They present a detailed analysis of the mechanical response properties of the nociceptors that is performed using a newly developed preparation for optical recordings from these cells. Using mechanical probes of varying tip diameters they are able to investigate the responses as they relate to force and pressure. Mutants in the cut gene, which show reduced branching of the nociceptors, are used to interrogate how the dendritic morphology structure might relate to their physiological responses. As well, the response profiles of the neurons that are mutant for mechanosensory channels ppk-26 and piezo are investigated. The ppk-26 mutant shows a more strongly impaired deficit in comparison to piezo mutants and double mutants show a nearly abolished mechanical response. The mechanosensory neurons are also found to respond to mechanical forces that are outside of the main dendritic fields which suggests that they are able to detect forces that are viscoelatically coupled through the overlying epidermis. Finally, a voltage activated calcium channel is found to be important which suggests that the dendrites of the nociceptive neurons are "active" (as opposed to passive). Overall, the study significantly advances our understanding of the response properties of the mechanical nociceptors of the Drosophila larva.

Questions for the authors:

The duration of the calcium responses long outlasts the force application. It has been previously proposed that dendritic breakage could be a contributing factor in the transduction mechanism in the Drosophila nociceptive neurons (Tracey, 2017). What is the relationship of dendritic breakage occur with the various force stimuli and probe sizes in the imaging setup? Sharp probes (smaller probes) may be more likely to break dendrites. Similarly, do the sharp probes that trigger rolling in Figure 2J break dendrites?

The results of experiments with cut are very interesting, and the authors are open and cautious in their interpretation when they state the caveats of unknown epistatic effects that may result from removal of a transcription factor. Even with the stated caveats, the unknown effect of cut removal causes the results to be very difficult to interpret. It cannot be concluded that the deficits are a consequence of reduced branching. For example, perhaps calcium-alpha1d is a target.

Equation 3 assumes that the glass rod is completely rigid, if the rod bends during the stimulus then then d becomes on overestimate. Does the glass rod bend in this setup?

Are force measurements in figure 1i made with a larval filet in the setup? Or is this the force applied to PDMS alone?

Where are the values for the elastic modulus for larval cuticle coming from in line 543? Experimental measurements of the stiffness of larval cuticle come in at 0.39 +/- 0.01 MPa which is an order of magnitude lower than the values input to the model (10Mpa). Kohane M, Daugela A, Kutomi H, Charlson L, Wyrobek A, Wyrobek J. Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument during development". J Biomed Mater Res A. 2003 Sep 1;66(3):633-42. doi: 10.1002/jbm.a.10028

Third instar larval cuticle is closer to 20 microns thick according to experimental measurements. (Christine E. Kaznowski, Howard A. Schneiderman, Peter J. Bryant, "Cuticle secretion during larval growth in Drosophila melanogaster", Journal of Insect Physiology, Volume 31, Issue 10, 1985, Pages 801-813, https://doi.org/10.1016/0022-1910(85)90073-3).

The reference provided for elastic modulus of muscle comes from a study on human muscles and is therefore not valid for a study performed on Drosophila larvae.

Is the force probe is compressing the entire larval filet such that there is an indentation into the PDMS (as depicted in figure 1A)? If so, then isn't it true that the forces on the filet (dendrites) are not coming solely from the force probe itself? If the larva is being squished between the probe and the PDMS, then the larva is also being exposed to an opposing force that is coming from the PDMS.

Figure 4D. Why "dendritic coverage threshold" instead of distance from dendrite? How do the predictions from this figure relate to real response data?

Could differences in distribution of Ca-alpha1D along the dendrite explain differences in proximal/distal sensitivities (ie. inhibition has less effect at proximal, maybe channels are less expressed/less important there?) Checking Ca-alpha1D localization would seem to be important.

Figure 6A, the top soma trace seems really low, compared to the responses shown earlier with this probe size and force.

Please provide details of the creation of the piezo::GFP line and better imaging of the distribution in the dendrites.

What is the frame rate of image acquisition? Are data from a single z-slice? Is pinhole wide open?

In Line 75 please do not refer to flies and worms as "lower animals." Recommend substituting a term such as "invertebrates. "

In line 85 cite Zhong et al. 2010.

In lines 152-153 it is stated that the c4da neurons use their entire dendritic field as the force-receptive field. However, the later experiments show that forces are detected through the viscoelastic coupling of the dendrites to the rest of the epidermis therefore this statement is not supported.

In line 163 Cite Cynthia Hughes et al. and he et al.

In line 166 please cite Tsubouchi et al. 2012 (which precedes Yan et al. 2013 in the demonstration of the gentle touch function for cIIIda neurons).

In Line 201 cite Robertson JL et al. 2013.

In line 296 cite Zhong et al. 2010, Mauthner et al. 2014, Gorczyca DA et al. 2014.

In line 385 calcium α 1- D is more precisely described as a calcium channel subunit.

Figure 1a cuticle is misspelled.

Reviewer #2 (Recommendations for the authors):

1. The citations are in some instances used somewhat loosely and do not appropriately reflect the literature. This should be amended in a revised version:

Lines 88-89: The selection of references cited for the downstream circuitry of c4da neurons seems random. Grueber et al. 2007 characterized axonal projections of c4da only, Burgos et al. and Yoshino et al. each described a class of c4da second order neurons that have not been characterized in regard to mechanical stimuli. If the authors wish to cite all studies then critical ones are missing including Ohyama et al. (Nature 2015), Kaneko et al. (Neuron 2017), Hu et al. (Nat Neurosci 2017) and Takagi et al. (Neuron 2017). The latter two studies actually characterized the c4da circuitry in regard to noxious mechanical stimuli, which seems most relevant here. Alternatively, Gerhard et al. (eLife 2017) provides the most complete description of the c4da second order circuitry.

Lines 201 and 230: Grueber et al. (Cell 2003) was the first study to show that cut determines c4da neuron morphology and should be cited here.

2. Some discussion is warranted regarding the force used here (<5 mN) compared to the 5-10x higher forces typically used in previous publications studying rolling responses in live larvae (e.g. Kim et al. Nature 2012, Zhong et al. Curr. Biol. 2012, Gorczyca et al. Cell Rep. 2014, Guo et al. Cell Rep. 2014, Hu et al. Nat Neurosci 2017, Lopez-Bellido J Neurosci. 2019). While this is likely in part linked to the small diameter probes here vs. the typically larger probes utilized in previous studies, both the neuronal as well as behavioral responses to low mN stimuli seem to be extremely strong. For the neuronal activation, this might be linked to several features unique to the semi-intact preparation used by the authors vs. an intact animal. For example, the PDMS stiffness is much higher than that of a larval body filled with hemolymph, which will resist force manipulation much less, i.e. indentation depth is likely higher for a given force/pressure. This point should be adequately discussed and put into context with the literature.

3. Relating to point 2, measuring larval rolling behavior using their different sized probes is interesting, yet the methodology was not so clearly explained in the methods or results. The authors indicate that the probes exerted a force between 10-20 mN, but do not indicate if the variability of individual probes depends on the diameter of the glass probe. As the pressure on the animal depends on probe size it might make sense to check if the measured force is dependent on the probe diameter and/or if response rates are just variable within the same range independently of the probe size.

4. Figure 2J: The legend is a bit confusing as the categories listed are <1, >1 and >2 turns. Previous studies typically identified a single roll while multiple rolling indicates severe tissue damage. Some of the tissue damage effects by different probe sizes and pressures have been studied in more detail suggesting that tissue damage is actually required for defensive behavior (Lopez-Bellido et al. J Neurosci 2019 and J Vis Exp 2020). The authors should check for tissue damage under their experimental conditions to exclude that excessive rupturing of the epidermis causes the observed strong behavioral reactions.

5. In line 195 and 717, the labeling for this panel should be "J" instead of G.

6. It should be considered that the relative contributions of ppk/ppk26 and piezo might not exclusively reflect their function in c4da neurons alone. While ppk/ppk26 expression is cell type-specific, piezo is widely expressed in multiple tissues. Using a piezoko allele thus does not allow to draw conclusions on its function in c4da neurons. This would require either a cell type-specific manipulation or a c4da neuron specific rescue experiment. It would help if the authors can model lateral tension and perpendicular pressure for the 30 micron probe. This might help to explain the different defects described in ppk26 vs. piezo loss of function. Correlating the differential reliance of ppk26 and piezo on Ca-alpha1D function might be another interesting aspect, given that distal function of piezo to some extent resembles the one of Ca-alpha1D.

7. The model in Figure 7 could be improved as the color code and labels are not intuitive and the assignment of piezo vs ppk function is not clear to me. The model might also have to be revised according to the criticism regarding the assigned functions of ppk26 vs piezo.

Reviewer #3 (Recommendations for the authors):

– Figure 2, legend: (G) should read (J).

– Line 160: "To further justify the validity" – change to "To further test the validity".

– Line 66: not sure whether e.g. proprioceptors controlling locomotion aren't equally important for the survival of an organism.

– Line 390 ff: use past tense when reporting on earlier findings.

https://doi.org/10.7554/eLife.76574.sa1

Author response

The reviewers have discussed at length their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. The reviewers were generally very impressed by the quality of your study and the novelty of your results. Nonetheless, a series of significant technical concerns were raised. We are confident that you will be able to address these concerns, and ask that you do so in a revised manuscript. Although the proposed revisions will entail additional work and new data, we encourage you to build on existing results. Specially:

1. Please check if dendrite/epidermal breakage occurs with the used probes. Ruling out such breakages is important to evaluate the data and its interpretation both at the level of the calcium signals and behavior. You should use ppk-tdtomato or ppk-mCD8GFP for this to make sure dendrites are bright enough and visible. Also, magnification higher than 20X is probably needed.

We thank the reviewers for this suggestion. In our experiments, we always recorded the dendritic morphology before and after the force stimulation to control dendritic breakage. To make this point clearer, we made two changes in the revised manuscript. First, in the revised Figure 1—figure supplement 1, we showed that in the condition of using a saturation force, no significant damage of dendritic morphology was observed. Second, we made it clearer now that it was our intention to stay with the small deformation constrain to focus on mechanosensory responses of c4da (see page 5, second paragraph, line 124-130).

2. Please use the existing set of recordings to evaluate the possibility of dendritic calcium signals. The idea is to analyze the spatial distribution of dendritic calcium responses to the pokes. If no or very weak dendritic calcium signals are found, this observation should be stated in the manuscript. If significant dendritic calcium signals are observed, you should determine the dependence of these signals on Ca-alpha1D.

In the original manuscript, we included the data on the dendritic calcium signal and showed that the dendritic signal was reduced when the activity of VGCCs were inhibited or in the Ca-α1D knockdown mutant (see Figure 6 A-B in the original manuscript). Inspired by the suggestion from the reviewers, we had a closer look at our data and performed additional experiments. In the revised Figure 6 A-B, we showed that the mechanical stimuli could evoke calcium responses not only in the soma, but also in the homolateral (i.e. between the soma and the force probe) and contralateral (i.e. opposite side of the force probe) dendrites, suggesting that the dendritic signals are propagating within the dendritic arbors. Moreover, in the revised Figure 6 A-B and Figure 6—figure supplement 1, we showed that these dendritic signals were reduced in the mutant strains of Ca-α1D or if the fillet preparation was treated with nimodipine, demonstrating a clear dependence on the activity of VGCCs. However, because our imaging speed is not fast enough to capture the dendritic flow of calcium signals, the dynamics of signal propagation remains undefined. This would be an interesting issue to study in the future. Along with the revised Figure 6, we also revised the text and legends accordingly.

3. Please validate the RNAi knockdown and mutant results, particularly for Ca-alpha1D and piezo. For the RNAi lines used, qPCR data on the knockdown-efficiency should be added. If the qPCR do not work, you should consider using piezo-GFP as a readout for assessing the knockdown efficiency.

First, we did not use RNAi mutant for Piezo. The PiezoKO line is a genomic mutant strain1.

Second, for Ca-α1D, because there are only a small number of c4da in each animal and Ca-α1D has a quite broad expression in various types of neurons (see our revised Figure 6—figure supplement 2), we expected that the reduction in the expression level of Ca-α1D in c4da would be very difficult to detect. Therefore, we knocked down the expression of Ca-α1D in the whole animal using the same uas-Ca-α1Di strain and the tub-gal4 strain. Using RT-PCR, we showed that the expression level of Ca-α1D was significantly reduced (revised Figure 6—figure supplement 2). In fact, the same RNAi strain was also used in other functional studies2,3.

For Ca-alpha1D, you should check the relevant mutants that could be used to provide better genetic evidence in support of a role for Ca-alpha1D.

We thank the reviewers for this suggestion. We have now performed additional functional experiments on Ca-α1DX10/AR66, a genomic mutant strain used in previous studies on Ca-α1D2,4. As said by the reviewer, the additional data strengthens the genetic evidence to support the role of Ca-α1D.

Data on protein localization is presented for the mechanosensitive channels, but not for Ca-alpha1D. Knowing the distribution of the Ca-alpha1D subunit is critical to the interpretation of the rest of your data. To do this, we recommend you use an antibody against Ca-alpha1D that was generated in I-Uen Hsu et al., PNAS 2020, PMID:33168737. Confirming expression, localization and RNAi knockdown should be feasible if the antibody proves to be specific enough. If the antibody's specificity is insufficient, you should check whether Ca-alpha1D is expressed in the cell using e.g. Mi{ET1}Ca-α1D[MB06807] that is available at Bloomington. For mutant alleles of Ca-alpha1D, combinations of available reagents were used in a previous study cited by the author (Terada et al. ELife 2016): Ca-α1DX7/AR66 and Ca-α1DX10/AR66. While we realize these extra experiments represent substantial work, their results will be important to clarify the role of Ca-alpha1D and piezo to support several or your statements. In case the experiments listed above are unsuccessful, we ask you to tone down the interpretation of your results, which would be unfortunate.

We thank the reviewers for these suggestions and agree that it is important to determine the cell biological properties of Ca-α1D. We performed all suggested experiments and additional ones to examine the expression and localization of Ca-α1D. However, most of the results are unfortunately negative, and we now summarize these results.

First, we performed the expression analysis using the existing RNA sequencing data. Our analysis showed that Ca-α1D had a broad expression in various types of neurons, including those expressing ppk1/ppk26 (e.g. c4da) (Figure 6—figure supplement 2). From the sequencing data, we noted that the expression level of Ca-α1D was generally low (Figure 6—figure supplement 2). To further confirm the expression of Ca-α1D in c4da, we used the Mi{ET1}Ca-α1DMB06807 line as suggested by the reviewers. However, no clear signal was observed.

Second, we generated the transgenic strain that could express mcherry-Ca-α1D under the endogenous promoter of Ca-α1D. However, no clear signal was observed. We also used the antibody as suggested by the reviewer 5 and no clear signal was observed in any part of c4da. We thought that it might be due to the low expression level of Ca-α1D, so we generated a uas-mcherry-Ca-α1D strain. Using the c4da specific ppk-gal4 strain to drive the expression of mCherry-Ca-α1D, we still could not see any signals. Because Ca-α1D is a subunit of the voltage-gated channel, we speculate that mCherry-Ca-α1D is probably not stable or is not able to assemble with the other subunits to form a functional VGCC. Future work is required to explore a good construct or other strategies to clarify the subcellular localization of Ca-α1D.

In all, although the cell biological evidence is lacking, we think that there is still good evidence from sequencing analysis and functional experiments to support the expression and the role of Ca-α1D in c4da. We have now summarized all our analysis of Ca-α1D in the revised manuscript (Figure 6—figure supplement 2), including all negative results, which would be useful not only as a set of supporting data but also for the information of other researchers. Finally, we also have added discussions on this issue and modified the text to ensure that the interpretation on the role of Ca-α1D is precise (see page 10, fourth paragraph, line 340-352, page 11, second paragraph, line 369-392 and the revised Discussion).

4. Please check/redo the calculations related the elastic modulus (PDMS vs. larval cuticle) and clarify the differences in the mechanical properties of the open filet preparation vs. the intact animal. In particular, transferring the elastic modulus of muscles characterized in humans to flies does not seem to be legitimate. The modeling should be repeated with a realistic value for the elastic modulus of the larva, along with an improved discussion of how the force distributions would compare to those in an intact larva. In the discussion, you should address the apparent discrepancy between the force used in your work (<5 mN) compared to the 5-10x higher forces typically used in previous publications studying rolling responses in live larvae.

First, we have now added a paragraph in the Discussion to clarify the mechanical differences between the semi-intact fillet preparation and the intact animal, in which we also discuss the possible reason underlying the discrepancy between the forces used in different studies (see Discussion, second paragraph, page 11). Second, we have checked the values for elastic modulus of insect muscle cells6, and it is similar to that of human muscle cells. We have now changed the reference for this point in the Methods. Moreover, because the muscle layer is expected to make little contribution to the force distribution, it is actually omitted in our simulations. Third, after looking into the literatures on the cuticle stiffness (including the one that the reviewer suggested), we think that there was still uncertainty on the modulus of insect cuticle (see below our discussion in the responses to the suggestion of reviewer 1). Therefore, we explored the parameter space by performing additional simulations using a lower value for the cuticle modulus (1 MPa). We showed that whether this modulus being 1 or 10 MPa had no effects on our conclusions. The new simulation results (Ecuticle=1 MPa) were now included in Figure 4—figure supplement 1 for comparison.

5. Please provide a minimal set of controls for behavioral data. This should include the driver control for the RNAi, the driver alone, and the driver plus RNAi. You have conducted these controls for the experiments related to cut but you are missing driver alone for Ca-alpha1D.

All checked and the missing controls were added.

6. Please address the points raised by the reviewers regarding the interpretation of the results, the statistics and grammatical errors. Among the missing references, it would be important to incorporate into the discussion the following paper from the Howard lab: Focal laser stimulation of fly nociceptors activates distinct axonal and dendritic Ca 2+ signals by Basak, Sutradhar and Howard (PMID: 34175294, DOI: 10.1016/j.bpj.2021.06.001). This paper shows that axonal activation of the c4da neurons can happen even in the absence of any dendritic calcium signals. This argues that any active properties of the dendrites only occur with very strong activation.

We thank the reviewers for all the suggestions. We have tried our best to improve the manuscript in these aspects. In particular, we have now added a paragraph to incorporate the discussion on the paper from the Howard lab (see Discussion, Line 457-481).

In the methodology, you should provide details about the creation of the piezo-GFP line (along with better imaging of its distribution in the dendrites). You should also add missing information about the regions of interest in which calcium responses were measured.

The Piezo-GFP line has been published7. We have now added information about how and where we measured the calcium responses in the figure legends.

Reviewer #1 (Recommendations for the authors):

Liu et al. present findings that significantly extend the understanding of molecular and cellular pathways of mechanical nociception in Drosophila larvae. They present a detailed analysis of the mechanical response properties of the nociceptors that is performed using a newly developed preparation for optical recordings from these cells. Using mechanical probes of varying tip diameters they are able to investigate the responses as they relate to force and pressure. Mutants in the cut gene, which show reduced branching of the nociceptors, are used to interrogate how the dendritic morphology structure might relate to their physiological responses. As well, the response profiles of the neurons that are mutant for mechanosensory channels ppk-26 and piezo are investigated. The ppk-26 mutant shows a more strongly impaired deficit in comparison to piezo mutants and double mutants show a nearly abolished mechanical response. The mechanosensory neurons are also found to respond to mechanical forces that are outside of the main dendritic fields which suggests that they are able to detect forces that are viscoelatically coupled through the overlying epidermis. Finally, a voltage activated calcium channel is found to be important which suggests that the dendrites of the nociceptive neurons are "active" (as opposed to passive). Overall, the study significantly advances our understanding of the response properties of the mechanical nociceptors of the Drosophila larva.

Questions for the authors:

The duration of the calcium responses long outlasts the force application. It has been previously proposed that dendritic breakage could be a contributing factor in the transduction mechanism in the Drosophila nociceptive neurons (Tracey, 2017). What is the relationship of dendritic breakage occur with the various force stimuli and probe sizes in the imaging setup? Sharp probes (smaller probes) may be more likely to break dendrites. Similarly, do the sharp probes that trigger rolling in Figure 2J break dendrites?

Please see our response to the first point in the above Editor’s summary. Briefly, within the range of forces and probe sizes used in this study, we did not observe significant dendrite breakage in both calcium imaging experiments and in the rolling behavior assays. We have now added data to make this point clearer (see Figure 1—figure supplement 1 in the revised manuscript). In addition, we showed that the responses of c4da to the force probes depends on Piezo and ppk1/ppk26, suggesting that mechanosensation, rather than chemosensation, is the major pathway underlying the calcium responses observed in this work.

We also noted that the duration of calcium responses was longer than that of force application. Our preliminary data suggest that intracellular calcium release (e.g. from ER) contributes to the calcium signal after the force application. The key molecule in this process is the ryanodine receptors (RyRs) in the membrane of ER. Drug treatment or knocking down the expression of the RyRs in c4da significantly could reduce the amplitude and duration of the force-evoked calcium responses (data not shown). This issue is currently under further investigation and we hope to clarify it in the near future.

The results of experiments with cut are very interesting, and the authors are open and cautious in their interpretation when they state the caveats of unknown epistatic effects that may result from removal of a transcription factor. Even with the stated caveats, the unknown effect of cut removal causes the results to be very difficult to interpret. It cannot be concluded that the deficits are a consequence of reduced branching. For example, perhaps calcium-alpha1d is a target.

We agree with the reviewer that the potential contribution of the unknown effect as a result of cut knockdown cannot be absolutely excluded. We now made this point clear in the revised Results and Discussion. On the other hand, the observations on the cut mutant are consistent with idea of the dendritic complexity being a contributing parameter underlying the signal-encoding mechanism of c4da. This argument is also supported by the observations in c3da. We have made open discussion on this issue (see Discussion, page 14-15).

Equation 3 assumes that the glass rod is completely rigid, if the rod bends during the stimulus then then d becomes on overestimate. Does the glass rod bend in this setup?

No. The glass probe does not bend as it is very short and has a strong base (see Author response image 1).

Author response image 1
The schematics for how the probe was mounted.

As shown in the picture, the force probe was firmly mounted in the double-layered holder. These glass probes were short and solid, so they would not be bent during the experiments.

Are force measurements in figure 1i made with a larval filet in the setup? Or is this the force applied to PDMS alone?

The forces were measured in the presence of a larval fillet.

Where are the values for the elastic modulus for larval cuticle coming from in line 543? Experimental measurements of the stiffness of larval cuticle come in at 0.39 +/- 0.01 MPa which is an order of magnitude lower than the values input to the model (10Mpa). Kohane M, Daugela A, Kutomi H, Charlson L, Wyrobek A, Wyrobek J. Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument during development". J Biomed Mater Res A. 2003 Sep 1;66(3):633-42. doi: 10.1002/jbm.a.10028

We think that the cuticle stiffness reported in the suggested literatures still has uncertainty for technical reasons. First, the two ends of the larvae were not fixed during the force application, which would lead to underestimation of cuticle stiffness. Second, the mechanical contribution of internal liquid pressure and the rigidity of the muscle layer are difficult to be separated from that of cuticle, which may lead to overestimation of cuticle stiffness. Based on these considerations, we decided to explore the parameter space of Ecuticle by performing additional simulations using a lower value for the cuticle modulus (1 MPa). We showed that whether this modulus being 1 or 10 MPa had no effect on our conclusions because the cuticle is thin in comparison to the PDMS pad. The new simulation results (Ecuticle=1 MPa) were now included in Figure 4—figure supplement 1 for comparison. Furthermore, we have added discussion on the mechanical differences between our preparation and the intact animals (see Discussion, second paragraph).

Third instar larval cuticle is closer to 20 microns thick according to experimental measurements. (Christine E. Kaznowski, Howard A. Schneiderman, Peter J. Bryant, "Cuticle secretion during larval growth in Drosophila melanogaster", Journal of Insect Physiology, Volume 31, Issue 10, 1985, Pages 801-813, https://doi.org/10.1016/0022-1910(85)90073-3).

10 μm is within the range of larval cuticle thickness based on our own EM observations (see Author response image 2) and others’ work, such as 8.

Author response image 2
Representative TEM micrographs on Drosophila larval fillet preparation.

The thickness of the cuticle was indicated. The samples were prepared using high pressure freezing and freeze substitution as previously described9.

The reference provided for elastic modulus of muscle comes from a study on human muscles and is therefore not valid for a study performed on Drosophila larvae.

We thank the reviewer for pointing out this mistake. We checked the values for the elastic modulus of insect muscle cells6, and it is similar to that of human cells. We have now changed the reference accordingly. It does not affect our simulation results because the muscle layer is expected to make little contribution to the force distribution, it was actually omitted in our simulations.

Is the force probe is compressing the entire larval filet such that there is an indentation into the PDMS (as depicted in figure 1A)? If so, then isn't it true that the forces on the filet (dendrites) are not coming solely from the force probe itself? If the larva is being squished between the probe and the PDMS, then the larva is also being exposed to an opposing force that is coming from the PDMS.

Yes, there is also a force from the PDMS. In physiological conditions, when the mechanical poking is applied to the larva, c4da also withstand forces from the other tissues, such as the muscle layers. The difference is that the rigidities of PDMS and the muscle layer are different and thereby would have different contributions to the overall tissue mechanics. We have now added discussions on the mechanical differences between our fillet preparation and the intact animals (see Discussion, second paragraph).

Figure 4D. Why "dendritic coverage threshold" instead of distance from dendrite? How do the predictions from this figure relate to real response data?

In our simulations, we tested different values for Cd (i.e. dendritic coverage threshold) and other parameters (data not shown) to explore the parameter space of our model. Figure 4D showed how the simulated probability of activation changes with the value of Cd. We think that this helps to understand how TL promotes the probability of neuronal activation and the robustness of this parameter in the model.

Regarding how cell activation changes with the distance from a dendrite, we have shown that the mechanosensitivity to TL would expand the radius of the force-receptive field of c4da for a few tens of micrometers (Figure 4B). When the probability of cell activation is calculated and plotted against the distance of force from a dendrite, similar conclusion can be made (Author response image 3). Both are consistent with the experimental observations (Figure 4A).

Author response image 3
The mechanosensitivity to lateral tension increases the probability of cell activation.

(A) The representative color map for the distance to the nearest dendrite, in which the distance was labeled by different colors. Scale bar, 100 μm. Random positions were chosen (the red crosses) as the force application points in the simulations (n=5 cells, 30 positions for each cell). (B-C) The plots of the cell activation probability versus the distance from a dendrite. Two types of probes were simulated, i.e. 30 μm (B) and 60 μm (C). Three conditions were considered: (1) only sensitive to PP; (2) only sensitive to TL; (3) sensitive to both PP and TL. The dendritic coverage threshold set was 20 μm. In panel B and C, data were presented as mean±std.

Could differences in distribution of Ca-alpha1D along the dendrite explain differences in proximal/distal sensitivities (ie. inhibition has less effect at proximal, maybe channels are less expressed/less important there?) Checking Ca-alpha1D localization would seem to be important.

Please see our response to the third point in the above Editor’s summary.

Figure 6A, the top soma trace seems really low, compared to the responses shown earlier with this probe size and force.

We checked the data and found that this trace was still within the range of responses. We now changed the images and curves to be more representative (see revised Figure 6A).

Please provide details of the creation of the piezo::GFP line and better imaging of the distribution in the dendrites.

The information about this strain has been published 7. We have now included this reference. This Piezo-GFP line is a knock-in strain, so the signal is quite weak. The image shown now in the revised manuscript is a representative one.

What is the frame rate of image acquisition? Are data from a single z-slice? Is pinhole wide open?

The frame rate is about 7.7 fps (130 ms per frame) and the images were taken using the confocal mode. The soma and dendrite usually get back to original vertical position with a brief latency (usually 1-2 s), which is shorter than the rising time of the calcium responses (~10 s).

In Line 75 please do not refer to flies and worms as "lower animals." Recommend substituting a term such as "invertebrates. "

Corrected.

In lines 152-153 it is stated that the c4da neurons use their entire dendritic field as the force-receptive field. However, the later experiments show that forces are detected through the viscoelastic coupling of the dendrites to the rest of the epidermis therefore this statement is not supported.

We have checked this issue throughout the revised manuscript and modified the text accordingly to be more precise.

In line 85 cite Zhong et al. 2010.

In line 163 Cite Cynthia Hughes et al. and he et al.

In line 166 please cite Tsubouchi et al. 2012 (which precedes Yan et al. 2013 in the demonstration of the gentle touch function for cIIIda neurons).

In Line 201 cite Robertson JL et al. 2013.

In line 296 cite Zhong et al. 2010, Mauthner et al. 2014, Gorczyca DA et al. 2014.

All cited. We thank the reviewer for these suggestions.

In line 385 calcium α 1- D is more precisely described as a calcium channel subunit.

Corrected.

Figure 1a cuticle is misspelled.

Corrected.

Reviewer #2 (Recommendations for the authors):

1. The citations are in some instances used somewhat loosely and do not appropriately reflect the literature. This should be amended in a revised version:

Lines 88-89: The selection of references cited for the downstream circuitry of c4da neurons seems random. Grueber et al. 2007 characterized axonal projections of c4da only, Burgos et al. and Yoshino et al. each described a class of c4da second order neurons that have not been characterized in regard to mechanical stimuli. If the authors wish to cite all studies then critical ones are missing including Ohyama et al. (Nature 2015), Kaneko et al. (Neuron 2017), Hu et al. (Nat Neurosci 2017) and Takagi et al. (Neuron 2017). The latter two studies actually characterized the c4da circuitry in regard to noxious mechanical stimuli, which seems most relevant here. Alternatively, Gerhard et al. (eLife 2017) provides the most complete description of the c4da second order circuitry.

Lines 201 and 230: Grueber et al. (Cell 2003) was the first study to show that cut determines c4da neuron morphology and should be cited here.

Corrected. We thank the reviewer for these suggestions.

2. Some discussion is warranted regarding the force used here (<5 mN) compared to the 5-10x higher forces typically used in previous publications studying rolling responses in live larvae (e.g. Kim et al. Nature 2012, Zhong et al. Curr. Biol. 2012, Gorczyca et al. Cell Rep. 2014, Guo et al. Cell Rep. 2014, Hu et al. Nat Neurosci 2017, Lopez-Bellido J Neurosci. 2019). While this is likely in part linked to the small diameter probes here vs. the typically larger probes utilized in previous studies, both the neuronal as well as behavioral responses to low mN stimuli seem to be extremely strong. For the neuronal activation, this might be linked to several features unique to the semi-intact preparation used by the authors vs. an intact animal. For example, the PDMS stiffness is much higher than that of a larval body filled with hemolymph, which will resist force manipulation much less, i.e. indentation depth is likely higher for a given force/pressure. This point should be adequately discussed and put into context with the literature.

Please see our response to the fourth point in the Editor’s summary. We have now added a paragraph in the Discussion (see Discussion, second paragraph) to clarify the mechanical differences between the semi-intact fillet preparation and the intact animal, including the discussion on the smaller forces used in the present study.

3. Relating to point 2, measuring larval rolling behavior using their different sized probes is interesting, yet the methodology was not so clearly explained in the methods or results. The authors indicate that the probes exerted a force between 10-20 mN, but do not indicate if the variability of individual probes depends on the diameter of the glass probe. As the pressure on the animal depends on probe size it might make sense to check if the measured force is dependent on the probe diameter and/or if response rates are just variable within the same range independently of the probe size.

We have now modified the corresponding part in Methods. We now included the data to show that the stimulating force was independent on the probe size (Figure 2—figure supplement 3 and page 6, line 189-191). Because the glass probes were all very short, they would not be deformed during the poking experiments. Therefore, all forces are reflected in the form of the bending of the Von Frey fiber, which are the same for all the probes.

4. Figure 2J: The legend is a bit confusing as the categories listed are <1, >1 and >2 turns. Previous studies typically identified a single roll while multiple rolling indicates severe tissue damage. Some of the tissue damage effects by different probe sizes and pressures have been studied in more detail suggesting that tissue damage is actually required for defensive behavior (Lopez-Bellido et al. J Neurosci 2019 and J Vis Exp 2020). The authors should check for tissue damage under their experimental conditions to exclude that excessive rupturing of the epidermis causes the observed strong behavioral reactions.

We have now used the same way as the previous studies to quantify the behavior responses. We have accordingly modified the figures and the texts in the Methods. Furthermore, we have checked the dendritic damage for both imaging and behavior experiments. Within the range of forces and probe sizes used in this study, we did not observe significant dendrite breakage (see Figure 1—figure supplement 1 in the revised manuscript).

5. In line 195 and 717, the labeling for this panel should be "J" instead of G.

Corrected.

6. It should be considered that the relative contributions of ppk/ppk26 and piezo might not exclusively reflect their function in c4da neurons alone. While ppk/ppk26 expression is cell type-specific, piezo is widely expressed in multiple tissues. Using a piezoko allele thus does not allow to draw conclusions on its function in c4da neurons. This would require either a cell type-specific manipulation or a c4da neuron specific rescue experiment.

We thank the reviewer for this suggestion. We have now performed c4da-specific rescue experiments for piezo to strengthen our conclusions (see revised Figure 5G and page 9, line 315-328).

It would help if the authors can model lateral tension and perpendicular pressure for the 30 micron probe. This might help to explain the different defects described in ppk26 vs. piezo loss of function.

We thank the reviewer for this suggestion. We have performed the additional simulations and the new data were now in the revised Figure 4.

Correlating the differential reliance of ppk26 and piezo on Ca-alpha1D function might be another interesting aspect, given that distal function of piezo to some extent resembles the one of Ca-alpha1D.

We thank the reviewer for the suggestion and feel that this is a very interesting issue to follow up. One key piece of information to understand this issue is the subcellular localization of Ca-α1D and the mutual dependence between Piezo and Ca-α1D. However, as we described in the responses to Editor’s summary, the subcellular localization of Ca-α1D in c4da was still not clear. Therefore, this issue would need to be further explored in future studies.

7. The model in Figure 7 could be improved as the color code and labels are not intuitive and the assignment of piezo vs ppk function is not clear to me. The model might also have to be revised according to the criticism regarding the assigned functions of ppk26 vs piezo.

In this model figure, what we would like to point out is that both Piezo and ppk1/ppk26 contribute to the mechanosensitivity to lateral tension. To make this point clear, we have modified this figure. In particular, we simplified the colors to avoid misunderstanding. We also edited the legends and the corresponding texts in Discussion accordingly. With respect to the roles of Piezo and ppk1/ppk26, we discussed their differential contributions in the Discussion.

Reviewer #3 (Recommendations for the authors):

– Figure 2, legend: (G) should read (J).

Corrected.

– Line 160: "To further justify the validity" – change to "To further test the validity".

Corrected.

– Line 66: not sure whether e.g. proprioceptors controlling locomotion aren't equally important for the survival of an organism.

Edited to be more precise.

– Line 390 ff: use past tense when reporting on earlier findings.

Checked.

References

1 Kim, S. E., Coste, B., Chadha, A., Cook, B. & Patapoutian, A. The role of Drosophila Piezo in mechanical nociception. Nature 483, 209-212, doi:10.1038/nature10801 (2012).

2 Terada, S. et al. Neuronal processing of noxious thermal stimuli mediated by dendritic Ca(2+) influx in Drosophila somatosensory neurons. Elife 5, doi:10.7554/eLife.12959 (2016).

3 Basak, R., Sutradhar, S. f. C. U. X. L. D.-s.-S.-m. p. & Howard, J. Focal laser stimulation of fly nociceptors activates distinct axonal and dendritic Ca(2+) signals. Biophys J 120, 3222-3233, doi:10.1016/j.bpj.2021.06.001 (2021).

4 Eberl, D. F. et al. Genetic and developmental characterization of Dmca1D, a calcium channel alpha1 subunit gene in Drosophila melanogaster. Genetics 148, 1159-1169, doi:10.1093/genetics/148.3.1159 (1998).

5 Hsu, I. U. et al. Stac protein regulates release of neuropeptides. Proc Natl Acad Sci U S A 117, 29914-29924, doi:10.1073/pnas.2009224117 (2020).

6 Zhang, Y., Pang, X., Yang, Y. & Yan, S. Effect of calcium ion on the morphology structure and compression elasticity of muscle fibers from honeybee abdomen. J Biomech 127, 110652, doi:10.1016/j.jbiomech.2021.110652 (2021).

7 Song, Y. et al. The Mechanosensitive Ion Channel Piezo Inhibits Axon Regeneration. Neuron 102, 373-389 e376, doi:10.1016/j.neuron.2019.01.050 (2019).

8 Kohane, M. et al. Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument during development. J Biomed Mater Res A 66, 633-642, doi:10.1002/jbm.a.10028 (2003).

9 Sun, L. et al. Ultrastructural organization of NompC in the mechanoreceptive organelle of Drosophila campaniform mechanoreceptors. Proc Natl Acad Sci U S A 116, 7343-7352, doi:10.1073/pnas.1819371116 (2019).

https://doi.org/10.7554/eLife.76574.sa2

Article and author information

Author details

  1. Zhen Liu

    School of Life Sciences, Tsinghua University, Beijing, China
    Contribution
    Conceptualization, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Meng-Hua Wu
    Competing interests
    No competing interests declared
  2. Meng-Hua Wu

    School of Life Sciences, Tsinghua University, Beijing, China
    Contribution
    Investigation
    Contributed equally with
    Zhen Liu
    Competing interests
    No competing interests declared
  3. Qi-Xuan Wang

    School of Life Sciences, Tsinghua University, Beijing, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  4. Shao-Zhen Lin

    Institute of Biomechanics and Medical Engineering, Department of Engineering Mechanics, Tsinghua University, Beijing, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Xi-Qiao Feng

    Institute of Biomechanics and Medical Engineering, Department of Engineering Mechanics, Tsinghua University, Beijing, China
    Contribution
    Supervision, Methodology
    Competing interests
    No competing interests declared
  6. Bo Li

    Institute of Biomechanics and Medical Engineering, Department of Engineering Mechanics, Tsinghua University, Beijing, China
    Contribution
    Supervision, Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  7. Xin Liang

    School of Life Sciences, Tsinghua University, Beijing, China
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    xinliang@tsinghua.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7915-8094

Funding

National Natural Science Foundation of China (31922018)

  • Xin Liang

National Natural Science Foundation of China (32070704)

  • Xin Liang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank Wei Zhang (Tsinghua University), Bing Zhou (Tsinghua University), Dan Tracey (Indiana University), Chun Han (Cornell University), Daniel Eberl (University of Iowa) and the Jan lab (UCSF) for sharing fly strains. We thank John Y Kuwada (University of Michigan) for sharing the antibody. Special thanks to the electron microscopy facility and fly facility in Tsinghua University. We acknowledge our funding from National Natural Sciences Foundation of China (31922018, 32070704), Tsinghua-Peking Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology and IDG/McGovern Institute for Brain Research (Tsinghua University).

Senior Editor

  1. Claude Desplan, New York University, United States

Reviewing Editor

  1. Matthieu Louis, University of California, Santa Barbara, United States

Reviewer

  1. Martin Göpfert

Publication history

  1. Received: December 21, 2021
  2. Preprint posted: January 5, 2022 (view preprint)
  3. Accepted: October 5, 2022
  4. Accepted Manuscript published: October 6, 2022 (version 1)
  5. Accepted Manuscript updated: October 7, 2022 (version 2)
  6. Version of Record published: November 21, 2022 (version 3)

Copyright

© 2022, Liu, Wu et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Zhen Liu
  2. Meng-Hua Wu
  3. Qi-Xuan Wang
  4. Shao-Zhen Lin
  5. Xi-Qiao Feng
  6. Bo Li
  7. Xin Liang
(2022)
Drosophila mechanical nociceptors preferentially sense localized poking
eLife 11:e76574.
https://doi.org/10.7554/eLife.76574
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    Maria Cecilia Martinez, Camila Lidia Zold ... Mariano Andrés Belluscio
    Research Article

    The automatic initiation of actions can be highly functional. But occasionally these actions cannot be withheld and are released at inappropriate times, impulsively. Striatal activity has been shown to participate in the timing of action sequence initiation and it has been linked to impulsivity. Using a self-initiated task, we trained adult male rats to withhold a rewarded action sequence until a waiting time interval has elapsed. By analyzing neuronal activity we show that the striatal response preceding the initiation of the learned sequence is strongly modulated by the time subjects wait before eliciting the sequence. Interestingly, the modulation is steeper in adolescent rats, which show a strong prevalence of impulsive responses compared to adults. We hypothesize this anticipatory striatal activity reflects the animals’ subjective reward expectation, based on the elapsed waiting time, while the steeper waiting modulation in adolescence reflects age-related differences in temporal discounting, internal urgency states, or explore–exploit balance.