Previously titled : A fruit fly model for studying paclitaxel-induced pain

Background: Paclitaxel-induced peripheral neuropathy is a common and limiting side effect of an approved and effective chemotherapeutic agent. The cause of this nociception is still unknown. Methods: To uncover the mechanism involved in paclitaxel-induced pain, we developed a Drosophila thermal nociceptive model to show the effects of paclitaxel exposure on third instar larvae. Results: We found that paclitaxel increases heat nociception in a dose-dependent manner, and at the highest doses also obstructs dendritic repulsion cues. Conclusions: Our simple system can be applied to identify regulators of chemotherapy-induced pain and may help to eliminate pain-related side-effects of chemotherapy.


Introduction
Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of many effective cancer treatments (Burton et al., 2007), and can have a lasting impact on the quality of life of cancer survivors (Hausheer et al., 2006 andShimozuma et al., 2012). A meta-analysis of 31 studies from over 4000 chemotherapy-treated patients revealed that CIPN was prevalent in 68.1% of patients in the first month following chemotherapy, in 60% of patients at 3 months, and in 30% at 6 months or more (Seretny et al., 2014).
While much knowledge has been gained about the genetics of pain from vertebrate systems, high-throughput dissection of pain is possible using the fruit fly Drosophila melanogaster (Neely et al., 2010). When challenged with a noxious thermal stimulus, third instar larvae exhibit an aversive escape response that has been utilised to identify conserved genes required for nociception (Babcock et al., 2009;Neely et al., 2010;Tracey et al., 2003). This nociceptive response is a result of activating class IV multidendritic-dendritic arborisation (md-da) sensory neurons at the site of stimulation (Hwang et al., 2007). Previously in Drosophila, paclitaxel has been reported to be toxic in somatic cells, and causes loss of axons in peripheral nerves. (Bhattacharya et al., 2012;Cunha et al., 2001). However, its effects on nociception have not yet been evaluated. Here, we examined the effects of paclitaxel exposure on the fruit fly larval nociception system, and observed a robust and dosedependent increase in pain perception. This system is amenable to high throughput screening and genetic manipulation (Honjo, et al., 2016), and may help define why chemotherapies such as paclitaxel cause pain.

Drosophila treatment
All flies were reared at 25°C and 65% humidity over a 12-hour light-dark cycle. Six female and two male Canton S Drosophila melanogaster were mated on food medium (5.4% sucrose, 3.6% yeast, 1% agar, 1.2% nipagin, and 0.6% propionic acid) treated with ethanol (vehicle), 0 µM, 0.1 µM, 0.5 µM, 2.5 µM, 5 µM or 10 µM paclitaxel (Taxol®; Catalog No. A4393) purchased from ApexBio (Houston, USA). A stock of 1000 µM paclitaxel in ethanol was prepared and diluted in food medium accordingly to create the different drug concentrated food. F0 Flies were discarded two days after mating and F1 larvae were left to grow for another three days. On the sixth day, early third instar were collected to assess nociception or dendritic morphology.

Behavioural assay
For the thermal nociceptive assay (Tracey et al., 2003), distilled water was added to experimental vials to soften the food and release the foraging third instar larvae. The softened, liquid food was then passed through mesh to catch the larvae to be transferred to a 100mm petri dish sprayed with distilled water. The larvae were touched laterally on abdominal segments four to six with a heat probe (soldering iron with narrow tip) set to 42°C or 46°C. The rolling response was measured in seconds with a cut-off of 10 seconds. For each drug concentration, five repeats were performed, with 30-40 larvae per repeat.
Live confocal microscopy and image analysis Third instar larvae (ppk-Gal4,20xUAS-mCD8-GFP) were collected, washed, and placed dorsal side up on a microscope slide, immobilized in 1:5 (v/v) diethyl ether to halocarbon oil and covered with a 22 × 50 mm glass coverslip (Das et al., 2017). A Nikon C2 Confocal microscope was used to image GFPexpressing class IV md-da sensory neurons at abdominal segment 2 (A2), under a 20x magnification. Images of Z-stack sections were captured at 1024 × 1024 pixel resolution and representative images were captured at 2048 × 2048 pixel resolution, both with 2x averaging. Z-stack images were converted to maximum intensity projection using ImageJ and automated Sholl analysis was performed on these images. Terminal branches were counted manually. 13 animals were imaged for each treatment. All experiments were conducted in a blinded manner.

Statistical analysis
Data represent mean ± SEM and are compared to vehicle control. Analysis was done using GraphPad Prism 5. Statistical analysis for response time was done using Krustal-Wallis, followed by Dunn's pairwise test for multiple comparisons. Statistical analysis for area under the curve mean, terminal branches,

Amendments from Version 1
This version of the article was revised to include new data on the effect of paclitaxel exposure on the morphology of peripheral pain sensing neurons. In version 1, we did this by dissecting, fixing and mounting the larvae, followed by confocal microscopy and image analysis ( Figure 2). However, the dissection method masked the intricate structural changes and we did not see a difference between paclitaxel treatment and vehicle control. In version 2, we instead used live confocal microscopy, and we found that paclitaxel obstructs dendritic repulsion cues at the highest doses (updated Figure 2).
Moreover, in this version, we have addressed the reviewer's comments and also updated all the figure to include all the data points.

Results
Our goal here was to develop a reproducible paradigm to investigate the effects of paclitaxel on nociception in the fly larvae. Based on previous studies for toxicity (Bhattacharya et al., 2012;Cunha et al., 2001), we selected paclitaxel doses below the lethal limit ( Figure 1A), and then tested larval nociception using a heat probe set to a low intensity noxious heat (42°C; Figure 1B), which is mildly nociceptive to fly larvae (Babcock et al., 2009). Our dose-response study revealed 2.5 µM paclitaxel was sufficient to induce significant hyperalgesia, with a maximal hyperalgesia effect observed at 10 µM ( Figure 1C, d = 0.54). Concentrations higher than 10 µM paclitaxel were 100% lethal (not shown). Paclitaxel did not significantly alter heat nociception latency to a 46°C heat stimulus across any of the doses ( Figure 1D, d = 0.17). Vehicle (ethanol) control and normal (no ethanol) control showed a response time of 5.71 sec (±0.23 SEM; n=173) and 5.62 sec (±0.20 SEM, n=180, not shown), respectively (42°C; Figure 1E). At low concertation's of 0.1 µM (5.21 sec ± 0.23 SEM; n=150) and 0.5 µM (5.44 sec ± 0.26 SEM; n=131) paclitaxel did not affect response profiles, however, concentrations of 2.5 µM paclitaxel (4.22 sec ± 0.19 SEM; n=180; p<0.001) and higher altered response distribution and significantly enhanced nociceptive latency (42°C; Figure 1E). The fastest latency response was observed at 10 µM paclitaxel (3.84 sec ± 0.24 SEM; n=140; p<0.001) with a 36.6% increase in response time relative to vehicle control ( Figure 1C).
To evaluate if paclitaxel exposure caused robust morphological differences in peripheral pain sensing neurons, we fed genetically labelled (ppk-Gal4,20xUAS-mCD8-GFP) larvae paclitaxel and imaged the sensory neuron structure (Figures 2A-B). Treating larvae with 10 µM paclitaxel affected its repulsive cues with like neurons, overlapping and forming a closed circular structure ( Figure 2B, orange box) compared to vehicle control (Observed in 5 paclitaxel treated animals compared to 0 control animals, Fisher's Exact Test p < 0.05). In some paclitaxel treated larvae we observed very short dendritic arbors with lower GFP intensity ( Figure 2B', open arrowhead). This was not observed in vehicle control larvae (Figure 2A'). We next used Sholl analysis to quantify branch distribution with a focus on number of intersections as a function of distance from the cell soma. This revealed increased branching closer to the cell soma in paclitaxel treated larvae compared to control ( Figure 2C). Area under the curve (AUC) was also calculated for each animal and mean AUC was also plotted for vehicle control (3894 ± 122, n=13) and 10 µM paclitaxel treatment (4329 ± 145.7, n=13) ( Figure 2D). Treatment with paclitaxel significantly increased the area under the curve compared to vehicle control ( Figure 2D, p < 0.05). We also determined maximum branch number and its critical radius and found paclitaxel treatment compared to vehicle control did not have a significant effect on maximum branch number (62.62 ± 2.69; n=13 control and 61.28 ± 2.72; n=13 paclitaxel) or critical radius (177.1 ± 6.78; n=13 control and 192.1 ±7.70; n=13 paclitaxel) ( Figures 2E-F). Finally, paclitaxel did not significantly affect terminal branch number compared with vehicle control ( Figure 2G). Paclitaxel fed larvae were touched with a 42°C heat probe and their response time was measured in seconds with a cut-off of 10 seconds. Different treatments were tested: food control, ethanol control, 0.1 µM, 0.5 µM, 2.5 µM, 5 µM, and 10 µM paclitaxel. Five repeats were performed (n = 130 -180). Paclitaxel fed larvae were touched with a 46°C heat probe and their response time was measured in seconds with a cut-off of 10 seconds. Different treatments were tested: food control, ethanol control, 0.1 µM, 0.5 µM, 2.5 µM, 5 µM, and 10 µM paclitaxel. Five repeats were performed (n = 130 -180). Confocal images of vehicle control and 10 µM paclitaxel treated larvae. Images represent class IV md-da neurons at abdominal segment A2. Images are at 20x magnification with 2x averaging. Scale bar represents 100 µm.

Discussion
Here we report a simple, high-throughput genetically tractable system to dissect the mechanisms of CIPN in Drosophila. Some effective and common chemotherapeutic agents such as paclitaxel cause peripheral neuropathy in a dose-dependent manner, limiting its therapeutic potential. Hyperalgesia, hypoalgesia and allodynia are some of the common side effects experienced by patients (Boland et al., 2010). By utilising a conserved hyperalgesia response, we performed a dose-finding study to determine the best drug dose to further investigate mechanisms for how paclitaxel causes pain. Our findings in Drosophila larvae are reminiscent of human patients, where paclitaxel increased pain sensitivity in a dose-dependent manner (Burton et al., 2007).
Drosophila experience a nociceptive response by activation of class IV md-da neurons at the site of stimulation. These neurons form extensive, space filling dendritic arbors that exhibit repulsive characteristics where they do not overlap with neighbouring dendrites but instead terminate projection or make abrupt turns (Grueber et al., 2007). In our system, we found that treatment with paclitaxel obstructs these dendritic guidance cues, leading to an overlap of dendritic arbors. This may be due to paclitaxel's effect on mitotic spindles where it binds to beta-tubulin, stabilizing its polymerization, leading to a disruption of the microtubule organization, and thus impacting microtubule-based dendritic guidance (De Brabander et al., 1981;Parness & Horwitz, 1981;Rowinsky et al., 1988;Schiff & Horowitz, 1980). Paclitaxel's unknown neuropathic mechanism may be related to its effects on microtubule function and axonal transport. Our simple system may be used with genomic Average nociceptive latency (in seconds) in response to a 42°C or 46°C thermal stimulus, respectively. Increased paclitaxel concentration significantly induces heat-hyperalgesia in third instar larvae at 42°C. Note concentrations higher than 10 µM paclitaxel were 100% lethal. E) Percentage response to each time point in seconds to 42°C thermal stimulus. All values represent mean ± SEM. p values were generated using Krustal-Wallis, followed by Dunn's pairwise test for multiple comparisons. Significance is relative to vehicle control. Five repeats were performed for each drug concentration with roughly 30 larvae each (n = 130-180 animals). approaches to dissect this mechanism and identify regulators of chemotherapy pain. Together this work can lead to a better understanding of how the pain arises, and potentially avoid these severe side effects while more effectively targeting the underlying disease.

Grant information
This work was supported in part through NHMRC project grants APP1026310, APP1029672, APP1028887, APP1046090, APP1042416, APP1086851, and by a NHMRC career development fellowship II CDF1111940.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
1. I found only two aspects of the work that are perhaps overlooked and could use a couple of notes in the discussion:

Open Peer Review
The work focuses on the effects of only one drug. The title appropriately refers to paclitaxel indeed, but it would be interesting to speculate on whether we could expect the same response using other drugs too. Larvae are developing organisms. Their neuronal network changes as they grow from instar to instar. CIPN, on the other hand, is normally observed in post-developmental conditions. Can we assume that the changes in synaptic structures reported in figure 2 would be observed in a fully developed nervous system too? I think adding a couple of lines of speculation regarding point 1 and 2 would strengthen the paper.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed. Competing Interests:

1.
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 09 Oct 2018 , University of Sydney, Australia Greg Neely Thank you for your comments.
This effect is not seen at 46°C. At this temperature intensity, larvae Response to comment #1: respond rapidly (~1.5 seconds) and it is difficult to see even faster responses. To look for hyperalgesia, we instead lowered the heat stimulus intensity to 42°C, which is at the threshold for nociception in this system, and where nociceptive responses take on average ~5 seconds to elicit.
The type IV multidendritic nociceptor neurons that transduce heat Response to comment #2: nociception also transduce mechanical nociception, as these neurons are multimodal. We have tried on numerous occasions to generate reproducible data for mechanical nociception but so far in our hands this assay does not work well enough for us to feel comfortable publishing. Given the multimodal nature of type IV multidendritic nociceptor neurons, we reasoned that thermal hyperalgesia is a good readout for the overall sensitization of these sensory neurons.
The animals are born into paclitaxel containing food, and then early Response to comment #3: third instar are collected at day 6 to assess nociception or dendritic morphology. This information was provided in the methods, however we have further clarified this aspect.
We have now written a short discussion, please see discussion Response to comment #4: section.
No competing interests were disclosed.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com