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Method Article

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

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DOI:

10.3791/57557

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May 5th, 2018

 ,  ,  , 
1Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, 2Department of Pathology and Laboratory Medicine, University of Pennsylvania

* These authors contributed equally

In This Article

Summary

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

Abstract

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron – dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

Introduction

The inability of axons to regenerate after an injury to the central nervous system (CNS), may lead to permanent disabilities in patients, and also plays a role in the irreversible neurological deficits in neurodegenerative diseases1,2,3,4,5. The CNS environment, as well as the intrinsic growth ability of neurons, determines whether axons are able to regenerate after trauma. Extracellular factors from oligodendrocyte, astroglial, and fibroblastic sources have been shown to impede neuronal growth4,6,7,8, but the elimination of these molecules only allows for limited sprouting5. Intrinsic regeneration signals can influence regenerative success5,9 and represent potential therapeutic targets, but these processes are still not well-defined at the molecular level. Increases in trophic factor signaling or elimination of endogenous brakes, such as the Pten phosphatase10, can result in axonal regeneration in certain circumstances. Combinations of different methods found to be individually effective also only provide limited overall recovery to date11,12,13,14. Therefore, there is a desperate need to identify additional pathways for targeted therapy. In addition to the initiation of axon regrowth, whether and how axons re-wire to the correct target, reform synapse specificity, and achieve functional recovery are important unanswered questions.

In summary, current understanding of the machinery dictating axon regeneration is still very fragmentary. Part of the problem is the technical difficulty of studying axon regeneration in mammals in real-time, an approach that is costly, time-consuming, and challenging for conducting large-scale genetic screens. Drosophila melanogaster, on the other hand, has proven to be an exceptionally powerful system for the study of complex biological questions. The fruit fly has been instrumental in defining genes and signaling pathways that are strikingly conserved in humans and has been a successful model for the study of human conditions, such as neurodegenerative diseases, through the vast molecular genetics tools available to manipulate gene function15. In particular, fruit flies are considered to be an ideal tool for the discovery of genes involved in neural injury and regrowth15,16. Several fly neural injury models have been developed, including adult-head or larval ventral nerve cord (VNC) stabbing with needles, larval VNC or nerve crush with forceps, larval neuron laser axotomy, olfactory receptor neuron removal, brain explants injury, and peripheral nerve lesion by wing severance15,17,18,19,20,21,22,23. Excitingly, recent work utilizing Drosophila injury models have advanced our understanding of the cellular and genetic pathways used by the nervous system to respond to neural injuries, some of which have been shown to be conserved in mammals24,25. Again, this emphasizes the utility of this model organism for identifying novel mechanisms of neural repair.

Described here is a two-photon laser-based Drosophila larval sensory neuron injury model. A two-photon laser was first used to cut axons in zebrafish in vivo in 200326. In the same year, the first laser dendriotomy was performed in Drosophila using a pulsed nitrogen laser27. Shortly afterward, several C. elegans labs used femtosecond lasers to establish models of axon regeneration28. In 2007, Wu and colleagues compared and reported the differences between laser injuries in C. elegans induced by various types of lasers29. In 2010, axon regeneration after laser axotomy was first shown to occur in Drosophila30. Building on this extensive laser injury literature, we have developed a fly neural injury model using the two-photon laser, which allows precise induction of injury to targeted sites with minimal perturbation of neighboring tissues, providing a relatively clean system to study both the intrinsic and extrinsic properties of neuroregeneration with single-cell resolution. Specifically, we have established a set of injury methods for dendritic arborization (da) sensory neurons in both the peripheral nervous system (PNS) and CNS. Da neurons can be grouped into four distinct classes distinguished primarily by their dendrite branching complexities: class I to IV31. Our published work shows that da neuron regeneration resembles mammalian injury models at the phenotypic and molecular level: da neurons display class specific regeneration properties, with class IV but not class I or III da neurons exhibiting regeneration in the PNS; class IV da neuron axons regenerate robustly in the periphery, but their regenerative potential is dramatically reduced in the CNS, thus resembling dorsal root ganglion (DRG) neurons in mammals; enhancing mTOR activity via Pten deletion or Akt overexpression enhances axon regeneration in the fly CNS19. Utilizing this injury model, we have been performing genetic screens and have identified the RNA processing enzyme Rtca as an evolutionarily conserved inhibitory factor for axon regeneration, linking axon injury to cellular stress and RNA modification20.

In the presented paradigm, the injury is induced via laser axotomy/dendriotomy of larval class IV or III da neurons, labeled by ppk-CD4-tdGFP or 19-12-Gal4, UAS-CD4-tdGFP, repo-Gal80, respectively. The injury is performed on 2nd to 3rd instar larvae at around 48 - 72 h after egg laying (h AEL). For PNS axotomy the lesion is targeted to the section of axon ~20 - 50 µm away from the cell body, for CNS axotomy to an area of ~20 µm in diameter at the commissure junction in the VNC, and for dendriotomy to the primary dendritic branch points. The same neuron is imaged at 8 - 24 h after injury (AI) to confirm complete transection, and at 48 - 72 h AI to assess regeneration. Through time-lapse confocal imaging, the degeneration and regeneration of individual axons/dendrites that have been injured in vivo can be monitored over time.

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Protocol

1. Preparation of Culture Plates and Bottles

  1. Preparation of grape juice agar plates
    1. Add 10 g of agar powder, 200 mL grape juice, and 192 mL ddH2O into a beaker and microwave for about 4-5 min, stirring intermittently until the agar is completely dissolved.
    2. In a fume hood, cool down the solution to approx. 60 °C. Add 4.2 mL 95% ethanol and 4.0 mL glacial acetic acid. Adjust the total volume of the solution to 400 mL with ddH2O. Mix well.
    3. For each 35-mm plate, add about 2-3 mL of the solution. Make approximately 120-150 plates for a total of 400 mL grape juice agar solution.
    4. Wait for 10 min to cool down the plates and solidify the agar solution. Pack the grape juice agar plates in self-sealed ziplock bags and store at 4°C for future use.
  2. Preparation of Drosophila culture bottles
    1. Use a blade to punch a 1.5 cm side length triangular hole on one wall of the Drosophila culture bottle and fill the hole with a 2-2.5 cm diameter ball of cotton for ventilation.
    2. Plug the bottle with a grape juice agar plate supplemented with about 0.5 cm3 of yeast paste.

2. Collection of Drosophila Larvae

  1. Set up crosses of adult flies
    1. Place 10 virgin females and 5 male adult flies together in a culture bottle plugged with a grape juice agar plate.
    2. Place the bottle bottoms up at 25°C, so that flies will lay eggs on the grape juice agar plate. Change the plate with yeast paste at least once a day. For a 2-h collection period, which permits the harvesting of larvae of a homogenous developmental stage, start with more than 20 virgin females and 10 males in Step 2.1.1.
    3. Culture the plate at 25°C in a 60-mm Petri dish with a wet tissue, for example, soaked in 0.5% propionic acid solution. Arrange the tissue so that it does not block oxygen flow.
      NOTE: The propionic acid solution is used to maintain humidity in the dish and avoid the growth of mold.
  2. Harvest larvae of a specific age
    1. Use a pair of forceps to transfer larvae of the desired stage, for example, 2nd to 3rd instar larvae at 48-72 h after egg laying (AEL), to a new grape juice agar plate without yeast paste.
    2. Remove the yeast from the larvae's skin by letting them crawl around on the new plate, to prevent from potential interfering of yeast with laser axotomy and imaging. Alternatively, thoroughly clean the larvae by washing them in a dish of PBS and drying briefly on a piece of tissue paper.

3. Two-photon Injury and Confocal Imaging

  1. Microscope setup
    NOTE: A confocal laser scanning microscope with a two-photon laser was used for this experiment, but other systems with an equivalent setup will also suffice. The two-photon laser (930 nm) was used for delivering injury and an Argon laser (488 nm) was used for confocal imaging of GFP.
    1. At the beginning of each session, turn on the two-photon laser and/or the confocal laser(s), and the microscope. Open the imaging software.
    2. For two-photon injury, set up the following parameters for imaging GFP with the two-photon laser at 930 nm (1,950 mW).
      1. Select line scan mode. Open up the pinhole all the way. Increase laser intensity to ~20% (390 mW).
      2. Select 512 x 512 as the frame scan. Use maximal scanning speed (typically with the pixel dwell time at 0.77 µs). Ensure that the average number is 1 and bit depth is 8 bits.
      3. Set gain to ~750, and the offset to 0.
      4. Save this pre-set experimental protocol as 2P GFP 930 Ablation, allowing for easy reuse in future experiments.
    3. For confocal imaging, set up the following parameters for imaging GFP with the Argon laser at 488 nm:
      1. Select the Acquisition tab and then Z-stack.
      2. Under "Laser", turn on the power for the 488 nm Argon laser.
      3. Go to Channels, select the 488 mm laser, and increase the laser power to 5-10%. For the pinhole, use the 1-2 airy unit (AU). Adjust the gain to 650.
      4. In Acquisition Mode, select 1024 x 1024 as the frame scan, use the maximum scan speed, an average number of 2, and bit depth of 8 Bit.
      5. Save this pre-set experimental protocol as GFP Imaging.
  2. Larvae anesthesia with diethyl ether and mounting
    1. In a fume hood, place a 60-mm glass dish in a 15-cm plastic Petri dish. Fold and lay a piece of tissue paper at the bottom of the glass dish, then place a grape juice agar plate on the tissue. Add diethyl ether into the glass dish, to the point where the tissue paper is soaked and there is a layer of liquid ether remaining in the dish. Keep the lid on at all times.
    2. Prepare a glass slide with one drop of halocarbon 27 oil in the center. Add 4 spots of vacuum grease onto the four corners of the slide, to later support the coverslip.
    3. Use forceps to pick up a cleaned larva and place it on the agar plate in the 60-mm glass dish. Cover the glass dish with its lid and wait until the larva stops moving. For PNS injury/imaging, take out the larva as soon as its tail stops twitching. For the CNS, wait until the entire larva becomes motionless, especially the head segments.
      NOTE: The timing of ether exposure is critical. See Discussion.
    4. Carefully pick up the anesthetized larva and place it head-upright into the drop of halocarbon oil on the slide. Add a coverslip on top of the slide. Use gentle pressure to push down on the coverslip, until it touches the larva (Figure 1A).
    5. Adjust the larva's position by gently pushing the coverslip towards the left or right to roll the larva, so that the neuron/axon/dendrite of interest is on the top and closest to the microscope lens.
    6. For PNS injury, mount the larva dorsal side up, so that both the tracheas are visible. Then roll the larva ~30 degrees to the left for injuring class III da neuron axons (Figure 1B and 1C), 90 degrees for injuring class IV da neuron axons (Figure 1B and 1E), or ~30 degrees for injuring class IV da neuron dendrites (Figure 2A).
    7. For CNS injury, position the larva to be perfectly ventral side up (Figure 3A), so that the region of interest is closest to the microscope lens in the z-plane.
  3. Injury by two-photon laser
    1. Place the slide with the larva under the microscope and secure it in place with the slide holder on the stage. Use the 10X (0.3 NA) objective to find the larva.
    2. Add 1 drop of objective oil onto the coverslip, switch to the 40X (1.3 NA) objective and adjust the focus.
    3. Switch to the scanning mode and reuse the experimental protocol 2P GFP 930 Ablation. Make sure the pinhole is opened all the way.
      NOTE: The configuration needs to be optimized based on individual systems.
    4. Start the Live mode to locate the region of interest (ROI), and fine-tune the settings to achieve good image quality with the appropriate zoom.
      NOTE: The purpose of this step is to find the neuron/axon/dendrite to injure, rather than taking the best quality image. Therefore, use the minimal settings sufficient to visualize the target area, in order to avoid overexposure or photobleaching.
    5. Stop Live scan, so that the Crop button will become available. Let the still image serve as the roadmap. Select the Crop function and adjust the scan window to focus on the target to be injured.
    6. Reduce the ROI to be the size of the prospective site of injury. For example, just cover the width of an axon or a dendrite, to ensure the precision of the injury and reduce damage to neighboring tissues. If desired, zoom in on the ROI before cropping, allowing for more precise injury.
    7. Open a new imaging window. Reduce the scan speed and increase laser intensity. Determine the increase in laser intensity based on the tissue fluorescence signal scanned in Live mode.
    8. Typically, set the two-photon laser intensity starting from 25% for PNS injury and 50-100% for VNC injury. For PNS axon injury, ensure that the laser intensity is ~480 mW and pixel dwell time is 8.19 μs. For the VNC axon injury, ensure that the laser intensity and pixel dwell time are usually 965-1930 mW and 8.19-32.77 μs, respectively.
    9. Start Continuous scan. Leave the cursor hovering over the Continuous button. Keep a close eye on the image and stop the scan as soon as a drastic increase in fluorescence is observed.
      NOTE: The appearance of the fluorescence spike is due to auto-fluorescence at the injury site.
    10. Switch back to the Live mode by reusing the settings. Find the region of interest that was just targeted by adjusting the focus.
      NOTE: A good indication of successful injury is the appearance of a small crater, ring-like structure, or localized debris right at the injury site.
    11. Move to the next neuron and repeat from Step 3.3.5, to injure multiple neurons in a single animal. Or repeat Step 3.3.5 while gradually increasing the power and/or reducing scan speed if the initial injury was insufficient.
      NOTE: In the case that the laser power is too high, a large damaged area will be visible in the post-injury live scan image. Too much injury may cause the death of the larva.
    12. Recover the larva by carefully removing the coverslip and transferring the injured larva onto a new plate with yeast paste. Ditch several caves on the agar plate with forceps; alternatively, make an island of agar in the plate instead of using the whole plate, to reduce the possibility of the larva crawling out of the plate.
    13. Put the plate in a 60-mm Petri dish with wet tissue (soaked with 0.5% propionic acid solution) and culture at room temperature or 25°C.
      NOTE: The larva will remain in the larval stage for approximately an extra day at room temperature (22°C) compared to at 25°C.
  4. Post-injury confocal imaging
    1. Image the injured larva at desired time points by preparing the larva using the same procedure of anesthesia and mounting as in Step 3.2, then imaging with the confocal laser.
      NOTE: Image the larva at 24 h after injury (AI) to confirm axonal injury and at 48 h AI (class IV da neurons) or 72 h AI (class III da neurons) to assess regeneration.
    2. Locate the larva using the 10X objective, then switch to a 25X (0.8 NA) objective. Reuse the experimental protocol GFP Imaging.
    3. Click the Live button and find the same neuron injured previously.
    4. Set the first and last Z positions in the live scan window. Press stop and click Start experiment to acquire a Z-stack image.
      NOTE: Make sure that a normalization point (the axon converging point) is included when capturing images so that quantification of regeneration is possible (Figure 1D, 1F) – this is discussed further in the data analysis section.
    5. Switch to Image Processing, select the image just taken, and generate a maximum intensity projection. Save both the z-stack and maximum intensity projection images.

4. Data Analysis

  1. Process and quantify images using either the imaging software or ImageJ.
  2. Quantification of axon regeneration in the PNS
    1. Calculate Regeneration percentage, which refers to the percent of regenerating axons among all the axons that were lesioned. Score an axon as regenerating as long as it regrows beyond the injury site.
    2. Measure Regeneration length, which is the increase in axon length. If quantifying with ImageJ, use the Segmented Line tool to trace the regenerated axon, and use Measure in the Analyze drop-down menu to obtain the length of the line representing the regenerated axon.
    3. Calculate Regeneration Index, which is the increase in normalized axon length.
      NOTE: Axon length is normalized by the distance between the cell body and the axon converging point (DCAC) – axon length/DCAC (Figure 1D and 1F). This value helps account for any axonal growth that is due to larval scaling. A positive value represents regeneration, a value of 0 means no regeneration, and a negative value means retraction.
  3. Quantification of dendrite regeneration
    1. Calculate Regeneration percentage, which is the percent of da neurons among all those severed that show obvious dendrite regrowth.
      NOTE: Dendrite regrowth is scored as positive if new dendrites regrow out from the retracted dendritic stem and beyond the injury site. The injury site is determined by landmarks, and in some cases, is readily visible due to the residual injury-induced autofluorescence.
    2. Calculate Increase of branch points, which counts the addition of new dendritic branch points after injury.
    3. Calculate Increase of total dendrite length, which is the cumulative length of all the new dendrites added after injury.
  4. Quantification of axon regeneration in the CNS
    NOTE: If a lesion site shows degeneration at the first time point imaged (Figure 3B), it will be included in the analysis of regeneration length and rate.
    1. Measure Regeneration length, which is the length of the regrowing axon.
      NOTE: The regrowing axon of a single lesion site is identified as it originates off the original axon route before the injury.
    2. Calculate Normalized regeneration length, which normalizes the regeneration length to the length of the commissure segment - the longitudinal distance between commissures ("Y" in Figure 3A and 3B).
      NOTE: This value helps correct for the effect of larval size differences.
    3. Calculate Regeneration rate, which is the percent of regenerating segments among all the segments that were lesioned, to reflect the regeneration capacity of a particular genotype.

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Results

Da neurons show differential regeneration potential between the peripheral and central nervous system, as well as class specificity. This provides unique opportunities to screen for novel factors that are required for axon regeneration (using class IV PNS injury), as well as those that are inhibitory for regeneration (with class IV CNS injury and class III PNS injury).

Axon regeneration in the PNS

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Discussion

When setting up fly crosses, the number of females and males used can vary depending on the genotypes and the number of larvae needed for specific experiments. For WT flies, typical cross uses 10 females and 5 males. The collection window may be narrowed, depending on the accuracy of the larvae age required. For example, a 2-h collection period will yield larvae of a more homogenous population. In this case, using 20 or more virgin females to set up the crosses will help yield sufficient eggs. The yield from the first da...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Jessica Goldshteyn for technical support. Work in the Song lab is funded by the NIH grant R00NS088211, and the Intellectual and Developmental Disabilities Research Center (IDDRC) New Program Development Award.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Diethyl ether, ACS reagent, anhydrousAcros OrganicsAC615080010
Halocarbon 27 OilGenesee Scientific59-133
Phosphate buffered saline (PBS), 20x Concentrate, pH 7.5, supplier # E703-1LVWR97062-948 
Agar powder, Alfa Aesar, 500GMVWRAAA10752-36
Grape juiceWelch’s
Ethanol 95% (Reagent Alcohol 95%)VWR64-17-5
Acetic acidSigma-AldrichA6283
Propionic AcidJ.T.BakerU33007
Cover Glasses: RectanglesFisher Scientific12-544-D50 mm X 22 mm
Zeiss LSM 880 laser scanning microscopeZeiss
Zen softwareZeiss
Chameleon Ultra IICoherent

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