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Behavioral and Functional Assays for Investigating Mechanisms of Noxious Cold Detection and Multimodal Sensory Processing in Drosophila Larvae
行为和功能分析实验研究蝇幼虫感知有害寒冷及处理多模态感知的机制   

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Abstract

To investigate cellular, molecular and behavioral mechanisms of noxious cold detection, we developed cold plate behavioral assays and quantitative means for evaluating the predominant noxious cold-evoked contraction behavior. To characterize neural activity in response to noxious cold, we implemented a GCaMP6-based calcium imaging assay enabling in vivo studies of intracellular calcium dynamics in intact Drosophila larvae. We identified Drosophila class III multidendritic (md) sensory neurons as multimodal sensors of innocuous mechanical and noxious cold stimuli and to dissect the mechanistic bases of multimodal sensory processing we developed two independent functional assays. First, we developed an optogenetic dose response assay to assess whether levels of neural activation contributes to the multimodal aspects of cold sensitive sensory neurons. Second, we utilized CaMPARI, a photo-switchable calcium integrator that stably converts fluorescence from green to red in presence of high intracellular calcium and photo-converting light, to assess in vivo functional differences in neural activation levels between innocuous mechanical and noxious cold stimuli. These novel assays enable investigations of behavioral and functional roles of peripheral sensory neurons and multimodal sensory processing in Drosophila larvae.

Keywords: Nociception(伤害感受), Noxious cold(有害寒冷), Multimodal sensory processing(多模态感知处理), Calcium imaging(钙成像), Optogenetics(光遗传学), Drosophila(果蝇)

Background

The capacity to sense and respond appropriately to environmental cues is one of the most fundamental aspects shared among the metazoans. Sensing potentially harmful stimuli, such as noxious temperature, chemical or mechanical insults, and responding appropriately is crucial for avoiding incipient damage that can lead to injury or death. Typically, upon sensing nociceptive stimuli an animal produces a set of avoidance behaviors that either mitigate or allow the animal to escape the noxious stimulus. Elucidating molecular, cellular, and behavioral level mechanisms in processing nociceptive stimuli is of great interest as there is potential for the identification and development of novel therapeutic interventions for aberrant sensory processing, which can lead to neuropathic pain. Sensory and behavioral responses to noxious chemical, mechanical and heat stimuli have been elucidated in Drosophila melanogaster larvae and adults, however, noxious cold detection has only recently been discovered in larvae (Im and Galko, 2012; Gorczyca et al., 2014; Guo et al., 2014; Mauthner et al., 2014; Turner et al., 2016). Drosophila larvae exhibit a distinct set of aversive behaviors in responses to noxious cold stimuli with the predominant cold-evoked response displaying as a bilateral anterior-posterior full body contraction (CT) (Turner et al., 2016). This behavioral response is mediated by class III md sensory neurons (Turner et al., 2016), which intriguingly have also been implicated in gentle touch mechanosensation revealing multimodality in these neurons (Tsubouchi et al., 2012; Yan et al., 2013). The Transient Receptor Potential (TRP) channels Pkd2, NompC, and Trpm are required for mediating noxious cold-evoked behavior and behavioral selection in response to innocuous mechanical vs. noxious cold stimuli is dependent upon class III neural activation levels providing insight into the mechanisms underlying cold nociception and multimodal sensory processing (Turner et al., 2016).

Materials and Reagents

  1. Kimwipe (KCWW, Kimberly-Clark, catalog number: 34155 )
  2. 25 x 75 mm microscope slide (Globe Scientific, catalog number: 1301 )
  3. 22 x 22 mm No.1 thickness coverslip (Globe Scientific, catalog number: 1401-10 )
  4. 24 x 50 mm No. 1 thickness coverslip (Genesee Scientific, catalog number: 29-118 )
  5. Pyrex 9 well glass spot plates (Fisher Scientific, catalog number: 13-748B)
    Manufacturer: Corning, PYREX®, catalog number: 7220-85 .
  6. Amber glass dropper bottles (Fisher Scientific, FisherbrandTM, catalog number: 02-983B )
  7. Bel-ArtTM SP SciencewareTM wide mouth color-code safety labeled wash bottles (Fisher Scientific, catalog number: 22-288654)
    Manufacturer: SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F11646-3739 .
  8. Bel-ArtTM SP Scienceware Trigger Sprayers with 53 mm adapters (Fisher Scientific, catalog number: 01-189-100)
    Manufacturer: SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F11620-0050 .
  9. Polypropylene vials (Genesee Scientific, catalog number: 32-120 )
  10. Droso-Plugs, Narrow vials (Genesee Scientific, catalog number: 59-200 )
  11. Drosophila stocks:
    1. ChETA: y1 w*; wgSp-1/CyO, P{Wee-P.ph0}BaccWee-P20; P{20XUAS-CHETA.YFP}attP2/TM6C, Sb1 Tb1 (Bloomington Drosophila Stock Center, catalog number: 36495 )
    2. CaMPARI: w*; P{UAS-CaMPARI}attP40 (Bloomington Drosophila Stock Center, catalog number: 58761 )
    3. GCaMP6 (medium variant): w1118; PBac{20XUAS-IVS-GCaMP6m}VK00005 (Bloomington Drosophila Stock Center, catalog number: 42750 )
    4. Class III md neuron driver: GAL419-12 and GAL4nompC (Bloomington Drosophila Stock Center, catalog numbers: 36369 and 36361 ) (Turner et al., 2016)
    5. Class IV md neuron driver: GAL4pp1.9 and GAL4477 (Turner et al., 2016)
    6. Control strain: w1118 (Bloomington Drosophila Stock Center, catalog number: 3605 )
  12. All trans-Retinal (ATR) (Sigma-Aldrich, catalog number: R2500 )
  13. Halocarbon oil #700 (LabScientific, catalog number: FLY-7000 )
  14. Ethyl ether anhydrous (Fisher Scientific, catalog number: E138-500 )
  15. NutriSoy, Soy Flour (Genesee Scientific, catalog number: 62-115 )
  16. Yellow cornmeal (Genesee Scientific, catalog number: 62-101 )
  17. Drosophila agar type II (Genesee Scientific, catalog number: 66-104 )
  18. Inactive dry yeast (Genesee Scientific, catalog number: 62-107 )
  19. Dry molasses (Genesee Scientific, catalog number: 62-119 )
  20. O-phosphoric acid (Fisher Scientific, catalog number: A242-212 )
  21. Propionic acid (Fisher Scientific, catalog number: A258-500 )
  22. Drosophila media (see Recipes)

Equipment

  1. Cold plate assay
    1. Brush (Craft Smart® round brush set golden taklon) (Michaels Stores, model: Size 3, catalog number: 10408282 )
    2. Nikon body plus lens combination (Nikon, model: D5300 ) and AF-S Nikkor 18-55 mm DX VRII (Nikon, model: AF-S DX )
    3. Tripod for mounting DSLR
    4. Cold plate cooler (TE Technology, model: CP-031 )
    5. Cold plate temperature controller (TE Technology, model: TC-720 )
    6. Cold plate power supply (TE Technology, model: PS-12-8.4A )
    7. Infrared thermometer (Fluke, model: Fluke 62 MAX )
    8. Aluminum plate–Laminated aluminum shim (Global Equipment, catalog number: WBB512969 )
      Notes:
      1. Cut the shim to 7.5 by 11.5 mm at 0.22 mm thickness.
      2. Paint the plate with black spray paint for high contrast.
    9. Black spray paint–12 oz. black flat general purpose spray paint (Rust-Oleum)

  2. In vivo GCaMP assay
    1. PE120 Peltier stage (Linkam Scientific Instruments, Linkam Scientific, model: PE120 )
    2. T95 system controller (including T95 linkpad and PE95) from (Linkam Scientific Instruments, Linkam Scientific, model: T95 )
    3. Laser confocal microscope capable of imaging GFP (Carl Zeiss, model: LSM 780 )

  3. Optogenetic dose response assay
    1. D5300 DSLR (Nikon, model: D5300 )
    2. Adapters for mounting Nikon DSLR onto Zeiss microscopes: T2-adapter for Nikon F (Carl Zeiss, model: T2-adapter, catalog number: 416009-0000-000 ) and Adapter 60N–T2 1.0x (Carl Zeiss, model: Adapter 60N, catalog number: 426103-0000-000 )
    3. Glass plate: 10 x 15 x 0.1 cm

  4. CaMPARI Ca2+ integrator assay
    1. Photo-conversion filter cube
      1. 612/69 BrightLine bandpass filter, 25 mm (IDEX Health & Science, Semrock, catalog number: FF01-612/69-25 )
      2. 440 nm BrightLine SWP edge filter, 25 mm (IDEX Health & Science, Semrock, catalog number: FF01-440/SP-25 )
      3. 562 BrightLine dichroic beamsplitter, 25.2 x 35.6 mm (IDEX Health & Science, Semrock, catalog number: FF562-Di03-25x36 )
    2. Axio Zoom.V16 (ZEISS, model: Axio Zoom.V16 ) with Illuminator HXP200c lamp (Carl Zeiss, model: HXP 200C )
    3. Light touch stimulus: Nickel plated pin holder (Fine Science Tools, catalog number: 26018-17 ) with mounted single fine paint brush bristle
    4. Noxious cold stimulus: TE technology cold plate cooler described in cold plate assay

  5. Drosophila media preparation
    1. Adventurer Pro II Analytical/Precision Balance (Ohaus, model: AX2202 )
    2. FastPette V2 Pipette Controller (Labnet International, catalog number: P2000 )
    3. 10ml Serological Pipets (Genesee Scientific, catalog number: 12-104 )
    4. Droso-Filler, Narrow (Genesee Scientific, catalog number: 59-168 )
    5. Avantco Induction Range (Avantco Equipment, model: IC3500 )
    6. Stainless Stock Pot with lid (Thunder Group, catalog number: SLSPS020 )
    7. Narrow Fly Vial Reload Tray (Genesee Scientific, catalog number: 59-207 )

Software

  1. ImageJ (https://imagej.nih.gov/ij/)
  2. Video to video converter (http://www.videotovideo.org/)
  3. Zeiss Zen Blue Lite (https://www.zeiss.com/microscopy/us/products/microscope-software/zen-lite.html)

Procedure

  1. Cold plate assay
    Note: The following procedure describes how noxious cold stimulus is delivered to the ventral surface of D. melanogaster larvae. Larvae are placed on a black metal plate, to enable high contrast imaging of larvae, allowed to resume locomotion and then exposed to noxious thermal stimulus by placing the black metal plate on a chilled Peltier plate. The benefit of using this assay to assess noxious cold evoked behavioral responses is that this assay exposes the entire ventral surface of the animal to noxious cold stimulus and the behavior can be quantitatively assessed (see Data analysis section).
    1. Cold plate assay
      1. Prepare genetic cross of interest with approximately 30-40 virgins and 20-25 males. Place the vial in 29 °C. Allow adult flies to mate for two days and then transfer to a new vial to perform a timed egg collection for 4-6 h at 29 °C. After 4-6 h remove adult flies and age the vial to 96-102 h after egg lay (AEL) corresponding to the third instar larval stage of development.
      2. Behavioral assays should be performed in a diffusely lit room.
      3. At 96-102 h AEL, turn on the Peltier cold plate and set to desired temperature (6 °C). Allow few minutes for the plate to reach desired temperature and critically, make sure that the plate is able to hold desired temperature as measured with an infrared thermometer.
        Note: At 6 °C make sure to wipe off excess condensation.
      4. Turn on recording equipment, Nikon DSLR and lens set to 55 mm placed roughly 18 cm above the focal plane of the plate.
      5. Select 6-9 third instar larvae (Figure 1A) at a time with a brush for cold plate assay and rinse them gently with tap water in a 9-well glass spot plate.


        Figure 1. Set up of cold plate assay. A. High magnification image of Drosophila third instar larva (scale bar represents 1.7 mm); B. 1. Nikon DSLR camera mounted on tripod, 2. TE Technology Peltier plate, 3. TE Technology temperature controller, and 4. TE Technology power supply. C. Cold plate assay of w1118 third instar larvae exposed 6 °C at 0 sec; D. Cold plate assay of w1118 third instar larvae exposed to 6 °C at 3 sec. Larvae exhibit contraction (CT) behavior in response to noxious cold stimulation.

      6. Quickly, transfer them to a wet Kimwipe.
      7. Apply a thin fine mist of water via a trigger sprayer on a black metal plate.
        Note: Too much water on the plate will create noise during the ImageJ/Fiji quantification steps.
      8. Using a brush gently pick up each larva and place them on the metal plate. After all the larvae have been placed on the plate, allow roughly 30 sec for the larvae to acclimate and resume peristaltic movement on the plate.
        Note: Make sure there are no residual food particles on larvae, excess water has not accumulated around the larvae, and there is enough space between larvae (Figure 1B).
      9. Place the metal plate on the Peltier plate very gently so that light touch stimuli do not confound noxious thermal assay. Record the animals for up to 30 sec at 30 frames per second (Figure 1B and Video 1).

        Video 1. Cold plate assay. Video of a cold plate assay setup.

      10. Repeat until desired number of larvae are recorded.
      11. Representative image stills (Figures 1C and 1D) and videos of baseline larval behavior at 25 °C (Video 2) and in response to noxious cold stimulation (6 °C) (Video 3). At baseline temperature (25 °C) larvae exhibit peristaltic locomotion and head turning behavior consistent with surveying their environment, whereas upon noxious cold stimulation (6 °C), larvae exhibit bilateral anterior-posterior contraction (CT) behavior.

        Video 2. Larval behavior at baseline temperature. Video of w1118 larvae at 25 °C.

        Video 3. Larval behavior in response to noxious cold. Video of w1118 larvae at 6 °C.

  2. In vivo calcium imaging via GCaMP
    Note: We designed an experimental paradigm for imaging transient calcium responses in live intact D. melanogaster larvae using modern confocal imaging and fluorescence technology. These novel in vivo analyses are facilitated by investigations of larval md sensory neurons that are located just beneath a semi-transparent cuticle. We use larval md neuron class specific GAL4 drivers to direct the expression of GCaMP6, a transient fluorescent intracellular Ca2+ sensor. To assess in vivo GCaMP responses to noxious cold exposure we delivered stimuli via a Linkam Peltier system. Using this method one can assess GCaMP calcium responses in any PNS neuron in a live animal preparation.
    1. Prepare before setting up genetic cross or stock for calcium imaging.
      1. Prepare an amber glass dropper bottle containing halocarbon oil #700. This will be used for microscope slide preparation for in vivo imaging.
        Note: Water will also work, but halocarbon oil #700 has greater viscosity that is ideal for mounting 22 x 22 mm coverslips not moving.
      2. Optimize laser confocal microscope time-lapse imaging settings.
        Notes:
        1. Make sure that laser power and gain settings do not saturate the neuron throughout time-lapse acquisition period.
        2. Set the highest possible image acquisition rate. At maximum image acquisition rate should be 1 frame per second for proper temporal resolution.
        3. Image resolution should be at least 256 x 256 pixel resolution. There will ideally have to be a balance between image resolution and image acquisition rate.
    2. Set up genetic cross or stock, as described above for the cold plate assay, prior to calcium imaging and place the vial in 29 °C.
    3. Age embryo collection in food vial to 96-102 h AEL and select third instar larvae for imaging.
    4. Set up water circulation system, Linkam PE 120 Peltier stage, mount Peltier stage onto the laser confocal microscope, and turn on confocal microscope (Figures 2A and 2B).
      Notes:
      1. Use double sided tape to mount the Peltier stage onto microscope stage.
      2. Allow time for lasers to stabilize and maximal water circulation.


        Figure 2. In vivo GCaMP set up, temperature profile and class III md neuron GCaMP response. A. View of water circulation tank (1), temperature programmer (2), temperature controller (3), and Zeiss laser confocal (4). B. Close up of laser confocal microscope stage (1) with Peltier stage (2); C. Close up of how larva is mounted on to the Peltier stage (1) and water circulation pipes (2) connected to Peltier stage; D. Sample temperature profile for GCaMP assay; E. Class III md neuron GCaMP response to noxious cold. Fluorescence intensity is represented as lookup table. There is a large increase in GCaMP fluorescence in response to noxious cold temperature (6 °C) compared to baseline (25 °C).

    5. Program the pre-determined temperature profile using the T95 linkpad.
      Ideally, 2-5 min of baseline at 25 °C, ramp down at 20 °C/min (maximal ramp speed) to desired temperature, hold at desired temperature for at least 10 sec and then ramp up at 20 °C/min to 25 °C and hold for up 1 min (Figure 2D).
      Note: For multiple noxious thermal stimulations allow at least 1 min at 25 °C for GCaMP signal to return baseline.
    6. Prepare microscope slide for calcium imaging
      1. Obtain one microscope slide, two 22 x 22 mm coverslips, and one 24 x 50 mm long coverslip.
      2. Place a small drop of halocarbon oil #700 on both ends of the slide.
      3. Place two small coverslips on top of the small droplets. One at a time shimmy the coverslips, and stop after the coverslip is difficult to move.
      4. Using a brush gently pick up one larva, expressing GCaMP in your desired cell type, and wash the larva in a 9-well glass spot plate. Place the larva on a wet Kimwipe.
      5. Place the semi-wet larva on the middle of the slide.
        Note: Depending on laser confocal microscope set up, the orientation of larva will differ either horizontal or vertical compared to the length of the slide.
      6. Place a drop of water on top of both the small coverslips. If the larva is not straight on the slide, then use forceps to gently reorient the larva to the preferred direction.
      7. Place a 24 x 50 mm coverslip on top of the larva and the two coverslips. Again, shimmy, this time gently, until the larva is completely flat, straight and the desired side of the animal facing up (Figure 2C).
    7. Mount the slide containing the larva onto the Linkam PE 120 Peltier stage.
    8. Focus onto the region of interest and acquire time-lapse images for duration of thermal cycle.
      Representative GCaMP response in class III md neuron at baseline (25 °C) and at noxious cold temperature (6 °C) (Figure 2E).

  3. Functional assays for multimodal sensory processing
    Recent studies have revealed class III md neurons function in multimodal sensory processing (Turner et al., 2016). Class III neurons act as noxious cold sensing nociceptors, where the cold evokes bilateral head and tail contraction (CT), and innocuous mechanical sensors, where light touch primarily evokes only a head withdrawal (HW) response (Tsubouchi et al., 2012; Yan et al., 2013; Turner et al., 2016).
    We developed an optogenetic dose response assay and implemented the use of the stable calcium integrator CaMPARI to investigate how a single class of md sensory neuron is able to mediate unique behaviors (HW and CT) in response to two distinct sensory stimuli. In the optogenetic dose response assay we titrate the amount of blue light intensity activating class III md sensory neurons, revealing that at high blue light intensities CT is the predominant behavior, whereas at lower blue light intensities HW is the predominant behavior and as expected at the lowest blue intensity, we failed to observe any CT behavior and only a few HW responders (Turner et al., 2016). Optogenetic dose response revealed that level of class III activation determines the behavioral output and suggested that class III neurons are high threshold cold nociceptors and low threshold mechanosensors.
    To functionally assess how different levels of class III activation lead to distinct sensory behaviors, we examined intracellular calcium responses to gentle touch and noxious cold stimuli. Assessing intracellular calcium dynamics upon light touch, in an intact larvae, posed a technical challenge in the in vivo GCaMP assay.
    Therefore, we implemented the use of class specific expression of CaMPARI, where the larva is freely behaving and either innocuous mechanical or noxious cold stimuli could be delivered. This technique allowed us to measure the amount of green-to-red fluorescence photo-conversion revealing that noxious cold evokes significantly greater photo-conversion than innocuous mechanical stimulus. Both of these techniques can be used to assess the multimodality of PNS neurons.

  4. Optogenetic dose response assay
    1. Optimize light delivery and recording systems.
      1. Using a Zeiss AxioZoom.V16 microscope vary the light intensity delivered to the animal.
        1. While keeping magnification and source of white light constant, change the aperture from 100-37% (Figure 3A).
          Note: Preferred aperture settings are 100%, 90%, 80%, 70%, 50% 40% and 37%.
        2. Use the dark field illumination setting on the microscope stage. Turn on the LED for bottom illumination, increase the brightness just enough to barely see the larva.
      2. Mount recording equipment onto the microscope.
    2. Prepare a genetic cross or stock as described above and age embryo collection to 96-102 h AEL prior to optogenetic dose response assay.
    3. Prepare ATR supplemented food for optogenetic experiments
      1. Final concentration of ATR needs to be 1,000 μM.
      2. In a dimly lit room, add appropriate amount of ATR in liquefied food, mix thoroughly and allow the food to solidify.
        Note: Maintain ATR and ATR supplemented food in the dark.
    4. For the ATR condition: add the genetic cross/stock to ATR supplemented food containing vial.
    5. For the no ATR control condition: add the genetic cross/stock to normal food containing vial.
    6. Place both types of crosses in the dark.
    7. On day of experimentation.
      1. Turn on Zeiss Axiozoom.V16 microscope system.
      2. Working in a dimly lit space, using a brush select third instar larvae and wash in a 9-well glass spot plate with tap water.
      3. Place the larva on a wet Kimwipe.
      4. Spray a thin fine mist of water via a trigger sprayer on a large piece of glass (10 x 15 x 0.1 cm).
      5. Place the larva on the center of glass piece.
        Note: Make sure there is not too much water around the animal, as it will make quantification difficult.
      6. Allow animal to acclimate to the plate and resume peristaltic movement.
      7. Use a timer for precise blue light delivery and expose the animal to the following blue light on/off cycles (Figure 3B).
        1. 10 sec off–5 sec on–10 sec off–5 sec on–10 sec off–5 sec on–10 sec off.
        2. Executing multiple stimulations per animal will allow within animal comparisons of multiple stimulations.
      8. Repeat the previous step until an N of 20 third instar larvae is achieved for each blue light dose.
      9. Quantify changes in length as described in Data analysis section. (Figure 3C and Video 4)


        Figure 3. Zeiss AxioZoom.V16 set up for optogenetic dose response and blue light exposure diagram. A. Optogenetic dose response set up in a brightly lit room for demonstration purpose, but conduct the assays in a dimly lit room. 1. Zeiss AxioZoom.V16, 2. Glass plate, 3. Blue light, 4. Aperture control and 5. Dark field illumination. B. Sample blue light on/off time course; C. Image stills of larvae expressing ChETA in class III md neurons being exposed 100% or 66% max blue light. 100% illumination elicits CT behavior (consistent with noxious cold evoked behavior), whereas 66% illumination elicits HW behavior (consistent with gentle touch evoked behavior).

        Video 4. Optogenetic dose response. Video of larvae expressing ChETA in class III md neurons being exposed to either 100% or 66% max blue light.

  5. CaMPARI Ca2+ integrator assay
    1. As with GCaMP analysis, optimize red and green fluorescence for live confocal imaging.
    2. Optimize photo-converting (PC) light intensity for consistent green to red photo-conversion.
      1. PC light is delivered via Zeiss AxioZoom.V16 using previously described filter cube set.
      2. Using Zeiss AxioZoom.V16 microscope and HXP200C lamp, deliver 84,000 lux for 20 sec of PC light, which reliably photo-converts CaMPARI from green to red in presence of high calcium.
    3. Prepare 1:5 (v/v) ratio of ethyl ether:halocarbon oil #700 solution in an amber glass dropper bottle at least one day prior to imaging.
    4. Prepare a genetic cross or stock as described above and age embryo collection to 96-102 h AEL prior to CaMPARI analysis.
    5. Prepare microscope slide for calcium imaging.
      1. Obtain one microscope slide, two 22 x 22 mm coverslips, and one 24 x 50 mm long coverslip.
      2. Place a small drop of ethyl ether:halocarbon oil #700 solution on both ends of the slide.
      3. Place two small coverslips on top of the small droplets. Shimmy the coverslips, one at a time, and stop after the coverslip is difficult to move.
    6. Turn on your PC light delivery system.
    7. Set up stimulus delivery equipment on the base of the microscope.
      1. For noxious cold stimulus, place the Peltier plate as shown in Figure 4A.
      2. For light touch stimulus, attach a fine brush bristle to nickel plated pin holder as shown in Figure 4A inset.


        Figure 4. Microscope slide preparation. A. A view of the Zeiss AxioZoom.V16 with Peltier plate placed on the microscope stage, where larvae can be stimulated via cold and delivered PC light. 1. TE tech cold plate, 2. Black metal plate, 3. TE tech. power supply, 4. TE tech. temperature controller, 5. PC light, 6. HXP200C white light source, 7. Zeiss AxioZoom.V16 microscope and 8. Inset shows image of fine brush bristle mounted to a nickel plated pin holder. B. For live confocal imaging, larval mounted on the microscope slide with dorsal side up and fully stretched. C. Representative images of class III neurons expressing CaMPARI. PC (photo-converting light) and NS (no stimulus).

    8. Using a brush gently pick up one larva, expressing CaMPARI in your desired cell type, and wash the larva in a 9-well glass spot plate. Place the larva on a wet Kimwipe.
    9. Place the larva under Zeiss AxioZoom.V16. Simultaneously deliver stimulus and 20 sec of PC light.
      1. For cold stimulus: place one larva on black metal plate. Gently place the metal plate on the Peltier plate (set to 6 °C) and turn on PC light.
      2. For light touch stimulus: place one larva on black metal plate. Deliver light touch stimulus first to the head of the larva, followed by a stroke along the length of the animal, while the animal is exposed to PC light.
    10. Mount larva on to a microscope slide (Figure 4B).
      1. Place the larva on the middle of the slide.
        Note: Depending on laser confocal microscope set up orientation of larva will differ either horizontal or vertical compared to the length of the slide.
      2. Place a drop of ethyl ether:halocarbon oil #700 solution on both small coverslips. If the larva is not straight on the slide, then use forceps to reorient the larva to the preferred direction. Generously add drops of ethyl ether:halocarbon oil #700 solution on to and around the larva.
      3. Place a 24 x 50 mm coverslip on top of the larva and the two coverslips. Again, shimmy, this time gently, until the larva is completely flat, straight and desired side of the animal facing up.
    11. Image z-stacks of neuronal cell bodies in the neural type of interest.
      Imaging multiple segments and/or both hemisegments will allow more detailed analysis of spatial CaMPARI response.

Data analysis

  1. Behavioral quantitation for cold plate and optogenetic dose response assays
    1. Uncompress video files using Video to video converter (videotovideo.org).
    2. Open uncompressed video file as gray scale in ImageJ.
    3. Use threshold function to make the larva black and white (Figure 5).
    4. Use ImageJ function Remove Outliers using the setting: radius = 2.0 pixels, threshold = 50, and which outliers: select dark.
    5. Apply the following functions: make binary and skeletonize. Watch the entire video to make sure that larval skeletons do not have any aberrant branches or protrusions (Figure 5).
    6. Using the analyze function, measure area, which will give the larval length for the duration of the video.
    7. Perform percentage change from max length calculation: % Max length = (Lmax - Ln)/Lmax
      1. Lmax is the maximum length of the larva.
      2. Ln is the length of larva in one frame.


        Figure 5. ImageJ screenshots for quantitative behavioral analysis

  2. In vivo calcium imaging data analysis conducted using ImageJ/Fiji
    1. Discard videos with excessive movement in either x, y, or z axis.
    2. Upload the time-lapse data file onto ImageJ.
    3. Run ImageJ plugin StackReg for lateral motion stabilization.
      Note: Rigid body and translation are two of the best options to use during image stabilization.
    4. Draw a region of interest around the cell body and using ImageJ function called Plot z-axis profile, which measures the mean fluorescence intensity normalized to area for each frame.
    5. Smooth the raw data using a 2 sec moving average.
    6. Calculate ΔF/F0 using the following equation: ΔF/F0 = (F - F0)/F0 x 100
      1. F0 is the average fluorescence during baseline
      2. F is the fluorescence at one frame
  3. CaMPARI Ca2+ integrator assay data analysis conducted using Zeiss Zen Blue Lite (Figure 4C)
    1. Draw region of interest around maximum intensity projections of neural cell bodies.
    2. Quantify red and green fluorescence.
    3. Calculate fluorescence change as previously described in Fosque et al. (2015).
      CaMPARI photo-conversion = Fred/Fgreen

Recipes

  1. Drosophila media
    1. Ingredients:
      18.4 g soyfluor
      132 g cornmeal
      12 g Drosophila agar
      36 g inactive dry yeast
      194 g dry molasses
      2.15 L water
      9 ml propionic acid
      1.56 ml O-phosphoric acid
      Note: Makes ~2 L of Drosophila media, enough for 200 vials.
    2. Mix the following ingredients in a large cooking vessel: soy, drosophila agar, cornmeal, inactive dry yeast, dry molasses, and water
    3. Cook at medium to high heat until food comes to boil. Mix thoroughly every 5-10 min
    4. Let the fly food low boil for 30 min and then turn off the stove
    5. Wait until cooking vessel is warm to touch and add appropriate amount of acids (O-phosphoric acid and propionic acid)
    6. Mix thoroughly and pour the fly food into Droso-Filler for making fly food vials
    7. Pour the desired volume in a vial loaded tray

Acknowledgments

We thank Kevin Armengol for initial designs of the cold plate assay, in vivo GCaMP assay and quantitative behavioral analysis described in Turner et al. (2016). The Cox laboratory is supported by NINDS R01 NS086082, NIMH R15 MH086928, Brains & Behavior Seed Grant awards and a 2CI Neurogenomics and Molecular Basis of Disease award (GSU) to D.N.C. A.A.P. is funded by 2CI Neurogenomics Fellowship (GSU).

References

  1. Fosque, B. F., Sun, Y., Dana, H., Yang, C. T., Ohyama, T., Tadross, M. R., Patel, R., Zlatic, M., Kim, D. S., Ahrens, M. B., Jayaraman, V., Looger, L. L. and Schreiter, E. R. (2015). Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators. Science 347(6223): 755-760.
  2. Gorczyca, D. A., Younger, S., Meltzer, S., Kim, S. E., Cheng, L., Song, W., Lee, H. Y., Jan, L. Y. and Jan, Y. N. (2014). Identification of Ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila. Cell Rep 9(4): 1446-1458.
  3. Guo, Y., Wang, Y., Wang, Q. and Wang, Z. (2014). The role of PPK26 in Drosophila larval mechanical nociception. Cell Rep 9(4): 1183-1190.
  4. Im, S. H. and Galko, M. J. (2012). Pokes, sunburn, and hot sauce: Drosophila as an emerging model for the biology of nociception. Dev Dyn 241(1): 16-26.
  5. Mauthner, S. E., Hwang, R. Y., Lewis, A. H., Xiao, Q., Tsubouchi, A., Wang, Y., Honjo, K., Pate Skene, J. H., Grandl, J. and Tracey Jr., W. D. (2014). Balboa (PPK-26) binds to Pickpocket in vivo and is required for mechanical nociception in Drosophila larvae. Curr Biol 24(24):2920-2925.
  6. Tsubouchi, A., Caldwell, J. C. and Tracey, W. D. (2012). Dendritic filopodia, Ripped Pocket, NOMPC, and NMDARs contribute to the sense of touch in Drosophila larvae. Curr Biol 22(22): 2124-2134.
  7. Turner, H. N., Armengol, K., Patel, A. A., Himmel, N. J., Sullivan, L., Iyer, S. C., Bhattacharya, S., Iyer, E. P., Landry, C., Galko, M. J. and Cox, D. N. (2016). The TRP channels Pkd2, NompC, and Trpm act in cold-sensing neurons to mediate unique aversive behaviors to noxious cold in Drosophila. Curr Biol 26(23): 3116-3128.
  8. Yan, Z., Zhang, W., He, Y., Gorczyca, D., Xiang, Y., Cheng, L. E., Meltzer, S., Jan, L. Y. and Jan, Y. N. (2013). Drosophila NOMPC is a mechanotransduction channel subunit for gentle-touch sensation. Nature 493(7431): 221-225.

简介

为了研究有害感冒检测的细胞,分子和行为机制,我们开发了冷板行为测定和评估主要有害冷诱发收缩行为的定量方法。为了表征响应于有害感冒的神经活动,我们实施了基于GCaMP6的钙成像测定,其能够在完整的果蝇幼虫中进行体内细胞内钙动力学研究。我们将果蝇III型多发感染(md)感觉神经元识别为无机械和有害冷刺激的多模态传感器,并解剖多模态感觉加工的机理基础,我们开发了两项独立的功能测定。首先,我们开发了一种光遗传剂量反应测定法,以评估神经激活水平是否有助于感觉感觉神经元的多模态方面。其次,我们利用CaMPARI,一种可开关的钙积分器,可以在高细胞内钙和光转换光的存在下,将荧光从绿色稳定转变为红色,以评估体内的神经激活水平之间的功能差异无害的机械和有害的冷刺激。这些新颖的测定能够研究在果蝇幼虫中外周感觉神经元和多峰感官处理的行为和功能作用。
【背景】感觉和适应环境线索的能力是后生动物共享的最根本的方面之一。感测潜在的有害刺激,如有毒温度,化学或机械侮辱和适当的反应对于避免可能导致人身伤害或死亡的初期损害至关重要。通常,在感测伤害感受刺激时,动物产生一组避免行为,其减轻或允许动物逃离有害刺激。阐明加工伤害性刺激的分子,细胞和行为水平机制是非常有意义的,因为鉴定和开发用于异常感觉加工的新型治疗干预的潜力可能导致神经性疼痛。在黑腹果蝇幼虫和成虫中已经阐明了对有毒化学,机械和热刺激的感觉和行为反应,然而,近来在幼虫中发现了有害的感冒检测(Im和Galko,2012; Gorczyca ,2014; Guo等人,2014; Mauthner等人,2014; Turner等人。 ,2016)。果蝇幼虫表现出一组独特的厌恶行为,在有害的冷刺激反应中,主要的冷诱发反应显示为双侧前后全身收缩(CT)(Turner et al。 ,2016)。这种行为反应由III类md感觉神经元介导(Turner等人,2016),其有趣地也涉及在这些神经元中揭示多模态的温和触摸机制显着(Tsubouchi等人, ,2012; Yan等人,2013)。瞬态受体电位(TRP)通道Pkd2,NompC和Trpm是调节有害的冷诱发行为所必需的,并且响应于无害机械与有害冷刺激的行为选择依赖于III类神经激活水平,从而提供对底层机制的了解冷伤害感受和多模态感觉处理(Turner等人,2016)。

关键字:伤害感受, 有害寒冷, 多模态感知处理, 钙成像, 光遗传学, 果蝇

材料和试剂

  1. Kimwipe(KCWW,Kimberly-Clark,目录号:34155)
  2. 25 x 75 mm显微镜载玻片(Globe Scientific,目录号:1301)
  3. 22 x 22毫米厚度盖玻片(Globe Scientific,目录号:1401-10)
  4. 24 x 50毫米1号盖玻片(Genesee Scientific,目录号:29-118)
  5. Pyrex 9孔玻璃斑点板(Fisher Scientific,目录号:13-748B)
    制造商:Corning,PYREX ® ,目录号:7220-85。
  6. 琥珀色玻璃滴管瓶(Fisher Scientific,Fisherbrand TM ,目录号:02-983B)
  7. Bel-Art TM SP Scienceware TM 广口色码安全标签洗瓶(Fisher Scientific,目录号:22-288654)
    制造商:SP Scienceware - Bel-Art产品 - H-B仪器,目录号:F11646-3739。
  8. Bel-Art TM SP科学触发式喷雾器,带53 mm适配器(Fisher Scientific,目录号:01-189-100)
    制造商:SP Scienceware - Bel-Art产品 - H-B仪器,目录号:F11620-0050。
  9. 聚丙烯小瓶(Genesee Scientific,目录号:32-120)
  10. Droso-Plugs,窄瓶(Genesee Scientific,目录号:59-200)
  11. 果蝇股票:
    1. ChETA:y 1 w *; wg Sp-1 / CyO,P {Wee-P.ph0} BaccWee-P20 ; p {20XUAS-CHETA.YFP} attP2 / TM6C,Sb 1 (布卢明顿果蝇库存中心,目录号:36495) />
    2. CaMPARI:w *; p {UAS-CaMPARI} attP40(Bloomington ia果蝇库存中心,目录号:58761)
    3. GCaMP6(中型): w 1118 ; PBac {20XUAS-IVS-GCaMP6m} VK00005(布卢明顿果蝇库存中心,目录号:42750)
    4. III类md神经元驱动程序:GAL4 19-12 和 GAL4 nompC (布卢明顿果蝇 Stock Center,目录号:36369和36361)(Turner等人,2016)
    5. IV类md神经元驱动程序:GAL4 pp1。 9 和 GAL4 (Turner等,2016)
    6. 控制菌株: w (Bloomington 果蝇库存中心,目录号:3605)
  12. 所有反式视黄醛(ATR)(Sigma-Aldrich,目录号:R2500)
  13. Halocarbon Oil#700(LabScientific,目录号:FLY-7000)
  14. 无水乙醚(Fisher Scientific,目录号:E138-500)
  15. NutriSoy,大豆面粉(Genesee Scientific,目录号:62-115)
  16. 黄色玉米粉(Genesee Scientific,目录号:62-101)
  17. 琼脂II型(Genesee Scientific,目录号:66-104)
  18. 非活性干酵母(Genesee Scientific,目录号:62-107)
  19. 干糖蜜(Genesee Scientific,目录号:62-119)
  20. O-磷酸(Fisher Scientific,目录号:A242-212)
  21. 丙酸(Fisher Scientific,目录号:A258-500)
  22. 果蝇媒体(见食谱)

设备

  1. 冷板测定
    1. 刷(Craft Smart ®圆刷设置金色Taklon)(Michaels商店,型号:Size 3,目录号:10408282)
    2. 尼康身体加镜头组合(尼康,型号:D5300)和AF-S Nikkor 18-55毫米DX VRII(尼康,型号:AF-S DX)
    3. 用于安装DSLR的三脚架
    4. 冷板冷却器(TE Technology,型号:CP-031)
    5. 冷板温控器(TE Technology,型号:TC-720)
    6. 冷板电源(TE Technology,型号:PS-12-8.4A)
    7. 红外线温度计(Fluke,型号:Fluke 62 MAX)
    8. 铝板 - 层压铝垫片(全球设备,目录号:WBB512969)
      注意:
      1. 将垫片以0.22mm厚度切割成7.5×11.5mm。

    9. 黑色喷漆-12盎司黑色通用喷漆(Rust-Oleum)

  2. 在体内GCaMP测定
    1. PE120珀耳帖级(Linkam Scientific Instruments,Linkam Scientific,型号:PE120)
    2. 来自(Linkam Scientific Instruments,Linkam Scientific,型号:T95)的T95系统控制器(包括T95连接板和PE95)
    3. 能够成像GFP的激光共焦显微镜(Carl Zeiss,型号:LSM 780)

  3. 光学剂量反应测定
    1. D5300数码单反相机(尼康,型号:D5300)
    2. 用于将尼康DSLR安装到蔡司显微镜上的适配器:Nikon F的T2适配器(Carl Zeiss,型号:T2适配器,目录号:416009-0000-000)和适配器60N-T2 1.0x(Carl Zeiss,型号:Adapter 60N,目录号:426103-0000-000)
    3. 玻璃板:10 x 15 x 0.1 cm

  4. CaMPARI Ca 2 + 积分仪测定
    1. 光转换滤镜立方体
      1. 612/69 BrightLine带通滤波器,25 mm(IDEX Health& Science,Semrock,目录号:FF01-612 / 69-25)
      2. 440 nm BrightLine SWP边缘滤波器,25 mm(IDEX Health& Science,Semrock,目录号:FF01-440 / SP-25)
      3. 562 BrightLine二向色分光镜,25.2 x 35.6 mm(IDEX Health& Science,Semrock,目录号:FF562-Di03-25x36)
    2. Axio Zoom.V16(ZEISS,型号:Axio Zoom.V16),带照明器HXP200c灯(Carl Zeiss,型号:HXP 200C)
    3. 轻触式刺激:镀镍针架(精细科学工具,目录号:26018-17),带有单个精细油漆刷毛刷
    4. 有害的冷刺激:TE技术冷板冷藏机在冷板测定中描述

  5. 果蝇媒体准备
    1. Adventurer Pro II分析/精密天平(Ohaus,型号:AX2202)
    2. FastPette V2移液器控制器(Labnet International,目录号:P2000)
    3. 10ml血清学针(Genesee Scientific,目录号:12-104)
    4. Droso-Filler,Narrow(Genesee Scientific,目录号:59-168)
    5. Avantco感应范围(Avantco设备,型号:IC3500)
    6. 不锈钢锅盖(雷霆集团,目录号:SLSPS020)
    7. 窄航空小瓶重装纸盘(Genesee Scientific,目录号:59-207)

软件

  1. ImageJ( https://imagej.nih.gov/ij/
  2. 视频到视频转换器( http://www.videotovideo.org/ )< br />
  3. Zeiss Zen Blue Lite( https ://www.zeiss.com/microscopy/us/products/microscope-software/zen-lite.html

程序

  1. 冷板分析
    注意:以下步骤描述了有毒的冷刺激如何传播到黑腹果蝇幼虫的腹侧表面。将幼虫放置在黑色金属板上,以实现幼虫的高对比度成像,允许恢复运动,然后通过将黑色金属板放置在冷冻的珀尔帖板上而暴露于有害的热刺激。使用此测定法评估有害的冷诱发行为反应的好处是该测定将动物的整个腹侧表面暴露于有害的冷刺激,并且可以定量评估该行为(参见数据分析部分)。
    1. 冷板测定
      1. 准备感兴趣的遗传十字架约30-40个童女和20-25个男性。将小瓶置于29°C。允许成年苍蝇交配两天,然后转移到一个新的小瓶,以在29℃下进行4-6小时的定时鸡蛋收集。 4-6小时后,去除成年苍蝇,并将小瓶养殖至96-102小时(AEL),对应于三龄幼虫发育阶段。
      2. 行为测定应在漫散的房间中进行。
      3. 在96-102 h AEL,打开珀尔帖冷板,并设置为所需温度(6°C)。允许板几分钟达到所需的温度,并严格确保板材能够保持用红外线温度计测量的所需温度。
        注意:在6°C时,请确保清除多余的冷凝。
      4. 打开录音设备,尼康数码单反相机和镜头设置为55毫米,放置在板的焦平面上方约18厘米处。
      5. 用刷子进行冷板测定,每次选择6-9只三龄幼虫(图1A),并在9孔玻璃斑点板上用自来水轻轻冲洗。


        图1.设置冷板测定。 :一种。果蝇的放大图像三龄幼虫(比例尺代表1.7毫米); B.尼康数码单反相机安装在三脚架上,2. TE技术珀耳帖板,3. TE技术温度控制器,4. TE技术电源。 C.第3龄幼虫在0秒暴露6℃的冷板测定 1118 D. 3℃下暴露于6℃的三龄幼虫的冷板测定。幼虫在有害的冷刺激反应中表现出收缩(CT)行为
      6. 快速转移到湿的Kimwipe。
      7. 通过触发式喷雾器在黑色金属板上施加细小的水雾。
        注意:在ImageJ / Fiji量化步骤期间,板上太多的水将产生噪音。
      8. 使用刷子轻轻拿起每只幼虫并将其放在金属板上。所有的幼虫都放在盘子上,让幼虫约30秒钟适应并恢复板上的蠕动运动。
        注意:确保幼虫上没有残留的食物颗粒,多余的水没有积累在幼虫周围,幼虫之间有足够的空间(图1B)。
      9. 非常轻轻地将金属板放在珀尔帖板上,使轻触摸刺激不会混淆有毒热测定。以30帧/秒记录动物长达30秒(图1B和视频1)。

        Video 1. Cold plate assay. Video of a cold plate assay setup.

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player


      10. 重复,直到记录所需数量的幼虫。
      11. (图1C和1D)和25°C(视频2)和响应于有害冷刺激(6°C)(视频3)的基线幼虫行为的视频。 在基线温度(25°C),幼虫表现出蠕动运动和头部旋转行为与测量环境一致,而在有害的冷刺激(6°C)时,幼虫表现出双侧前后收缩(CT)行为。
        Video 2. Larval behavior at baseline temperature. Video of w1118 larvae at 25 °C.

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player


        Video 3. Larval behavior in response to noxious cold. Video of w1118 larvae at 6 °C.

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player


  2. 通过GCaMP进行体内钙成像
    注意:我们设计了一个实验范例,用于使用现代共聚焦成像和荧光技术在活的完整的黑腹果蝇幼虫中成像瞬时钙反应。通过对位于半透明角质层正下方的幼虫md感觉神经元的研究来促进这些新的体内分析。我们使用幼虫md神经元类特异性GAL4驱动器来指导GCaMP6的表达,即瞬时荧光细胞内Ca 2 + 传感器。为了评估活体GCaMP对有害感冒暴露的反应,我们通过Linkam Peltier系统传递刺激。使用这种方法可以评估活体动物制剂中任何PNS神经元中的GCaMP钙反应
    1. 在建立钙成像的遗传交叉或股票之前准备。
      1. 准备一个含有卤代烃油#700的琥珀色玻璃滴管瓶。这将用于体内成像的显微镜载片准备。
        注意:水也可以工作,但是#700的卤代烃油具有更大的粘度,这对于安装不能移动的22×22mm盖玻片是理想的。
      2. 优化激光共焦显微镜延时成像设置。
        注意:
        1. 确保激光功率和增益设置不会在延时采集期间饱和神经元。
        2. 设置图像采集速率最高。对于适当的时间分辨率,最大图像采集速率应为每秒1帧。
        3. 图像分辨率应至少为256 x 256像素分辨率。理想情况下,图像分辨率和图像采集率之间必须要平衡。
    2. 在钙成像之前,如上所述设置冷板测定的基因交叉或库存,并将小瓶置于29℃。
    3. 将食物小瓶中的年龄胚胎收集至96-102小时AEL并选择第三龄期幼虫进行成像。
    4. 设置水循环系统,Linkam PE 120珀耳帖级,将Peltier级安装到激光共聚焦显微镜上,打开共聚焦显微镜(图2A和2B)。
      注意:
      1. 使用双面胶带将帕尔帖舞台安装到显微镜平台上。
      2. 允许激光器的时间稳定和最大限度的水循环。


        图2. 体内 GCaMP设置,温度曲线和III类md神经元GCaMP响应。水循环箱(1),温度编程器(2),温度控制器(3)和蔡司激光共聚焦(4)。激光共焦显微镜阶段(1)与珀尔帖阶段(2)的关系; C.关闭如何将幼虫安装在与珀尔帖阶段连接的珀尔帖阶段(1)和水循环管道(2) D. GCaMP测定的样品温度曲线; E. III类md神经元GCaMP对有害感冒的反应。荧光强度表示为查找表。与基线(25°C)相比,响应于有害的冷温度(6°C),GCaMP荧光增加很大。

    5. 使用T95连接板编程预定温度曲线。
      理想地,在25℃下2-5分钟基线,以20℃/ min(最大斜坡速度)下降至所需温度,保持所需温度至少10秒,然后以20℃/ min升高至25℃并保持1分钟(图2D)。
      注意:对于多次有害的热刺激,允许在25°C下至少1分钟使GCaMP信号返回基线。
    6. 准备显微镜幻灯片进行钙成像
      1. 获得一个显微镜载玻片,两个22 x 22毫米盖玻片和一个24 x 50毫米长的盖玻片。
      2. 在幻灯片的两端放上一滴卤碳油#700。
      3. 将两个小盖玻片放在小滴的顶部。一次一片地盖上盖玻片,盖玻片难以移动后停止。
      4. 使用刷子轻轻拿起一只幼虫,将GCaMP表达为您想要的细胞类型,并在9孔玻璃斑点板中洗涤幼虫。将幼虫置于湿的Kimwipe上。
      5. 将半湿的幼虫放在幻灯片的中间。
        注意:根据激光共焦显微镜的设置,与幻灯片的长度相比,幼虫的方向与水平或垂直方向不同。
      6. 在小盖玻片的顶部放一滴水。如果幼虫在幻灯片上不直,请使用镊子轻轻地将幼虫重新调整到首选方向。
      7. 在幼虫的顶部和两个盖玻片上放置一个24 x 50毫米的盖玻片。再次,这一次轻轻地摇摆,直到幼虫完全平坦,直到动物面向上方的想要的一面(图2C)。
    7. 将含有幼虫的幻灯片放在Linkam PE 120 Peltier舞台上。
    8. 专注于感兴趣的区域,并获得热循环持续时间的延时图像。
      在基线(25°C)和有害的低温(6°C)下,III类md神经元的代表性GCaMP反应(图2E)。

  3. 多模态感官处理的功能测定
    最近的研究已经揭示III型md神经元在多模式感觉加工中的功能(Turner等人,2016)。 III类神经元作为有害的感冒伤害感受器,其中冷引起双侧头部和尾部收缩(CT)以及无害的机械传感器,其中轻微的触觉主要仅唤起头部退缩(HW)反应(Tsubouchi等人, / em>,2012; Yan等人,2013; Turner等人,2016)。
    我们开发了一种光遗传剂量反应测定,并实施了稳定的钙积分器CaMPARI的使用,以研究单一类md感觉神经元如何能够针对两种不同的感觉刺激来介导独特的行为(HW和CT)。在光遗传剂量反应测定中,我们滴定激活III级md感觉神经元的蓝光强度的量,显示在高蓝光强度下,CT是主要行为,而在较低的蓝光强度下,HW是主要行为,如最低的蓝色强度,我们没有观察到任何CT行为和只有几个HW响应者(Turner等人,2016)。光学剂量反应显示III级激活水平决定行为输出,并表明III类神经元是高阈值冷伤害感受器和低阈值机械传感器。
    为了功能评估III级活化的不同程度如何导致不同的感觉行为,我们检查了细胞内钙对温和触感和有害冷刺激的反应。在完整的幼虫中,通过轻触摸评估细胞内钙动力学,在体内GCaMP测定中提出了技术挑战。
    因此,我们实施了使用类别特异性表达的CaMPARI,其中幼虫自由行为,并且可以传递无害的机械或有害的冷刺激。这种技术允许我们测量绿 - 红荧光光转换的量,显示有害的冷引起比无害机械刺激显着更大的光转化。这两种技术都可用于评估PNS神经元的多态性
  4. 光学剂量反应测定
    1. 优化光传送和记录系统。
      1. 使用Zeiss AxioZoom.V16显微镜可以改变交付给动物的光强度。
        1. 在保持放大倍数和白光源恒定的同时,将光圈从100-37%改变(图3A) 注意:首选光圈设置为100%,90%,80%,70%,50%40%和37%。
        2. 使用显微镜舞台上的暗场照明设置。打开LED进行底部照明,增加亮度,足够勉强看到幼虫。
      2. 将记录设备安装在显微镜上。
    2. 准备如上所述的遗传交叉或股票,并在发光剂量反应测定之前将胚胎收集至96-102小时AEL。
    3. 准备ATR补充食物进行光遗传实验
      1. ATR的最终浓度需要为1,000μM
      2. 在昏暗的房间里,在液化食品中加入适量的ATR,彻底混合,使食物凝固。
        注意:在黑暗中维持ATR和ATR补充的食物。
    4. 对于ATR条件:将基因交叉/库存添加到ATR补充食物的小瓶中。
    5. 对于无ATR控制条件:将基因交叉/库存添加到正常的含有食物的小瓶中。
    6. 将两种类型的十字架放在黑暗中。
    7. 实验当天。
      1. 打开Zeiss Axiozoom.V16显微镜系统。
      2. 在昏暗的空间工作,使用刷子选择三龄幼虫,并在带有自来水的9孔玻璃斑点板中洗涤。
      3. 将幼虫置于湿的Kimwipe上。
      4. 通过触发式喷雾器在大块玻璃(10 x 15 x 0.1 cm)上喷洒一层薄薄的水雾。
      5. 将幼虫放在玻璃片的中心。
        注意:确保动物周围没有太多的水,因为它会使量化困难。
      6. 让动物适应平板并恢复蠕动运动。
      7. 使用定时器进行精确的蓝光传输,并将动物暴露于以下蓝色光的开/关循环(图3B)。
        1. 10秒关闭5秒10秒,关闭5秒-10秒,关闭5秒。
        2. 每个动物执行多次刺激将允许在多次刺激的动物比较中。
      8. 重复上一步骤,直到每个蓝光剂量达到20只三龄幼虫的N。
      9. 量化长度变化,如数据分析部分所述。 (图3C和视频4)


        图3. Zeiss AxioZoom.V16设置用于光遗传剂量反应和蓝光曝光图。 A.在明亮的房间内设置用于示范目的的光致剂量反应,但是以明暗的方式进行测定房间。 1.蔡司AxioZoom.V16,2玻璃板,3.蓝光,4.光圈控制和5.暗场照明。 B.样品蓝光开/关时间过程; C.在III类md神经元中表达ChETA的幼虫的图像残余物暴露于100%或66%的最大蓝光。 100%照明引起CT行为(与有害的冷诱发行为一致),而66%的照明引发HW行为(与温柔的触觉诱发行为一致)。

        Video 4. Optogenetic dose response. Video of larvae expressing ChETA in class III md neurons being exposed to either 100% or 66% max blue light.

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player


  5. CaMPARI Ca 2 + 积分仪测定
    1. 与GCaMP分析一样,优化红色和绿色荧光的活体共焦成像
    2. 优化光转换(PC)光强度,实现一致的绿光转换。
      1. PC灯通过Zeiss AxioZoom.V16使用之前描述的过滤器立方体集。
      2. 使用Zeiss AxioZoom.V16显微镜和HXP200C灯,提供84,000勒克斯的20秒的PC灯,在高钙的情况下,可靠的将CaMPARI从绿色转换为红色。
    3. 在成像前至少一天,在琥珀色玻璃滴管瓶中准备1:5(v / v)乙醚:卤代烃油#700溶液比例。
    4. 准备如上所述的遗传交叉或股票,并在CaMPARI分析之前将胚胎收集至96-102小时AEL。
    5. 准备显微镜幻灯片进行钙成像。
      1. 获得一个显微镜载玻片,两个22 x 22毫米盖玻片和一个24 x 50毫米长的盖玻片。
      2. 在玻片两端放置一滴乙醚:卤代烃油#700溶液。
      3. 将两个小盖玻片放在小滴的顶部。盖上盖玻片,一次一张,盖玻片难以移动后停下来。
    6. 打开您的电脑发光系统。
    7. 在显微镜的基础上设置刺激输送设备。
      1. 对于有害的冷刺激,放置如图4A所示的珀耳帖板。
      2. 对于轻触式刺激,请将细毛刷刷到镀镍针座上,如图4A所示

        图4.显微镜幻灯片准备。A.蔡司AxioZoom.V16的视图,佩尔贴板放置在显微镜平台上,幼虫可以通过冷和递送的PC光刺激。 1. TE技术冷板,2.黑色金属板,3. TE技术。电源,4. TE技术。温度控制器,5. PC灯,6.HXP200C白光源,7.蔡司AxioZoom.V16显微镜和8.插图显示精细刷毛的图像,安装到镀镍针座上。 B.对于活的共聚焦成像,将幼体安装在显微镜载玻片上,背面朝上并完全拉伸。 C.表达CaMPARI的III类神经元的代表性图像。 PC(光转换灯)和NS(无刺激)。

    8. 使用刷子轻轻拿起一只幼虫,将其表达为所需细胞类型的CaMPARI,并在9孔玻璃斑片中洗涤幼虫。将幼虫置于湿的Kimwipe上。
    9. 将幼虫置于蔡司AxioZoom.V16下。同时提供刺激和20秒的PC灯。
      1. 对于冷刺激:将一只幼虫置于黑色金属板上。轻轻将金属板放在珀尔帖板上(设定为6°C),然后打开PC灯。
      2. 对于轻触摸刺激:在黑色金属板上放置一只幼虫。将轻触摸刺激首先提供给幼虫的头部,然后沿着动物的长度进行中风,同时将动物暴露于PC灯。
    10. 将幼虫安装在显微镜载玻片上(图4B)。
      1. 将幼虫放在幻灯片的中间。
        注意:根据激光共聚焦显微镜的设置,幼虫的方向与幻灯片的长度不同,水平或垂直方向不同。
      2. 在两个小盖玻片上放一滴乙醚:#7卤代烃油溶液。如果幼虫在幻灯片上不直,则使用镊子将幼虫重新定向到首选方向。大量添加乙醚滴液:卤代烃油#700溶液到幼虫周围。
      3. 在幼虫的顶部和两个盖玻片上放置一个24 x 50毫米的盖玻片。再次,这一次轻轻地摇摆,直到幼虫完全平坦,直到动物面向上的一侧。
    11. 神经元类型感兴趣的神经元细胞体的z-stack图像。
      成像多个片段和/或两个半数将允许更详细地分析空间CaMPARI响应。

数据分析

  1. 冷板和光遗传剂量反应测定的行为定量
    1. 使用视频到视频转换器解压缩视频文件( videotovideo.org )。
    2. 在ImageJ中打开未压缩的视频文件作为灰度级。
    3. 使用阈值功能使幼虫成为黑色和白色(图5)。
    4. 使用ImageJ函数使用以下设置删除离群值:radius = 2.0像素,阈值= 50,以及哪些异常值:选择暗。
    5. 应用以下功能:使二进制和骨架化。观看整个视频,以确保幼虫骨骼没有异常的分支或突起(图5)。
    6. 使用分析功能,测量区域,这将给出视频持续时间的幼虫长度。
    7. 执行从最大长度计算的百分比变化:%最大长度=(L> max -L n )/ L max
      1. L max 是幼虫的最大长度。
      2. L 是一帧中的幼虫长度。


        图5.用于定量行为分析的ImageJ截图

  2. 使用ImageJ / Fiji进行的体内钙成像数据分析
    1. 在x,y或z轴上丢弃具有过度移动的视频。
    2. 将延时数据文件上传到ImageJ上。
    3. 运行ImageJ插件用于横向运动稳定的StackReg 注意:刚体和平移是影像稳定期间使用的两种最佳选择。
    4. 在细胞体周围绘制感兴趣的区域,并使用称为Plot z轴轮廓的ImageJ函数,测量每帧的标准化面积的平均荧光强度。
    5. 使用2秒移动平均值平滑原始数据。
    6. 使用以下等式计算ΔF/ F 0 =(F -F 0)/ F 0 x 100
      1. F 0 是基线期间的平均荧光
      2. F是一帧的荧光
  3. 使用Zeiss Zen Blue Lite进行的CaMPARI Ca 2+ 积分仪测定数据分析(图4C)
    1. 在神经细胞体的最大强度投影周围绘制感兴趣区域。
    2. 量化红色和绿色荧光。
    3. 计算荧光变化,如先前在Fosque等人(2015)中所述。
      CaMPARI photo-conversion = F red / F green

食谱

  1. 果蝇媒体
    1. 成分:
      18.4克大豆油 132克玉米粉
      12克果蝇琼脂
      36 g无活性干酵母
      194克干糖蜜
      2.15升水
      9毫升丙酸
      1.56ml O-磷酸 注意:制作约2升的果蝇介质,足够200瓶。
    2. 将以下成分混合在一个大型烹饪容器中:大豆,果糖琼脂,玉米粉,无活性干酵母,干糖蜜和水
    3. 在中等至高温烹饪,直到食物沸腾。每5-10分钟彻底混合一下
    4. 让飞行食物低沸腾30分钟,然后关掉炉子
    5. 等到烹饪容器温热接触并加入适量的酸(O-磷酸和丙酸)
    6. 彻底混合,将飞行食物倒入Droso-Filler,以制作飞行食物小瓶
    7. 在小瓶装载的托盘中倾倒所需的体积

致谢

我们感谢Kevin Armengol在Turner等人描述的GCaMP测定和定量行为分析中的冷板测定的初始设计。 (2016)。 Cox实验室由NINDS R01 NS086082,NIMH R15 MH086928,Brains&amp;行为种子授予奖和2CI神经元学和分子基础疾病奖(GSU)到D.N.C. A.A.P.由2CI神经元学研究员(GSU)资助。

参考

  1. Fosque,BF,Sun,Y.,Dana,H.,Yang,CT,Ohyama,T.,Tadross,MR,Patel,R.,Zlatic,M.,Kim,DS,Ahrens,MB,Jayaraman, Looger,LL和Schreiter,ER(2015)。 神经电路。使用设计的钙积分器在体内标记活性神经回路。 科学 347(6223):755-760。
  2. Gorczyca,DA,Younger,S.,Meltzer,S.,Kim,SE,Cheng,L.,Song,W.,Lee,HY,Jan,LY and Jan,YN(2014)。&lt; a class = ke-insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/25456135“target =”_ blank“> Ppk26的识别,Ppk1以相互依赖的方式起作用的DEG / ENaC信道指导 中的运动行为 Cell Rep 9(4):1446-1458。
  3. Guo,Y.,Wang,Y.,Wang,Q. and Wang,Z.(2014)。&nbsp; PPK26在果蝇中的作用幼虫机械伤害感受。 Cell Rep 9(4):1183-1190。
  4. Im,SH和Galko,MJ(2012)。&nbsp; Pokes ,晒伤和热辣酱:果蝇作为伤害感受生物学的一个新兴模式。 Dev Dyn 241(1):16-26。 >
  5. Mauthner,SE,Hwang,RY,Lewis,AH,Xiao,Q.,Tsubouchi,A.,Wang,Y.,Honjo,K.,Pate Skene,JH,Grandl,J。和Tracey Jr.,WD(2014) 。巴尔博亚(PPK-26)绑定到体内的Pickpocket ,并且是在果蝇幼虫中进行机械伤害感应所需的。 24卷(24):2920-2925。
  6. Tsubouchi,A.,Caldwell,JC和Tracey,WD(2012)。树枝状丝状伪足,Ripped Pocket,NOMPC和NMDARs有助于果蝇的幼体感。 Curr Biol 22(22):2124 -2134。
  7. Turner,HN,Armengol,K.,Patel,AA,Himmel,NJ,Sullivan,L.,Iyer,SC,Bhattacharya,S.,Iyer,EP,Landry,C.,Galko,MJ和Cox,DN(2016) 。 TRP频道Pkd2,NompC和Trpm行为冷感知神经元,以介导果蝇中有害感冒的独特的厌恶行为。 26(23):3116-3128。
  8. Yan,Z.,Zhang,W.,He,Y.,Gorczyca,D.,Xiang,Y.,Cheng,LE,Meltzer,S.,Jan,LY and Jan,YN(2013)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/23222543”target =“_ blank”> 果蝇 NOMPC是一种机械转导通道亚基,触摸感觉。自然 493(7431):221-225。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Patel, A. A. and Cox, D. N. (2017). Behavioral and Functional Assays for Investigating Mechanisms of Noxious Cold Detection and Multimodal Sensory Processing in Drosophila Larvae. Bio-protocol 7(13): e2388. DOI: 10.21769/BioProtoc.2388.
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