The aim of this procedure is to apply a stabbing injury to the ventral nerve cord of the drosophila larval CNS, and induce a cellular response to this injury. This is accomplished by first staging the larvae up to 96 hours after reg laying. Then the larval CNS is dissected out and the ventral nerve cord is stabbed using a fine tongue stern needle.
The final steps are to culture and then fix the stabbed ventral nerve cords for immuno staining or to just culture them for time-lapse recordings. Ultimately, confocal microscopy of the samples can show the dynamics of wound progression and changes in glial cell proliferation, apoptosis, and morphology. The main advantage of this technique over 16 methods like injury to the adult fly, is that while it is still a paradigm for a fo functional central nervous system, the larval CNS is technically more accessible.
Generally, individuals new to this method will struggle because the benal nerve code can degenerate if the dissection step has not been Begin by ensuring sanitary conditions. Clean the bench and your hands with 70%ethanol. The instruments should be wrapped in 70%ethanol soaked tissue and set aside.
When ready to handle the larvae, unwrap the tissue and allow the instruments to air dry. For these protocols, it is vital that the forceps tips meet perfectly. If they do not adjust them, using Arkansas stones are equivalent.
Load one staining block with distilled water and three blocks with two milliliters of M three PS medium. Select a vial with 96 hour old lava and add some water to it. Then using a spatula, gently take food with larvae from the vial and spread it on a wet paper towel.
Transfer 10 larvae from the spread to the staining block with water. Replace the water six times to fully clean the lava. Then replace the water with M three PS media.
Transfer one of the larvae to a staining block of M three PS media and place it under a dissecting microscope. Remove the VNC with minimal tissue damage. First position the lava dorsal side up.
Then at one third of the distance from the anterior end, grasp the dorsal side with two pairs of forceps. Then pull away the posterior end with the forceps tearing the epidermis. Next, cut the gut where it is closest to the brain because not much food should be contained there.
The brain and the VNC can now be seen from the posterior edge of the anterior half. Carefully remove the fat body, gut, and epidermis carefully. Serve the peripheral nerves by the thoracic VNC with two pairs of forceps placed close to each other and by pulling them apart.
Do not pull or tear discs or peripheral nerves from the thoracic VNC or it will degenerate the mouth. Parts can also be left attached to the VNC imaginal discs should be left attached to the brain. Now wetter widened P 20.
TIP in M three PS medium and use it to transfer the VNC to a staining block containing fresh M three PS medium before injuring the vnc. Make sure enough of being gathered. Four to five are needed for time lapse, but over 24 are needed for immuno staining.
Transfer one VNC to a clean staining block containing M three PS under a dissecting microscope, put the dorsal neuro pill in view. This is the most natural orientation for VNC with attached optic lobes. Now only once use a sten needle to manually stab the VNC from the dorsal side at a virtually right angle Aim for the abdominal half of the VNC.
The stab will gently hit the glass surface below the tissue. It is therefore important to routinely check whether the needle tip is still sharp enough for the next go. If it is blunted, sharpen the tip.
Using an Arkansas stone or equivalent Stabbing vent, a nerve guard with a tango stain needle is the most important step in this procedure since this is injury paradigm. If the needle is too blunt, the wound be will be far too big and the vent a nerve guard can de generate. When stabbing prevent the stem of the needle holder from touching the medium when completed, the wound will not be visible even after fluorescence immuno staining.
No sign of degeneration can be observed in fixed healthy VNC after 22 hours of culture. This procedure can result in cellular degeneration within the VNC unrelated to the stab 22 hours after stabbing. Look for rough surfaces, especially at the lateral ventral area of the thorax as pointed out by the arrowheads.
In extreme cases, the VNC is protruded. Also look for holes or vacuoles in the thoracic neuro pill, which can be visible as holes after fluorescent immuno staining. Notice the holes at the arrowhead.
Samples with any of these signs of degeneration in the neuro pill must be discarded. To visualize the axonal neuro pill in the living VNC, use a GFP protein trap called G nine, which labels all axons. To visualize all the glial cells except the midline gl, use the glial driver repo G four with for instance, the U-A-S-D-S red S 1 97 Y reporter flies carrying both reporters will have green axons and red GL that can be recorded in living tissue in time lapse.
After stabbing the VNC of animals carrying the requisite reporters transfer the stabbed VNC to a polyol lysine coated 3.5 millimeter glass bottom dish with one milliliter of M three PS media Position the VNC dorsal side down next, gently press the VNC to the dish using the flat side of the forceps so it sticks there. Now carefully add one milliliter of M three PS with 15%FPS in a 25 degree Celsius controlled imaging chamber. Acquire images of the VNC using confocal microscopy on the somewhat limited LIR SP two.
Use a 20 x objective with four times zoom. Set the scanning mode to XYZT. Set the pixel resolution to 512 square.
Set the Z steps to one micron and collect images on a one or two hour interval. These settings will allow eight to nine time points to be scanned on other confocal microscopes. Adjust the settings to acquire image stacks for longer time.
Points up to 24 hours. A VNC lesion was visualized in time lapse as a GFP negative area within the neuro pill of specimens bearing the G nine axonal marker shortly after stabbing small GFP negative areas which looked like holes or vacuoles started to appear. Such GFP negative areas generally enlarged up to six to eight hours after stabbing.
Subsequently, the GFP negative areas shrunk more and some even disappeared by 22 hours after stabbing. The area occupied by the wound was generally smaller than the maximum area seen at six to eight hours after stabbing. Similarly, the DS red negative area in glial processes initially increased.
However, they shrunk by 22 hours after stabbing. Interestingly, often DS red positive glial processes filled the GFP negative holes in the neuro pill prior to their disappearance. Immuno staining of fixed specimens bearing repo GAL four and U-A-S-M-C-D-H-G-F-P to label glial cell membranes with anti GFP revealed that dorsal side stabbing injury resulted in a dent.
Ventrally stabbing appeared to affect the neuro pill and neuro pill associated glial cells more severely than glial cells at the surface and in the cortex. Glial processes appeared disorganized in the neuro pill, whereas in the cortex, they still in sheath the VNC and maintained their mesh like organization. Injury resulted in GS two positive cellular debris as pointed out by the arrowheads.
The debris is distinct from normal cells. It is much smaller and is not connected to a cell body. This revealed damage to neuro pill associated glial cells, anti cleaved caspase.
Three positive apoptosis. Staining was observed in both neurons and glial cells showing they underwent apoptosis upon stabbing injury. Once mastered, this technique can be done in one to two hours.
It takes one hour to dissect the vent nerve code, stab them and set them up for time-lapse confocal microscopy and in two hours 24 vent nerve code can be dissected, stabbed, and set up for culturing after which the sample can be fixed at the desired time point.
