Plan your experiment for the week that the fish turn 4 weeks old. Early in the week, check them out in the baby farm to see how big they are. You can even Tricaine a few to measure them and make sure they are between 1.5 cm and 1.8 cm long. Prepare the following supplies:
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• Tungsten needle in needle holder (0.37mm sharpened using KOH electrode bath in the Bashaw lab // Melissa Baxter is the expert on doing this)
• 0.2% Tricaine in brown bottle
• 2.5% low-melt agarose in Ringer’s (aliquot ~700µl in an eppendorf tube; 4 tubes total); melted and stored in a 42ºC heat block
You will need to do work on the following days during this experiment:
• Day 0: precut imaging and cutting
• Day 1: 24 hour post-cut imaging (to make sure the nerves are fully cut)
• Day 4: 4 day post-cut imaging (to observe regeneration)
Protocol Day 0
• Put fish to sleep in 0.2% Tricaine diluted 1:10 in system water (final concentration 0.02%)
• Transfer fish using spoon to a slide that has a border of vacuum grease (or the new acrylic slide that Owen made on the laser cutter!); mount the fish so it is lying on its right side, with anterior to the left and dorsal up.
• Measure the fish and record your measurement (ideal size for a fish is between 1.5 cm and 1.8 cm).
• Remove as much liquid from around the fish as possible, and add a few drops of 2.5% agarose to the tail and snout to immobilize the fish. Let dry for 10 seconds and then add back a few drops of system water + Tricaine so the fish can breathe. You should be able to see the fish breathing regularly or else it will die!
• Put the fish under the fluorescent dissecting scope and turn on the 488 lamp. Focus on the pectoral fin nerve. Change the viewfinder so the camera is receiving the light. Focus the image on the screen and take a picture - 83ms exposure works well. This is your precut image.
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• Change the viewfinder back to the microscope. Grab your tungsten needle and insert it just above the left pectoral fin, where the adductor superficialis branch extends. Pull the needle across the nerve, in a roughly-vertical SSW to NNE direction. See Figure
• The nerve is quite stretchy, so pulling on it with the tungsten needle might not initially sever it. Stay persistent and keep pulling! The agarose should keep the fish mounted so you can apply enough force to sever the nerve.
• When it is transected, you should be able to see the distal part degenerate almost immediately.
• When you think the nerve is cut, put the whole fish on the slide into its single box so it starts to wake up, then gently unmount it from the agarose. The fish should wake up within 2 minutes (one time it took 5 minutes, but we thought for sure the fish was dead).
• Clean and dry your slide with a kimwipe, and put the next fish to sleep to start the process again.
Day 1 (24 hours post-transection)
• At 24 hours post-transection, verify that the nerve was fully cut. Repeat steps 1 and 2 from Day 0, putting the fish to sleep and mounting it on a slide in the same way. Note: you do not need to immobilize the fish with agarose, since you won’t be cutting anything!
• Once the fish is mounted on the slide, take a picture of its adductor superficialis nerve. It should look like a nerve stump, with no obvious branches distal to the cut site. See Figure
• Put the fish back in its single box and feed it a little bit of brine shrimp. Day 4 (4 days post-transection)
• Repeat the same protocol as on Day 1. The fish doesn’t need to survive any longer, so it’s okay to wait until the fish stops breathing to capture the best image!
87 Troubleshooting
The biggest hurdle is capturing a good image on a slow camera with a fish that is actively breathing. If you are having trouble getting a good image using the camera, you can try taking a screenshot of the nerve (keyboard shortcut Fn + PrintScreen) and pasting the image into Microsoft Paint (Ctrl + V).
Figure 5-3: Peripheral motor nerves in 4-week-old fish regenerate after transection injury
(A) Lateral, precut view of the zebrafish fin and gill area, with all motor nerves expressing
Tg(mnx1:GFP).In the field of view is the motor nerve that innervates the adductor superficialis
muscle. Gill and fin are indicated. Transection site and direction of cut are indicated with a white arrow. (B) Immediately after cutting, the distal part of the nerve degenerates while the proximal part of the nerve retracts (arrowheads). (C) 1 day post-cut, the proximal part of the nerve has
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begun extending branches that explore the environment (arrowheads). (D) By 4 days post-cut, the proximal part of the nerve has re-extended axons that grow across the injury gap and return to the original muscle target (arrowheads).
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