The overall goal of this procedure is to measure force generation during a maximal contraction of zebra fish, larva, trunk muscle. This is accomplished by first making suture loops that will be used to hold the larvae in place during force measurements. Next, the anesthetized larvae is positioned inside the experimental chamber.
Once in place, the force is recorded during a maximal twitch contraction. The final step is to measure the dimensions of the trunk musculature so that peak force can be normalized to muscle cross-sectional area. Ultimately, measuring force generation during contraction is used to show changes in muscle function and overall muscle health.
Although we demonstrate this method using wild type zebrafish larvae, it can also be used with genetically modified larva or with larva treated with drugs or toxicants. The method can then be used to characterize muscle disease models and evaluate treatments or to study muscle development, injury or chemical toxicity. The first step is to prepare the suture loops that will be used to hold the larvae in place.
During force testing, use forceps to separate nonsterile suture into three strands. Once separated, begin to tie a double overhand knot in one of the strands, but stop before tightening the knot completely to make a small loop. Instead to finish the loop, use scissors to cut excess suture from the tails.
Place the loop on the sticky side of a sticky note for later use. Make two suture loops for each larva that will be tested. First, place the testing apparatus on the stage of a stereo microscope.
Next, connect the force transducer and length controller cables to the testing apparatus. Turn on the force transducer and the length controller and flip the switch on the front of the length controller. That makes the length controller tube bridged with a disposable transfer.Pipette.
Fill the experimental chamber with testing solution. Use forceps to pick up a suture loop by one of the tails as gripping. The loop on the curved part can kink the suture and cause it to break during subsequent steps.
Hang one loop on the force transducer tube and another on the length controller With a disposable transfer pipette transfer a zebrafish larvae to a small Petri dish filled with testing solution. Once the anesthetic has taken effect, gently nudge the tail to verify that there is a lack of touch evoked swimming. Next, use a glass pipette to transfer the larvae to the experimental chamber with forceps.
Gently guide the anterior portion of the larva through the suture loop on the force transducer tube, grasping both tails of the suture loop. Pull them simultaneously to tighten the loop posterior to the oak sac or swim bladder with forceps. Hold one suture tail and pull, causing the larvae to swivel 90 degrees around the tube until the lateral side of the larva faces up.
Using the X, Y, Z positioning device attached to the length controller, move the length controller tube along the x axis and under the trunk and tail of the larvae. Leave space between the ends of the length controller tube and the force transducer tube. Next, guide the suture loop over the tail of the larvae and tighten the suture loop.
As previously demonstrated, you may need to swivel the posterior part of the larvae so that the lateral side faces up. Once in place, trim the suture loop tails. Use the XY, Z positioning devices to slowly lower the tubes until they just touch the bottom of the chamber.
Then raise the larvae to an appropriate distance from the chamber bottom. This should be within the working distance of an inverted microscope objective used later. Finally, adjust the length controller tube to align the long axis of the larva with the long axis of the force transducer tube.
To begin, move the testing apparatus to the stage of an inverted microscope. Next, connect the water bath circulator thermometer, and temperature controller to the testing apparatus. Turn on the necessary components and adjust the setting on the temperature controller to the desired value.
Connect cables from the stimulator to the testing apparatus. Turn on the power to the stimulator, but do not stimulate the larvae at this point. To check that the larvae is parallel to the chamber bottom view the portion of the larvae between the ends of the tubes.
If parallel, both ends of the larvae will be in focus if needed. Adjust the force transducer tube until both ends are in focus and the larva is parallel. Turn on the video sarcomere length system and rotate the video camera such that the striations are parallel to the sides of the video frame.
The striation spacing is used as an indicator of sarcomere length. Adjust the microscope to focus on peripheral fibers and note the indicated sarcomere length. If necessary, adjust the length controller to shorten the larvae until the sarcomere length is less than optimal To optimize the twitch force.
First, set the output current on the stimulator to low magnitude, and then program specialized software to deliver current pulses. 0.2 milliseconds in duration. Deliver the first pulse and use the oscilloscope to measure the peak twitch force.
Increase the current by 50 milliamp increments and measure the peak twitch force at each new current level. Wait 30 seconds between twitches to prevent fatigue as stimulation current increases. Peak twitch force typically increases to a maximum and then gradually decreases the current at which the larva generates.
The greatest force is the optimal stimulation current once identified, set the current amplitude to the optimal stimulation current and adjust the length of the larvae in order to elicit maximum twitch force. Finally, elicit a twitch of the larva muscles. Use the oscilloscope to record the force response and save the record for subsequent analysis.
To measure the musculature dimensions, move the testing apparatus back to the stereo microscope. Using the IP scale, measure the height of the musculature as viewed from the side. Take the measurements at an anatomical landmark.
For example, the urogenital opening marked here in red. Then taking care not to change the length of the larvae. Swivel the larvae by 90 degrees using the suture loop tails in order to view the larvae from the bottom.
Measure the width of the musculature as viewed from the bottom. Finally cut the suture loops with a micro blade to release the larvae from the testing equipment in healthy wild type zebrafish larvae, the muscle fibers should be parallel to one another without large gaps between them and have evidence striations wild type zebrafish larvae that do not exhibit these features or with evident damage such as detached fibers as seen here should be discarded here. The peak twitch force versus stimulation current is plotted for a single zebrafish larvae.
The optimal stimulation current is typically between 400 to 600 milliamps. A representative force response collected during a maximal twitch contraction of a single larvae is shown here.Here. Peak force values from wild type larvae of different ages are shown to range from 0.9 to 1.7.
Milli newtons with older larvae generating more force than younger larvae. Older larvae generate more force than younger larvae even after peak forces are normalized to cross-sectional muscle area. While attempting this procedure, it's important to tighten the suture loops enough so that the larvae cannot swivel easily around the force transducer and length controller tubes.
If the suture loops are too loose, the force generated by the larva will not be fully transmitted to the force transducer and the maximum twitch force will be underestimated.
