The overall goal of this procedure is to activate single somatosensory neurons in larval zebra fish. This is accomplished by first preparing an optic cable and connecting it to A-D-P-S-S blue laser. The second step is to inject the transgene into one to two cell stage embryos.
Next screen fat embryos expressing C-H-E-F-T-D tomato in somatosensory neurons and mount them inus. The final step is to deliver a short pulse of blue light for single neuron activation and to record the resulting behavior. The main advantage of this technique of existing methods using channel root Option two is that this approach could be used to elicit behavioral responses from single neurons activated up to at least four days post-hospitalization.
Though this method is being used to provide insight into the function of specific somatosensory neurons in larval zebrafish. This approach could also be adapted to study behaviors elicited by any zebrafish neuronal population such as inner neurons or motor neurons. This genetic approach could be used to activate specific neurons in vivo, characterize functional development, assess the effects of mutations or phenomenological blockade of specific proteins on behavior and map downstream neuronal networks leading to distinct behavioral responses.
To begin this procedure, create a storage unit for the optic cable by melting the tapered neck of a glass. Pasta pipette over a bunts and burner to make a 150 degree angle. Next, using a wire cutter or razor blade, carefully cut the optic cable into two pieces.
Each piece should have one exposed end and one end with an FC PC adapter. Then strip the optic cable down to the cladding by removing the fiber jacket, strengthening fibers and coating from 5.0 centimeters of the cut end of the cable. Afterwards, insert the optic cable into the prepared pasta pipette.
Make sure the cable can easily move in and outta the pipette tip using a diamond glass cutter or a fine tipped forcep. Nick the glass fiber and break off the end to create a clean cut at the end of the fiber. Next, retract the optic cable into the pasta pipette.
Then position the optic cable in the pasta pipette using a micro manipulator under the microscope manually COATE 24 to 48 HPF embryos using a pair of forceps afterwards, anesthetize the larvae using 0.02%trica using a fluorescent dissecting microscope. Identify the larvae with Ron Beard neuron or trigeminal neuron expression Embryos with sparse expression in easily identifiable cells are optimal, but individual neurons will be targeted for activation with a fiber optic cable, so a wide range of expression density is acceptable. Transfer the larvae to a new dish with fresh PTU blue embryo water.
Store the embryos at 28.5 degrees Celsius in the dark until the desired experimental stage. In this section, make 1.5%low maltase and double distilled water and store in a 42 degrees Celsius heat block to prevent it from solidifying. Using a glass pasta pipette transfer one of the pre-screen larvae into a tube of 1.5%low melt agarose with as little blue embryo water as possible.
Then transfer the larva in a drop of agarose onto a small Petri dish. Under a dissecting microscope position the lava dorsal side up when the aros is solidified. Use a thin razor blade to cut away aros from both sides of the lava.
Fill the area surrounding the aros with embryo blue water. Next, make two diagonal cuts at both sides of the yolk, being careful not to nick the lava. Afterwards, pull the agrow away from the trunk and the tail of the lava.
Now mount the high speed camera onto the dissecting scope and connect the camera to the computer. Then turn on the computer and the high speed camera. Open the video imaging software and adjust the camera settings.
Next, connect the optic cable, the laser and the stimulator together. Then turn on the stimulator and set it to a maximum of five volts and the pulse duration of five milliseconds. Subsequently turn on the laser.
Next, use the fluorescent dissecting microscope to position the tip of the optic cable near a neuron cell body with C-H-E-F-T-D tomato expression. Deliver a pulse of blue light to activate the sensory neuron. Then record the responses using a high speed camera.
Set at 500 or 1000 frames per second, and repeat the experiment with at least one minute between activations to avoid habituation, to release the lava prior part, the Aris with forceps, and be careful not to inre the animal. Then transfer it into fresh blue embryo water. The animals can be allowed to develop further and the procedure can be repeated at older stages.
The embryo could also be remounted for high resolution confocal imaging of the activated cell in order to correlate the behavior with cellular structure. The zebrafish lava diagrammed here is partially mounted in 1.5%low malt Aris represented by the dash lines surrounding its rostral portion. A trigeminal neuron and a Rohan beard neuron are depicted in red mouther cells are outlined by the dash lines in the illustration.
Here is a movie showing that the activation of a single Rohan beard neuron expressing C-H-E-F-T-D tomato in an approximately 35 HPF lava elicits a behavioral response. This movie shows the activation of C-H-E-F-T-D tomato expressing Rohan beard neurons in a 60 HPF lava, and this movie shows the activation of C-H-E-F-T-D tomato expression Rohan beard neurons in a four DPF lava Once mastered. This genic approach can be performed in under one hour.
After watching this video, you should have a good understanding of how to activate individual neurons and larval Libra fish at various time points. Don't forget that working with lasers can be extremely hazardous and proper safety precautions should always be taken while performing this procedure.
