In this Video, we will show how to inject liter quantities of a compound while obtaining electrophysiological recordings from the giant fiber system and how to monitor the effects of the compound on the function of this circuit. Before beginning this video, it is critical that you carefully watch and become familiar with the Joe video titled Electrophysiological Recordings from the Giant Fiber Pathway of the Melanogaster for Augustine Etal. As the video presented here is intended as an expansion to this existing Technique, The require equipment for the electrophysiology rig setup is described in detail by Augustine Al.In this journal modify the previously described electrophysiology rig setup by adding a sixth micro manipulator, which holds an ano injector.
For easy access to the head of the animal, it should be placed between the two stimulating electrode micro manipulators. The nano injection setup requires a nanoliter 2000 or similar type of injector prepare injection needle using the glass micro pipettes supplied with the injector by pulling them to a resistance of 80 to 100 megas. With an electrode puller for smooth injections, it is required to bevel the micro pipettes to an 11 to 17 micrometer opening at a 45 degree Angle.
Slowly backfill the injection micro pipette with synthetic oil using a Hamilton syringe as instructed by the Nanoliter 2000 manual, ensuring that no air bubbles are present. Carefully secure the micro pipette on the nano injector and prepare it to load the compound by emptying excess oil as instructed by the Nanoliter 2000 manual load. The compound as instructed in the Nanoliter 2000 manual.
Make sure that the tip of the micro pipette does not break during this procedure. Set the desire amount of nanoliters to be injected in the injector's control box as instructed in the NANOLITER 2000 manual. Please note that the total amount injected should not exceed 100 nanoliters.
Place a Nanoliter 2000 on the micro manipulator between the two stimulating electrode micro manipulators. Unplug the injector from the control box during recording acquisition with the exception of the injection itself. However, to not disconnect the power supply to the control box as it will reset it anesthetized two to six day old flies with CO2 or eyes as previously described.
Once in mobile, use a pair of tweezers to transfer the animal to a small plate with soft dental wax by picking it up from its legs as previously described. Carefully mount the fly dorsal side up and ensure that the thorax and head are immobilized with soft dental wax placed around the body. Carefully spread the wings out so that they lay perpendicular to the thorax.
Place the mounted Fly on the electrophysiology rig with its head towards the stimulating electrodes impale the animal with the corresponding stimulating ground and recording electrodes As previously Described, the DLM muscle is Located in the thorax between the anterior dorsal central hairs and the midline of the fly. The TTM muscle is located near the wing attachments between the posterior and anterior interior and interior supra allar hairs of the Fly align the Injection micro pipette containing the compound with the center of the three osli situated on the medial posterior portion of the head, but do not inject yet before compound injection. Obtain a baseline recording of the giant fiber to TTM and giant fiber to DLM pathways of the giant fiber system via brain stimulation by activating the giant fibers with 10 trains of 10 stimuli at 100 hertz.
With a one second delay between the trains. A wild type fly should be able to follow one-to-one at this rate of stimulation for both DLM and TTM pathways. Discard the fly.
If the giant fiber to DLM and giant fiber to TTM pathways, do not follow the 100 hertz stimulation at a one-to-one ratio. Switch to continuous stimulation of the giant fiber with single pulses at one hertz. Quickly plug the injector into the control box even though background noise will interfere with the one hertz stimulation recordings.
Do not discontinue it carefully. Insert the injection micro pipette into the head capsule of the fly just below the surface. Inject the desire amount of compound while maintaining the one hertz stimulation.
Immediately remove the injection micro pipette from the set of injection and unplug the injector from the control box while continuing the one hertz giant fiber stimulation up to one minute after injection. In order to reveal more subtle effects of compounds on the giant fiber system, stress the giant fibers with 10 trains of 10 stimuli at 100 hertz with a one second delay between the trains. Continue to test the function of the giant fiber pathways with this paradigm every five minutes up to 15 minutes.
In the example shown here, the giant fiber to TTM pathway remained unaffected after compound injection, whereas the giant fiber to DLM pathway was affected In order to test whether the compound has an effect at the neuromuscular junctions of the giant fiber system and possibly narrow down the effects of the compound, proceed to activate the motor neurons directly by thoracic stimulation for this. Remove the stimulating electrodes from the eyes and replace them on the anterior sides of the thorax in order to stimulate the motor neurons with 10 trains of 10 stimuli at 100 hertz. In this Example, when the stimulation is delivered via the brain, the DLM pathway fails to follow reliably.
However, the DLM neuromuscular junction response one to one at 100 hertz with thoracic stimulation excluding the neuromuscular junction as a particular target of the compound injected the compound's target can then be narrowed down to the connections between the giant fiber to PSI or PSI to DLM motor neuron.
