The overall goal of this procedure is to monitor synaptic transmission through the giant fiber pathway by recording post-synaptic potentials from a dorsal longitudinal muscle or DLM and from a Togo tro cantoral muscle or TTM following direct stimulation of the giant fiber into neurons. This is accomplished by first properly securing an anesthetized fly in a dish containing a dab of wax. The next step of the procedure is to insert stimulating ground and recording electrodes into the fly.
The final step of the procedure is to stimulate the brain and record responses from the jump and flight muscles. Ultimately, results can be obtained that show changes in the functional properties of the giant fiber pathway as a function of a genotype age, temperature, et cetera. By recording the amount of time between the stimulus and the response, and by counting the number of successful responses following a high frequency train of stimuli.
This method can help some of the key questions in the field of neurophysiology and help examine the functional properties of neuronal pathways. This technique can be employed either, either as an introductory experiment that is followed by genetic or molecular analysis, or as a way to assess physiological and functional properties in animals carrying a known mutation. Visual demonstration of this method is critical.
As the preparation and impairment steps take some time to learn. cause flies are comparatively small, a certain level of steadiness and coordination that needs to be acquired before experiments can be done correctly. These experiments use a standard electrophysiology setup comprised of a stimulator, a stimulus isolation unit, two micro electrode amplifiers, a data acquisition system, and a computer with collection software.
Additional equipment includes a Faraday cage, a stereo microscope on a boom stand, a vibration isolation table, a light source, and a recording platform. Five micro manipulators are used. Two micro manipulators require fine controls for positioning the recording electrodes, while the other three micro manipulators only require gross controls.
To position the two stimulation electrodes and the ground electrode, the manipulator for the DLM recording electrode is placed at the tail end of the preparation. The micro manipulator for the TTM recording electrode is placed between the experimenter and the side of the preparation. The two micro manipulators for the stimulation electrodes are placed at the head of the preparation.
The micro manipulator for the ground electrode is placed at the far side of the preparation. Pull glass recording micro electrodes with resistances of 40 to 60 mega ohms and store flat in a dish supported by wax. Use two electromagnetically sharpened tungsten electrodes for stimulation.
Use a stem wire or a third electromagnetically fabricated electrode. As the ground attach the stimulating and ground electrodes to the micro manipulators before the start of the experimental session. They will not need to be replaced for the duration of the session.
Once your equipment is set up, it's time to prepare the flies. Anesthetize the flies by cooling them on ice for 10 to 15 minutes. Use forceps to transfer the fly gently by its legs to a dish containing a platform of soft wax sloped at an angle of approximately 45 degrees.
The next step is to secure the fly-in wax. Orient the fly ventral side down with its anterior facing upwards on the slope. Using a pair of fine forceps, extend the legs outwards in pairs and push them into the wax.
Once you be familiar with the location of the muscles to be recorded from before beginning this experiment, the dorsal longitudinal muscle or DLM and the turga TRO cantoral muscle or TTM, the subcuticular attachment sites of the dlms correspond with the region between the thoracic midline and the anterior dorsal bristles. The TTM attachment sites are located dorsally of the posterior and anterior supra alar bristles, making sure that the wings will not obstruct access to the DLM or TTM fibers, hold the wings outward and glue them to the wax. Using a pair of fine forceps, pull the psis outward carefully and secure it by immersing into the wax.
This is a critical step that requires some practice since the probos is soft and is easily detached from the rest of the head. If that happens, discard the fly and start over. Failure to secure the head in this way leads to problems when inserting the stimulating electrodes through the eyes.
Once the fly is anchored to the wax, place the preparation under the stereo microscope Inside the faraday cage, orient the fly sideways with the head of the fly to the right of the experimenter. The next step is to insert the electrodes. Bring the electrodes close to the sites of insertion with the help of the micro manipulators to facilitate their proper placement and subsequent recordings without looking through the scope, lower the ground electrode into the posterior end of the abdomen using the adjustment wheels on the micro manipulator to place the sharpened tungsten stimulating electrodes in the brain.
Use the micro manipulator to position the tip of one of the electrodes, so it just touches one of the fly's eyes. Do the same with the other electrode, so both electrodes are just touching the outside of each eye. Then push the electrodes in.
Turn through each eye about two to three millimeters so the tips of the electrodes reach the brain situated at the back of the head capsule. Correctly placed electrodes will activate the giant fiber system to test that the stimulating electrodes are placed correctly. Apply a short stimulus of 30 to 60 volts across the stimulating electrodes and look for movement of the wings and twitches of the flight or leg muscles.
The next step is to backfill the glass micro electrodes with three molar potassium chloride using a Hamilton or heat pulled plastic syringe, and place them into the fine control micro manipulators. Properly inserted micro electrodes can be used for several rounds of experiments. The first recording electrode will be inserted into A DLM fiber.
There are two bilaterally symmetric dlms. Each one is composed of six individual muscle fibers. The recordings can be done from any of the six fibers, however, the most commonly used are the DLM fibers 45 A and 45 B, due to their good accessibility through the dorsal side of the thoracic cuticle, and the fact that both fibers are innovated by the same motor neuron.
Using the micro manipulator placed at the tail of the preparation inserter, recording electrode into DLM fiber 45 A or B on the side of the fly that is farthest away. The slope of the platform allows the DLM electrode to enter the dorsal cuticle at a 60 to 90 degree angle, which aids penetration using the software in oscilloscope mode. Look at the computer monitor while inserting the recording electrode into the thorax.
When the electrode has entered a muscle, the baseline drops to near or a negative value test with a single stimulus to see if you can observe the muscle response. Insert the other recording electrode into the TTM on the side of the fly that is closest to you. This electrode is inserted laterally in front of you due to the location of the muscle attachment site.
Again, observe the monitor while doing this. Once the trace indicates the electrode is in the muscle test with a single stimulus to elicit a muscle response. To begin stimulating the brain and recording responses from the leg and flight muscles, apply a 0.03 millisecond stimulus across the stimulating electrodes, starting at 30 volts and increasing to 60 volts until you observe a muscle twitch and a muscle cell depolarization on the computer monitor.
For the remainder of the experiment, set the voltage to five to 10 volts above the response threshold. To measure response latency. Give at least five single stimuli with a five second rest period between each stimulus.
Determine the frequency of following by providing trains of stimuli at different rates. Typically, 10 trains of 10 stimuli are given at 100 hertz, 200 hertz, and 300 hertz allow a rest period of two seconds between each train of stimuli. This figure shows responses recorded from the TTMs and dlms following a single stimuli to the brain.
The response latency is the time between stimulation of the brain and depolarization of the muscle. A healthy preparation and proper recording technique are indicated by latencies between 0.7 and 1.2 milliseconds for the G-F-T-T-M pathway and between 1.2 and 1.7 milliseconds for the GF DLM pathway. The latencies can vary with genotype, genetic background, temperature, and age as shown here.
Recordings from the TTM show more variability in terms of amplitude and shape of the post-synaptic potential compared to those from the large DLM fibers. This increased variability is due to the small size of the TTM muscle fibers. This variability, however, does not affect the response latency values for the giant fiber to TTM pathway.
This figure shows TTM and DLM stimulation at 100 hertz and 200 hertz at 100 hertz. Both TTMs and dlms respond to all 10 stimuli at 200 hertz. The TTM responses remain one-to-one, however, the DLM responses start to fail.
The reason for the DLM failure to follow at stimulation frequencies above 100 hertz is because the intermediary chemical synapse between two inter neurons does not have sufficient time to recover between stimuli. Once mastered, this technique can be done in approximately 30 minutes. After watching this video, you should have a good understanding of how to monitor neurotransmission through the giant fiber pathway.
This is done by stimulating the giant fiber into neurons in the brain, and then recording from a jump muscle and flight muscles in the thorax.
