To produce stable electrophysiological recordings from Joof larva neuromuscular junctions. First dissect open a larvae to expose the body wall, muscles and nervous system prior to recording. The axons at the base of the CNS are cut posterior to the ventral ganglion.
Then a recording electrode is placed on the muscle of interest and a stimulating electrode is placed near the innervating motor neurons. The severed axons of the motor neurons are sucked into the stimulating pipette. Excitatory junction potentials can then be recorded.
Hi, I'm Wendy MLA from the McCabe Lab in the Department of Physiology and Cellular biophysics at Columbia University. Today we'll show you a procedure for measuring tic transmission at the neuromuscular junction of GE lava. We use this procedure in our laboratory to study synaptic physiology and neuro transmission.
So let's get started. To begin the experiment, dissect wandering third and star larvae to expose the body wall. This protocol is modified for electrophysiological experiments by using short dissection pins.
These are about two to four millimeters in length and are less likely to hit the microscope objective in electrodes during the experiment. After exposing the body wall, use forceps to hold the CNS and raise it slightly. Then cut the peripheral nerve motor axons and remove the brain.
After removing the brain wash the dissected prep twice with HL 3.1 buffer with one millimolar calcium. Now that dissection is complete, intracellular recordings can be made from the larval muscle cells. Place the dissection plate on the electrophysiology microscope and submerge the prep with HL 3.1 buffer plus calcium.
Then fix the bath electrode so it is in contact with the solution. The suction electrode is used to stimulate the peripheral nerves that innervate the muscle. Position the suction electrode in the dish first to avoid vibrations when the intracellular electrode is placed on the muscle.
Once the electrode is in the dish, position it close to the motor neuron that innervates muscle six. In the third abdominal segment, apply gentle suction until the cut nerve is inside the glass pipette. Be careful not to stretch the nerves when suction is applied.
Raise the pipette slightly so it is not touching the muscle. And then position it so it is not pulling on the cut nerve fibers. Once the suction electrode is in place, position the recording electrode for the intracellular recordings.
Use a sharp micro electrode filled with three molar potassium chloride. Position the recording electrode above the center of muscle six. Adjust the input offset so it reads zero for the bath solution.
Slowly lower the electrode and approach the muscle under high optical magnification until it touches the muscle surface. To confirm that the muscle has been penetrated, watch the oscilloscope. The resting membrane potential should be at least negative 60 millivolts.
Sporadic miniature nplate potential should now be visible. Once the recording electrode is in place, let the cells stabilize for one minute. Before starting the recordings, use an axon HS two, a head stage in an axle clamp two B amplifier to detect changes in the membrane potential.
The axle clamp two B is interface to a computer that runs P clamp software through the digit data. 1322 A, which digitizes the signal. Record the membrane potential on a PC.With clamp X software, apply stimuli to the motor axon to generate excitatory membrane potentials or ejs.
Once the ejs are generated, record the miniature ejs over a three minute period with no stimuli synapsis. Soft mini analysis software can be used to analyze the traces and determine mini EJP amplitude and frequency. To generate ejs.
Use a master eight pulse generator to stimulate to the severed end of a motor neuron program. The generator to deliver 0.3 millisecond square voltage pulses program. A five second interval time between stimuli to allow synaptic recovery.
At the NMJ record at least 10 evoked potentials from each muscle and average the amplitudes. This figure shows a typical trace recorded from muscle six of a wild type sal larva with evoked EJP responses and spontaneous mini EJ.PS We've just shown you how to record cy potentials from the neuromuscular junction of drosophila lava. When doing this procedure, it's important to remember to complete the dissection within three to 10 minutes and to start the electrophysiological recordings immediately while the cells are still healthy and responsive.
If the dissection takes too long, the resting membrane potential of muscle cells will be less than minus 60 millivolts, which is too low to record. So that's it. Thanks for watching and good luck with your experiments.
