The overall goal of this technique is to monitor synaptic activity of visualized synaptic boutons at the drosophila larval neuromuscular junction. An excellent model system readily amendable to genetic manipulations. This technique can address key questions in your science.
Such as how mutations of synaptic proteins regulate synaptic transmission. The main advantage of this technique is that the activity of a limited number of release sites can be monitored and recorded currents of fast kinetics and can be easily resolved. To begin the experiment:prepare the microelectrode puller.
Use a microscope at 35X magnification to ensure the inner diameter of the pulled electrode is in the range of seven to ten micrometers. Store the capillaries in tightly closed containers to prevent dust accumulation. Next, fire polished capillaries using a micro forge.
Ensure the final inner diameter of the polished electrode is five micrometers. Then, move on to bending. Fix the electrode in the manipulator and position the tip over the filament not touching it.
Set the heat value to 60-70%of the maximum and press the pedal of the micro forge for one to two seconds to heat the filament. Use an L-shaped needle to gently pull down the tip of the electrode and make the bend at approximately 90 degrees. Hold the electrode over the flame of the torch by hand and make the second bend at seven to ten millimeters from the first bend and at an angle of approximately 120 degrees.
Prepare a hemolymph-like, or HL3 solution. Keep the solution in the refigerator and make it fresh every week. Prepare stimulation pipettes the same way as recording glass pipettes without bending them.
Next, insert a stimulation pipette in a microelectrode holder connected to a syringe. Manufactured dissection plates from small Petri dishes coated with silicone rubber epoxy. Then, using forceps pick a wandering third instar larvae from a vial.
Pin the larvae placing the first pin in the posterior and another pin anterior close to mouth hooks. Add HL3 solution. On the dorsal side of the larvae make a cut using spring scissors from the top pin to the bottom pin.
Pin the larvae filet placing two additional pins on the left and right sides of the larvae. Remove the guts and trachea using forceps. Cut the nerves just outside the ventral nerve cord using spring scissors.
Place the petri dish with the preparation on the microscope stage. Insert the reference electrode in the bath. Fill the recording electrode with HL3 solution.
Under the 10X objective immerse the electrode into the bath and place it over muscles six and seven of the abdominal segements of two, three or four using the micro manipulator. Next, switch the objective to 60X and focus on the area of interest using either epi-florescence or DIC optics. Place the tip of the electrode on top of the synaptic bouton then press the electrode very gently onto the muscle as excessive pressure may damage the NMJ or induce an increase in spontaneous synaptic activity.
Next, switch on the amplifier, AD board and the computer. Then, choose the voltage clamp mode on the amplifier. Start the acquisition software and choose the gap-free mode and observe the appearance of MEJCs on the computer screen.
Ensure the amplitude of the MEJCs is in the range of 0.2 to 0.7 nanoamperes. Using a micro manipulator under a visual control place the stimulation electrode near the axon innervating abdominal segments two through four. Apply negative pressure by pulling the piston of the syringe connected to the electrode holder so that the axon is pulled inside of the electrode.
Next, turn on the stimulator. Turn the knob on the isolation unit to set a zero current and then gently increase it until the EJCs appear or until the threshold is reached. Perform the stimulation in a superthreshold regime.
With the stimulation current increase approximately twice compared to the threshold for the observation of EJCs. Measure the seal resistance of the recording macro-patch electrode by turning the electrode resistance switch of the amplifier to seal test position. Observe the value of seal resistance in giga ohms which will be displayed in the current window.
Focal macro patch recordings enable monitoring synaptic activity from selected synaptic boutons. When the electrode is positioned on top of a synaptic bouton the recorded MEJCs have amplitudes significantly exceeding the noise level and sharp rising phases at a sub millisecond range. When the recording electrode is moved away from the synaptic bouton by several microns the amplitudes of recorder MEJCs decline almost to the noise level.
The recorded EJCs can barely be distinguished from the noise and they have prolonged rising phases. Focal recordings enable accurate detection of MEJCs in the complex and gnome mutant. While the intercellular recordings from this mutant exhibit release events that overlap and can not be reliably detected.
Adjustment of the angles of electro chip is critical for positioning of the electrode. Following this procedure other methods like checking imaging of individual release events can be performed in order to answer additional questions like the integration of the activity of individual active zones.
