recording synaptic currents at the drosophila larval neuromuscular junction

Recording Synaptic Currents at the Drosophila Larval Neuromuscular Junction

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 segments 2, 3, or 4 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 boutons, 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 that 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 2 through 4. 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 suprathreshold regime, with the stimulation current increased approximately twice compared to the threshold for the observation of EJCs.

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1. Fabrication of Recording Electrodes
<ol>
	<li>Pulling the glass electrodes
	<ol>
		<li>Use the following protocol for the microelectrode puller (see Table of Materials):
		<ol>
			<li>Line 1: Heat 510 Pull - Velocity 30 Time 250; Line 2: Heat 490 Pull - Velocity 30 Time 250. 
			NOTE: Time units correspond to 0.5 ms per unit; the other units are relative. The value of the heat should be adjusted for every filament after the ramp test is performed.</li>
		</ol>
		</li>
		<li>Use a microscope (35x magnification) to ensure that the inner diameter of the pulled electrode is in the range of 7 - 10 &#181;m (Figure 1A). Store the capillaries in tightly closed containers to prevent dust accumulation.</li>
	</ol>
	</li>
	<li>Fire polishing
	<ol>
		<li>Fire polish capillaries (80 - 90% of the maximum heat value for 1 - 2 s) using a micro-forge (see Table of Materials). Ensure the final inner diameter of the polished electrode is 5 &#181;m (Figure 1A).</li>
	</ol>
	</li>
	<li>Bending 
	NOTE: Two bends are made in order to position the electrode on the top of the muscle under a high magnification objective.
	<ol>
		<li>Use the apparatus shown in Figure 1B. Fix the electrode in the manipulator and position the tip over the filament, not touching it.</li>
		<li>Set the heat value to 60 - 70% of the maximum and press the pedal of the micro-forge for 1 - 2 s (see Table of Materials) to heat the filament. Use an L-shaped needle to gently pull down the tip of the electrode (Figure 1B enlarged). Make the bend at approximately 90o (Figure 1C).</li>
		<li>Hold the electrode over the flame of the torch by hand using forceps and make the second bend at a distance of 7 - 10 mm from the first bend and at an angle of approximately 120o (Figure 1C).</li>
	</ol>
	</li>
</ol>
2. Additional Preparatory Steps
<ol>
	<li>Prepare hemolymph-like (HL3) solution (in mM): 70 sodium chloride (NaCl), 5 potassium chloride (KCl), 20 magnesium chloride (MgCl2), 10 sodium hydrogen carbonate (NaHCO3), 5 trehalose, 115 sucrose, 5 N-2-hydroxyethylpiperazine-N&#39;-2-ethanesulfonic acid (HEPES), and 1 mM calcium chloride (CaCl2); adjust the pH to 7.3 - 7.4. Keep the solution in the refrigerator and make it fresh every week.</li>
	<li>Make stimulation pipets the same way as recording glass pipets, except there is no need to bend them. 
	NOTE: The preparation of stimulation electrodes is described in detail in 28 and 27. The final diameter after fire polishing should be in the range of 5 - 7 &#181;m.</li>
	<li>Insert a stimulation pipette in a microelectrode holder connected to a syringe.</li>
</ol>
3. Electrical recordings of excitatory junctional currents (EJCs)
<ol>
	<li>Recording
	<ol>
		<li>Place the petri dish with the preparation on the microscope stage (Figure 2). Insert the reference electrode in the bath.</li>
		<li>Fill the recording electrode with HL3 solution. Under the 10x objective (see Table of Materials), immerse the electrode into the bath and place it over muscles 6 and 7 of the abdominal segments 2, 3, or 4 using the micromanipulator (see Table of Materials) (Figure 3 A.1 and A.2).</li>
		<li>Switch the objective to 60x (see Table of Materials). Focus on the area of interest using either epi-fluorescence or DIC optics. Place the tip of the electrode on top of the synaptic bouton (Figure 3 B.1-3).</li>
		<li>Press the electrode very gently onto the muscle. Excessive pressure may damage the neuromuscular junction (NMJ) or induce an increase in spontaneous synaptic activity. Make sure the tip of the electrode is not clogged- if it is, replace it.</li>
		<li>Switch on the amplifier, A/D board, and the computer.</li>
		<li>Choose the voltage clamp mode on the amplifier.</li>
		<li>Start acquisition software and choose the &#39;gap-free&#39; mode.</li>
		<li>Observe the appearance of miniature excitatory junctional currents (mEJCs) on the computer screen.</li>
		<li>Ensure that the amplitude of the mEJCs is in the range of 0.2 - 0.7 nA. 
		NOTE: Smaller EJCs indicate that the recording electrode has defects or that it is not positioned properly.</li>
	</ol>
	</li>
	<li>Stimulation
	<ol>
		<li>Using a micromanipulator, under visual control, place the stimulation electrode near the axon innervating abdominal segments 2 - 4.</li>
		<li>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 (Figure 2).</li>
		<li>Turn on the stimulator. Turn the knob on the isolation unit (see Table of Materials) to set a zero current, and then gently increase it until EJCs appear (or until the threshold is reached).</li>
		<li>Perform the stimulation in a suprathreshold regime, with the stimulation current increased approximately twice, compared to the threshold for the observation of EJCs. 
		NOTE: In our experience, such stimulation intensity is optimal to avoid action potential failures and action potential firing. For example, if EJCs appear at the stimulation current of 0.2 mA, use a current of 0.4 mA throughout the experiment.</li>
	</ol>
	</li>
</ol>

Source: Vasin, A., et al. <a href="https://app.jove.com/v/56493">Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for recording synaptic currents from a synaptic bouton at the Drosophila larva&#39;s neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton, which is important for the study of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

<img alt="Figure 1" src="/files/ftp_upload/22836/22836_Figure1.jpg" /> 
Figure 1. Final steps of micropipette fabrication. (A) Electrode tips after pulling and fire polishing. (B.1) The setup for tip bending. (B.2) The boxed area is shown enlarged on the right. The electrode and the filament are fixed so that the electrode tip is positioned slightly above the wire and not touching. (C.1) The recordin...

1. Fabrication of Recording Electrodes

	Pulling the glass electrodes
	
		Use the following protocol for the microelectrode puller (see Table of ...

Place a Petri dish containing a dissected Drosophila larva with exposed muscles and nerve branches onto the microscope stage.
Insert a reference electrode into a bath solution within the Petri dish.
Fill a recording electrode with an isotonic solution.
Using a micromanipulator, position this electrode over an abdominal muscle innervated by motor neurons.
Place the electrode tip on a synaptic bouton, a structure at the axon tip containing neurotransmitters.
Continuously record miniature excitatory junctional currents or mEJCs, which are responses to spontaneous neurotransmitter release.
The correct range of mEJC amplitude confirms the proper electrode positioning.
Next, position a stimulation electrode near the axon that innervates the targeted muscle.
Apply negative pressure to pull the axon inside the electrode.
Gradually increase the stimulation current to induce an action potential.
This triggers the axon to release neurotransmitters at the neuromuscular junction, which are recorded as synaptic currents.

9 Views. Recording Synaptic Currents at the Drosophila Larval Neuromuscular Junction

Recording Synaptic Currents at the Drosophila Larval Neuromuscular Junction

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