Recording Synaptic Currents at the Drosophila Larval Neuromuscular Junction

Samenvatting

Source: Vasin, A., et al. Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction. J. Vis. Exp. (2017)

This video demonstrates the technique for recording synaptic currents from a synaptic bouton at the Drosophila larva'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.

Protocol

1. Fabrication of Recording Electrodes

1. Pulling the glass electrodes
   1. Use the following protocol for the microelectrode puller (see Table of Materials):
      1. 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.
   2. Use a microscope (35x magnification) to ensure that the inner diameter of the pulled electrode is in the range of 7 - 10 µm (Figure 1A). Store the capillaries in tightly closed containers to prevent dust accumulation.
2. Fire polishing
   1. 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 µm (Figure 1A).
3. Bending
   NOTE: Two bends are made in order to position the electrode on the top of the muscle under a high magnification objective.
   1. Use the apparatus shown in Figure 1B. Fix the electrode in the manipulator and position the tip over the filament, not touching it.
   2. 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).
   3. 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 120^o (Figure 1C).

2. Additional Preparatory Steps

1. 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'-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.
2. 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 µm.
3. Insert a stimulation pipette in a microelectrode holder connected to a syringe.

3. Electrical recordings of excitatory junctional currents (EJCs)

1. Recording
   1. Place the petri dish with the preparation on the microscope stage (Figure 2). Insert the reference electrode in the bath.
   2. 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).
   3. 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).
   4. 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.
   5. Switch on the amplifier, A/D board, and the computer.
   6. Choose the voltage clamp mode on the amplifier.
   7. Start acquisition software and choose the 'gap-free' mode.
   8. Observe the appearance of miniature excitatory junctional currents (mEJCs) on the computer screen.
   9. 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.
2. Stimulation
   1. Using a micromanipulator, under visual control, place the stimulation electrode near the axon innervating abdominal segments 2 - 4.
   2. 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).
   3. 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).
   4. 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.

Resultaten

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...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Microtorch 1WG61	Grainer	1WG61	Microtorch
Olympus BX61WI	Olympus	BX61WI	Upright microscope
Olympus Lumplan FL N 60x	Olympus	UPLFLN 60X	Microscope objective 60X
Olympus UPlan FL N 10x	Olympus	Uplanfl N 10X	Microscope objective 10X
Narishige Micromanipulator	Narishige	MHW-3	Three-axis Water Hydraulic Micromanipulator
npi Electronic GmbH ELC-03XS	npi Electronic GmbH	ELC-03XS	Electrophysiological amplifier
A.M.P.I Master 8	A.M.P.I.	Master 8	Electrical stimulator
A.M.P.I Iso-Flex	A.M.P.I.	Iso-Flex	Stimulus isolator
TMC antivibration table	TMC	63-9090	Antivibration table
TMC Faraday cage	TMC	81-333-90	Faraday cage
Digidata 1322A	Axon Instruments	Digidata 1322A	Digidata
Computer	Dell	Dell Dimension 5150	Computer with Win XP OS
Electrode holder	WPI	MEH3SW	Electrode holder
Optical filter	Omega optical	XF 115-2	Filter cube for Green Fluorescent Protein (GFP) detection
pCLAMP 8	Axon Instruments	8.0.0.81	Software for signal recording
Quantan	In-house software	-	Software for signal processing
Canton-S (Wildtype)	Bloomington Stock Center	64349	Control fly line
cpx SH1	Generous Gift of J.T. Littleton	-	Complexin knock-out fly line with increased spontaneous exocytosis
CD8-GFP	Bloomington Stock Center	5137	Fly line with neuronal fluorescent (GFP) Tag