Name: Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction (Video) | JoVE Journal
Uploaded: 2017-09-25T14:00:00.000+00:00
Duration: 7 min 1 s
Description: Learn focal macropatch recordings of synaptic currents from Drosophila larval neuromuscular junctions. This technique enables monitoring of miniature excitatory junctional currents at single boutons to study genetic effects on synaptic transmission.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Supported by the NIH grant R01 MH 099557

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 µm (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 µm (Figure 1A)27.</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 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>
<img alt="Capillary tube preparation, microscope setup, glass manipulation; lab experiment process." class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
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 recording electrode. (C.2) The boxed area is shown enlarged on the right. The first bend has an angle of approximately 90°, and the distance between the first bend and the tip of the electrode is approximately 1 mm. Scale bar = 3 mm. <a href="//cloudfront.jove.com/files/ftp_upload/56493/56493fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Additional Preparatory Steps
<ol>
	<li>Prepare heamolymph-like (HL3) solution (in mM): 70 NaCl, 5 KCl, 20 MgCl2, 10 NaHCO3, 5 trehalose, 115 sucrose, 5 HEPES, and 1 mM 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 µm.</li>
	<li>Insert a stimulation pipette in a microelectrode holder connected to a syringe.</li>
	<li>Manufacture dissection plates from small petri dishes (35 x 10 mm) coated with silicone rubber epoxy (see Table of Materials) as described in 28. 
	NOTE: Silicone rubber should be completely hardened before use.</li>
	<li>Pick a wandering third-instar larvae and dissect it as described in27,28,29,30. 
	NOTE: To visualize boutons, use the fly strain CD8-GFP (see Table of Materials)
	<ol>
		<li>Using forceps (see Table of Materials), pick the 3rd instar larvae from a vial.</li>
		<li>Pin the larvae, placing the first pin posterior and another pin anterior (close to mouth hooks).</li>
		<li>Add HL3 solution.</li>
		<li>Make a cut using spring scissors (see Table of Materials) all the way from the top pin to the bottom one on the dorsal side of the larvae.</li>
		<li>Pin the larvae fillet, placing 2 additional pins on the left and right sides of the larvae.</li>
		<li>Remove the guts and tracheas using forceps.</li>
		<li>Cut the nerves just outside the ventral nerve cord using spring scissors.</li>
	</ol>
	</li>
</ol>
3. Electrical recordings of EJCs
<img alt="Microscope setup for optical excitation in spectroscopy; includes stage, lenses, and wired controls." class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg" data-altupdatedat="1788250015000" data-alt="Figure 2"> 
Figure 2. The recording setup. The sample NMJ pinned to the silicon coated petri dish (arrow) is positioned over the movable stage of the upright compound microscope equipped with epifluorescence capabilities, a high magnification objective, and two micromanipulators. The microscope is stationed on an anti-vibration table. <a href="//cloudfront.jove.com/files/ftp_upload/56493/56493fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<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 synaptic bouton (Figure 3 B.1-3).</li>
		<li>Press the electrode very gently onto the muscle. Excessive pressure may damage the 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 'gap-free' mode.</li>
		<li>Observe the appearance of 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>
</ol>
<img alt="Electrode insertion in neural tissue; optical and microscopic imaging; experimental setup and results." class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg" data-altupdatedat="1788250015000" data-alt="Figure 3"> 
Figure 3. Visualization of synaptic boutons. (A) A brightfield image of a hemi-segment under 10x magnification (A.1) and the enlarged boxed area showing muscles 6 and 7 (A.2, the arrow marks the recording electrode). Scale bar = 50 µm. (B) Synaptic boutons are visualized in a Drosophila line with a genetically encoded neuronal marker (CD8-GFP) using epi-fluorescence imaging (B.1) or DIC optics (B.2). Images are taken with the 60x objective and the filter cube for GFP imaging (see Table of Materials). Synaptic boutons are marked with arrows, and an overlay of fluorescent and DIC images is shown in B.3. Scale bar = 10 µm. <a href="//cloudfront.jove.com/files/ftp_upload/56493/56493fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Stimulation
	<ol>
		<li>Using a micromanipulator, under a 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>
	<li>Seal resistance
	<ol>
		<li>Measure the seal resistance of the recording macropatch electrode by turning the electrode resistance switch of the amplifier to "seal test" position. Observe that the value of seal resistance in GΩ will be displayed in the "current" window.</li>
		<li>Make sure the seal resistance is in the range of 0.5 - 2 MΩ. Values out of this range indicate that the electrode is not properly polished or not properly positioned.</li>
		<li>Monitor the seal resistance throughout the recording, making sure it remains constant throughout the experiment.</li>
	</ol>
	</li>
</ol>
4. Analysis
<ol>
	<li>Analyze recordings employing customized software for analysis (see Table of Materials). 
	NOTE: Our lab uses in-house software Quantan31, which is customized for the detection of EJCs and mEJCs recorded focally (Figure 4). This software includes a Gaussian digital filter and allows the detection of quantal peaks in overlapping multi-quantal events (Figure 4A). Other approaches are described in17.</li>
</ol>
<img alt="Electrophysiology diagram showing mEJC and EJC amplitude over time at 0.2nA and 0.5nA scales." class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg" data-altupdatedat="1788250015000" data-alt="Figure 4"> 
Figure 4. Quantal analysis. Detection of mEJCs (A) and EJCs (B) by Quantan software. The event area is marked in green, and peaks are marked by red arrowheads. <a href="//cloudfront.jove.com/files/ftp_upload/56493/56493fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol><li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Properties+of+the+larval+neuromuscular+junction+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Properties of the larval neuromuscular junction in Drosophila melanogaster.</a> J Physiol. 262 (1), 189-214 (1976).</li>
<li>Katz, B., Miledi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+temperature+on+the+synaptic+delay+at+the+neuromuscular+junction%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">The effect of temperature on the synaptic delay at the neuromuscular junction.</a> J Physiol. 181 (3), 656-670 (1965).</li>
<li>Macleod, G. T., Gan, J., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicle-associated+proteins+and+quantal+release+at+single+active+zones+of+amphibian+%28Bufo+marinus%29+motor-nerve+terminals%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve terminals.</a> J Neurophysiol. 82 (3), 1133-1146 (1999).</li>
<li>Macleod, G. T., Farnell, L., Gibson, W. G., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantal+secretion+and+nerve-terminal+cable+properties+at+neuromuscular+junctions+in+an+amphibian+%28Bufo+marinus%29%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus).</a> J Neurophysiol. 81 (3), 1135-1146 (1999).</li>
<li>Zefirov, A., Benish, T., Fatkullin, N., Cheranov, S., Khazipov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+of+active+zones%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Localization of active zones.</a> Nature. 376 (6539), 393-394 (1995).</li>
<li>Macleod, G. T., Lavidis, N. A., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+dependence+of+quantal+secretion+from+visualized+sympathetic+nerve+varicosities+on+the+mouse+vas+deferens%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens.</a> J Physiol. 480 (Pt 1), 61-70 (1994).</li>
<li>Samigullin, D., Bill, C. A., Coleman, W. L., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+transmitter+release+by+synapsin+II+in+mouse+motor+terminals%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Regulation of transmitter release by synapsin II in mouse motor terminals.</a> J Physiol. 561 (Pt 1), 149-158 (2004).</li>
<li>Coleman, W. L., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rab3a-mediated+vesicle+recruitment+regulates+short-term+plasticity+at+the+mouse+diaphragm+synapse%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">Rab3a-mediated vesicle recruitment regulates short-term plasticity at the mouse diaphragm synapse.</a> Mol Cell Neurosci. 41 (2), 286-296 (2009).</li>
<li>Atwood, H. L., Parnas, H., Parnas, I., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantal+currents+evoked+by+graded+intracellular+depolarization+of+crayfish+motor+axon+terminals%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Quantal currents evoked by graded intracellular depolarization of crayfish motor axon terminals.</a> J Physiol. 383, 587-599 (1987).</li>
<li>Parnas, H., Dudel, J., Parnas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotransmitter+release+and+its+facilitation+in+crayfish.+I.+Saturation+kinetics+of+release%2C+and+of+entry+and+removal+of+calcium%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">Neurotransmitter release and its facilitation in crayfish. I. Saturation kinetics of release, and of entry and removal of calcium.</a> Pflugers Arch. 393 (1), 1-14 (1982).</li>
<li>Wojtowicz, J. M., Marin, L., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity-induced+changes+in+synaptic+release+sites+at+the+crayfish+neuromuscular+junction%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Activity-induced changes in synaptic release sites at the crayfish neuromuscular junction.</a> J Neurosci. 14 (6), 3688-3703 (1994).</li>
<li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crayfish+neuromuscular+facilitation+activated+by+constant+presynaptic+action+potentials+and+depolarizing+pulses%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Crayfish neuromuscular facilitation activated by constant presynaptic action potentials and depolarizing pulses.</a> J Physiol. 241 (1), 69-89 (1974).</li>
<li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+the+statistics+of+transmitter+release+during+facilitation%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Changes in the statistics of transmitter release during facilitation.</a> J Physiol. 229 (3), 787-810 (1973).</li>
<li>Worden, M. K., Bykhovskaia, M., Hackett, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Facilitation+at+the+lobster+neuromuscular+junction%3A+a+stimulus-dependent+mobilization+model%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Facilitation at the lobster neuromuscular junction: a stimulus-dependent mobilization model.</a> J Neurophysiol. 78 (1), 417-428 (1997).</li>
<li>Bykhovskaia, M., Hackett, J. T., Worden, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Asynchrony+of+quantal+events+in+evoked+multiquantal+responses+indicates+presynaptic+quantal+interaction%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Asynchrony of quantal events in evoked multiquantal responses indicates presynaptic quantal interaction.</a> J Neurophysiol. 81 (5), 2234-2242 (1999).</li>
<li>Bykhovskaia, M., Polagaeva, E., Hackett, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechnisms+underlying+different+facilitation+forms+at+the+lobster+neuromuscular+synapse%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse.</a> Brain Res. 1019 (1-2), 10-21 (2004).</li>
<li>Cooper, R. L., Stewart, B. A., Wojtowicz, J. M., Wang, S., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantal+measurement+and+analysis+methods+compared+for+crayfish+and+Drosophila+neuromuscular+junctions%2C+and+rat+hippocampus%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus.</a> J Neurosci Methods. 61 (1-2), 67-78 (1995).</li>
<li>Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+stability+of+Drosophila+larval+neuromuscular+preparations+in+haemolymph-like+physiological+solutions%5BTitle%5D)+AND+%22J+Comp+Physiol+A%22%5BJournal%5D)">Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions.</a> J Comp Physiol A. 175 (2), 179-191 (1994).</li>
<li>Pawlu, C., DiAntonio, A., Heckmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postfusional+control+of+quantal+current+shape%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Postfusional control of quantal current shape.</a> Neuron. 42 (4), 607-618 (2004).</li>
<li>Akbergenova, Y., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synapsin+maintains+the+reserve+vesicle+pool+and+spatial+segregation+of+the+recycling+pool+in+Drosophila+presynaptic+boutons%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Synapsin maintains the reserve vesicle pool and spatial segregation of the recycling pool in Drosophila presynaptic boutons.</a> Brain Res. 1178, 52-64 (2007).</li>
<li>Akbergenova, Y., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancement+of+the+endosomal+endocytic+pathway+increases+quantal+size%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">Enhancement of the endosomal endocytic pathway increases quantal size.</a> Mol Cell Neurosci. 40 (2), 199-206 (2009).</li>
<li>Vasin, A., Volfson, D., Littleton, J. T., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+the+Complexin+Accessory+Helix+with+Synaptobrevin+Regulates+Spontaneous+Fusion%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.</a> Biophys J. 111 (9), 1954-1964 (2016).</li>
<li>Wong, K., Karunanithi, S., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantal+unit+populations+at+the+Drosophila+larval+neuromuscular+junction%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Quantal unit populations at the Drosophila larval neuromuscular junction.</a> J Neurophysiol. 82 (3), 1497-1511 (1999).</li>
<li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+reduced+calcium+on+quantal+unit+current+and+release+at+the+crayfish+neuromuscular+junction%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction.</a> Pflugers Arch. 391 (1), 35-40 (1981).</li>
<li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contribution+of+Ca2%2B+inflow+to+quantal%2C+phasic+transmitter+release+from+nerve+terminals+of+frog+muscle%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">Contribution of Ca2+ inflow to quantal, phasic transmitter release from nerve terminals of frog muscle.</a> Pflugers Arch. 422 (2), 129-142 (1992).</li>
<li>Marrero, H. G., Lemos, J. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMarrero%2C+HG+%22Loose-Patch-Clamp+method%22">Loose-Patch-Clamp method</a>. , 2 ed, Humana Press. (2007).</li>
<li>Wu, W. H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiological+recordings+of+high+and+low+output+NMJs+on+the+crayfish+leg+extensor+muscle%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Physiological recordings of high and low output NMJs on the crayfish leg extensor muscle.</a> J Vis Exp. (45), (2010).</li>
<li>Verstreken, P., Ohyama, T., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FM+1-43+labeling+of+synaptic+vesicle+pools+at+the+Drosophila+neuromuscular+junction%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.</a> Methods Mol Biol. 440, 349-369 (2008).</li>
<li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/19229190">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li>
<li>Imlach, W., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+methods+for+recording+synaptic+potentials+from+the+NMJ+of+Drosophila+larvae%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.</a> J Vis Exp. (24), (2009).</li>
<li>Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+quantal+analysis+more+convenient%2C+fast%2C+and+accurate%3A+user-friendly+software+QUANTAN%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN.</a> J Neurosci Methods. 168 (2), 500-513 (2008).</li>
<li>Huntwork, S., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+complexin+fusion+clamp+regulates+spontaneous+neurotransmitter+release+and+synaptic+growth%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth.</a> Nat Neurosci. 10 (10), 1235-1237 (2007).</li>
<li>Zhong, Y., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.</a> Science. 251 (4990), 198-201 (1991).</li>
<li>Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Size+of+vesicle+pools%2C+rates+of+mobilization%2C+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant%2C+shibire%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.</a> Neuron. 28 (3), 941-953 (2000).</li>
<li>Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+and+evoked+release+are+independently+regulated+at+individual+active+zones%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Spontaneous and evoked release are independently regulated at individual active zones.</a> J Neurosci. 33 (44), 17253-17263 (2013).</li>
</ol>

The authors have nothing to disclose.

Drosophila represents an advantageous model organism to study synaptic transmission. Several recording configurations have been used at the larval NMJ, including intracellular recordings of synaptic potentials, recordings of synaptic currents with two electrode voltage clamp33,34, and focal macropatch recordings of synaptic currents described here. The latter technique allows the precise quantification of synaptic transmission at visualized boutons.
...

Drosophila is an excellent model system to study the molecular mechanisms controlling synaptic transmission. The neuromuscular system in Drosophila is glutamatergic, and therefore the Drosophila neuromuscular junction (NMJ) can be used to study the conserved features of glutamatergic release. Since Jan and Jan&#39;s study1, the third instar larvae has been broadly used to study evoked and spontaneous synaptic transmission by monitoring excitatory junction potentials (EJPs) or currents (EJCs). EJPs are commonly recorded intracellularly with a sharp glass micro-electrode, and they reflect the activity of the entire NMJ, including all the boutons making synapses at the given muscle fiber.
In contrast, the activity of a limited number of the sites of release can be recorded focally by positioning a micropipette tip near neuronal terminals or synaptic varicosities. This technique was originally employed by Katz and Miledi2, and focal extracellular recordings have been successfully employed at several NMJ preparations, including frog3,4,5, mouse6,7,8, crustacean9,10,11,12,13,14,15,16, and Drosophila17,18,19,20,21,22,23. This approach was further developed by Dudel, who optimized macropatch recoding electrodes24,25. In Dudel&#39;s implementation, this technique closely matched the loose-patch-clamp method26.
The Drosophila larval NMJ has clearly defined synaptic boutons, and transgenic lines with genetically encoded neuronal fluorescent tags (see Table of Materials) are readily available. These advantages enabled us to record EJCs and mEJCs from a selected synaptic bouton20,21,22. Here, we describe this technique in detail.

<table><tbody><tr><td>Sutter P-97</td><td>Sutter instrument</td><td>P-97</td><td>Microelectrode puller</td></tr><tr><td>Narishige MF-830</td><td>Narishige</td><td>MF-830</td><td>Microforge</td></tr><tr><td>WPI MF200</td><td>WPI</td><td>MF200</td><td>Microforge</td></tr><tr><td>Glass capilaries</td><td>WPI</td><td>B150-86-10</td><td>Glass capilaries</td></tr><tr><td>Microtorch 1WG61</td><td>Grainer</td><td>1WG61</td><td>Microtorch</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>SYLGARD 184</td><td>Silicone for dissection plates preparation</td></tr><tr><td>Dissection pins</td><td>Amazon</td><td>B00J5PMPJA</td><td>Pins for larvae positioning</td></tr><tr><td>Tweezers</td><td>WPIINC</td><td>500342</td><td>Tweezers for placing pins, removing the guts and tracheas.</td></tr><tr><td>Scissors</td><td>WPIINC</td><td>501778</td><td>Scissors for cutting the cuticula of the larvae and nerves.</td></tr><tr><td>Olympus BX61WI</td><td>Olympus</td><td>BX61WI</td><td>Upright microscope</td></tr><tr><td>Olympus Lumplan FL N 60x</td><td>Olympus</td><td>UPLFLN 60X</td><td>Microscope objective 60X</td></tr><tr><td>Olympus UPlan FL N 10x</td><td>Olympus</td><td>Uplanfl N 10X</td><td>Microscope objective 10X</td></tr><tr><td>Narishige Micromanipulator</td><td>Narishige</td><td>MHW-3</td><td>Three-axis Water Hydraulic Micromanipulator</td></tr><tr><td>npi Electronic GmbH ELC-03XS</td><td>npi Electronic GmbH</td><td>ELC-03XS</td><td>Electrophysiological amplifier</td></tr><tr><td>A.M.P.I Master 8</td><td>A.M.P.I.</td><td>Master 8</td><td>Electrical stimulator</td></tr><tr><td>A.M.P.I Iso-Flex</td><td>A.M.P.I.</td><td>Iso-Flex</td><td>Stimulus isolator</td></tr><tr><td>TMC antivibration table</td><td>TMC</td><td>63-9090</td><td>Antivibration table</td></tr><tr><td>TMC Faraday cage</td><td>TMC</td><td>81-333-90</td><td>Faraday cage</td></tr><tr><td>Digidata 1322A</td><td>Axon Instruments</td><td>Digidata 1322A</td><td>Digidata</td></tr><tr><td>Computer</td><td>Dell</td><td>Dell Dimension 5150</td><td>Computer with Win XP OS</td></tr><tr><td>Electrode holder</td><td>WPI</td><td>MEH3SW</td><td>Electrode holder</td></tr><tr><td>Optical filter</td><td>Omega optical</td><td>XF 115-2</td><td>Filter cube for Green Fluorescent Protein (GFP) detection</td></tr><tr><td>pCLAMP 8</td><td>Axon Instruments</td><td>8.0.0.81</td><td>Software for signal recording</td></tr><tr><td>Quantan</td><td>In-house software</td><td>-</td><td>Software for signal processing</td></tr><tr><td>Canton-S (Wildtype)</td><td>Bloomington Stock Center</td><td>64349</td><td>Control fly line</td></tr><tr><td>cpx SH1</td><td>Generous Gift of J.T. Littleton</td><td>-</td><td>Complexin knock-out fly line with increased spontaneous exocytosis</td></tr><tr><td>CD8-GFP</td><td>Bloomington Stock Center</td><td>5137</td><td>Fly line with neuronal fluorescent (GFP) Tag</td></tr></tbody></table>

focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal macropatch recordings enable monitoring synaptic activity from selected synaptic boutons (Figure 5). When the electrode is positioned on the top of a synaptic bouton (Figure 5A, site 1), the recorded mEJCs (Figure 5C, site 1) have a amplitudes significantly exceeding the noise level and sharp rising phases (at a sub-millisecond range). When the recording electrode is moved away from the synapti...

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

focal-macropatch-recordings-synaptic-currents-from-drosophila-larval

Research

JoVE Journal

Neuroscience

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants.   This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Electrophysiological Recording in the Drosophila Embryo

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

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.

Recording Synaptic Currents at the Drosophila Larval Neuromuscular Junction

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

The zebrafish larval neuromuscular junction is an attractive model for studying synaptic physiology. It is amenable to many experimental techniques, including electrophysiology and optical imaging. Here, we describe a protocol for imaging synaptic transmission using a pHluorin-based probe under an upright epifluorescence microscope.

Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish

Learn focal macropatch recordings of synaptic currents from Drosophila larval neuromuscular junctions. This technique enables monitoring of miniature excitatory junctional currents at single boutons to study genetic effects on synaptic transmission.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Explore More Videos