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 &#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)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="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.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 recording electrode. (C.2) The boxed area is shown enlarged on the right. The first bend has an angle of approximately 90&#176;, 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.remotexs.ntu.edu.sg/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 &#181;m.</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="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg" /> 
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.remotexs.ntu.edu.sg/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 &#39;gap-free&#39; 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="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg" /> 
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 &#181;m. (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 &#181;m. <a href="//cloudfront-jove-com.remotexs.ntu.edu.sg/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 &#34;seal test&#34; position. Observe that the value of seal resistance in G&#937; will be displayed in the &#34;current&#34; window.</li>
		<li>Make sure the seal resistance is in the range of 0.5 - 2 M&#8486;. 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="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg" /> 
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.remotexs.ntu.edu.sg/files/ftp_upload/56493/56493fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.
The success of the described protocol critically depends on the ability to clearly visualize the area of interest and to customize the preparation of recording electrodes. Thus, quality DIC optics, a high magnification water immersion objective with a long working distance, and customized equipment for fire polishing and bending of recording electrodes are critically important.
The advantage of this approach is that it allows monitoring the activity of a few synapses that are positioned under the recording electrode. It should be noted, however, boutons positioned near the rim of the electrode may also contribute to recorded activity. It is critical, therefore, that the position of the electrode, as well as the seal resistance, does not change in the course of the experiment.
The ability to monitor activity of a single bouton can be potentially combined with recent imaging technologies. For example, optical detection of activity at individual active zones35 can be combined with focal recordings of EJCs and mEJC, and this could couple the spatial resolution of optical detection with temporal resolution of electrical recordings.

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 width="1251">
	<tbody>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Sutter P-97</td>
			<td style="width:288px;">Sutter instrument</td>
			<td style="width:180px;">P-97</td>
			<td style="width:495px;">Microelectrode puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narishige MF-830</td>
			<td style="width:288px;">Narishige</td>
			<td style="width:180px;">MF-830</td>
			<td>Microforge</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">WPI MF200</td>
			<td style="width:288px;">WPI</td>
			<td>MF200</td>
			<td>Microforge</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Glass capilaries</td>
			<td style="width:288px;">WPI</td>
			<td>B150-86-10</td>
			<td style="width:495px;">Glass capilaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtorch 1WG61</td>
			<td style="width:288px;">Grainer</td>
			<td style="width:180px;">1WG61</td>
			<td>Microtorch</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:288px;">Dow Corning</td>
			<td style="width:180px;">SYLGARD 184</td>
			<td>Silicone for dissection plates preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Dissection pins</td>
			<td style="width:288px;">Amazon</td>
			<td>B00J5PMPJA</td>
			<td>Pins for larvae positioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Tweezers</td>
			<td style="width:288px;">WPIINC</td>
			<td>500342</td>
			<td>Tweezers for placing pins, removing the guts and tracheas.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Scissors</td>
			<td style="width:288px;">WPIINC</td>
			<td>501778</td>
			<td>Scissors for cutting the cuticula of the larvae and nerves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus BX61WI</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">BX61WI</td>
			<td>Upright microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus Lumplan FL N 60x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">UPLFLN 60X</td>
			<td>Microscope objective 60X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus UPlan FL N 10x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">Uplanfl N 10X</td>
			<td>Microscope objective 10X</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Narishige Micromanipulator</td>
			<td style="width:288px;">Narishige</td>
			<td style="width:180px;">MHW-3</td>
			<td>Three-axis Water Hydraulic Micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">npi Electronic GmbH ELC-03XS</td>
			<td style="width:288px;">npi Electronic GmbH</td>
			<td style="width:180px;">ELC-03XS</td>
			<td>Electrophysiological amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Master 8</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Master 8</td>
			<td>Electrical stimulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Iso-Flex</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Iso-Flex</td>
			<td>Stimulus isolator</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">TMC antivibration table</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">63-9090</td>
			<td>Antivibration table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMC Faraday cage</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">81-333-90</td>
			<td>Faraday cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Digidata 1322A</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">Digidata 1322A</td>
			<td>Digidata</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Computer</td>
			<td style="width:288px;">Dell</td>
			<td style="width:180px;">Dell Dimension 5150</td>
			<td>Computer with Win XP OS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Electrode holder</td>
			<td style="width:288px;">WPI</td>
			<td>MEH3SW&#160;</td>
			<td style="width:495px;">Electrode holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Optical filter</td>
			<td style="width:288px;">Omega optical</td>
			<td style="width:180px;">XF 115-2</td>
			<td style="width:495px;">Filter cube for Green Fluorescent Protein (GFP) detection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">pCLAMP 8</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">8.0.0.81</td>
			<td>Software for signal recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Quantan</td>
			<td style="width:288px;">In-house software</td>
			<td style="width:180px;">-</td>
			<td>Software for signal processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Canton-S (Wildtype)</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td>64349</td>
			<td>Control fly line</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">cpx SH1</td>
			<td style="width:288px;">Generous Gift of J.T. Littleton</td>
			<td style="width:180px;">-</td>
			<td>Complexin knock-out fly line with increased spontaneous exocytosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">CD8-GFP</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td style="width:180px;">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

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 synaptic bouton by several microns (Figure 5A, site 2), the amplitudes of recorded mEJCs decline almost to the noise level (Figure 5B, site 2). The recorded EJCs can barely be distinguished from the noise and they have prolonged rising phases (Figure 5C, site 2 versus site 1).
The limited number of release sites contributing to recorded EJCs and mEJCs, as well as rapid kinetics of synaptic currents recorded focally, enables accurate detection of release events in mutants with elevated synaptic activity. This can be clearly illustrated by recordings of mEJCs from complexin null mutant (Figure 6A). Spontaneous activity is drastically elevated in this mutant32, and therefore mEJPs recorded intracellularly overlap and cannot be clearly distinguished from each other (Figure 6B), while focal recordings22 enable accurate detection of spontaneous release events (Figure 4A).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56493/56493fig5.jpg" /> 
Figure 5. Recordings of EJCs and mEJCs from a selected bouton. (A) Placing the electrode over a selected bouton (site 1) and moving it away from the bouton (site 2). The images show the CD8-GFP tagged NMJ visualized with epi-fluorescence (A.1), recording electrode over the muscle fiber (A.2), and overlays (A.3, A.4), with the electrode positioned over site 1 (A.3) or site 2 (A.4). Scale bar = 10 &#181;m. (B) mEJCs recorded from the bouton (site 1) are clearly distinguished from the recording noise and have rapid rising phases. In contrast, mEJCs recorded from site 2 cannot be reliably distinguished from the recording noise, their amplitudes are reduced several-fold, and they have a slower time-course. (C) The Amplitudes of EJCs recorded from the bouton (site 1) exceed by many-fold the amplitudes of EJCs recorded from site 2, and they also have more rapid kinetics. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56493/56493fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56493/56493fig6.jpg" /> 
Figure 6. Focal macropatch versus intracellular recordings. Focal recordings enable accurate detection of mEJCs in the complexin null mutant (A), while intracellular recordings (B) from this mutant exhibit release events that overlap and cannot be reliably detected. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56493/56493fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.0K Views.  Wayne State University. 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.

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

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.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...