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...

<ol>
	<li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+temperature+on+the+synaptic+delay+at+the+neuromuscular+junction.">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. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+currents+evoked+by+graded+intracellular+depolarization+of+crayfish+motor+axon+terminals.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitter+release+and+its+facilitation+in+crayfish.+I.+Saturation+kinetics+of+release,+and+of+entry+and+removal+of+calcium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-induced+changes+in+synaptic+release+sites+at+the+crayfish+neuromuscular+junction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crayfish+neuromuscular+facilitation+activated+by+constant+presynaptic+action+potentials+and+depolarizing+pulses.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+statistics+of+transmitter+release+during+facilitation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+at+the+lobster+neuromuscular+junction:+a+stimulus-dependent+mobilization+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asynchrony+of+quantal+events+in+evoked+multiquantal+responses+indicates+presynaptic+quantal+interaction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechnisms+underlying+different+facilitation+forms+at+the+lobster+neuromuscular+synapse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+measurement+and+analysis+methods+compared+for+crayfish+and+Drosophila+neuromuscular+junctions,+and+rat+hippocampus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+stability+of+Drosophila+larval+neuromuscular+preparations+in+haemolymph-like+physiological+solutions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postfusional+control+of+quantal+current+shape.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapsin+maintains+the+reserve+vesicle+pool+and+spatial+segregation+of+the+recycling+pool+in+Drosophila+presynaptic+boutons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+endosomal+endocytic+pathway+increases+quantal+size.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+the+Complexin+Accessory+Helix+with+Synaptobrevin+Regulates+Spontaneous+Fusion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+unit+populations+at+the+Drosophila+larval+neuromuscular+junction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+reduced+calcium+on+quantal+unit+current+and+release+at+the+crayfish+neuromuscular+junction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Ca2++inflow+to+quantal,+phasic+transmitter+release+from+nerve+terminals+of+frog+muscle.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Loose-Patch-Clamp method. , (2007).</li><li>Wu, W. H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+recordings+of+high+and+low+output+NMJs+on+the+crayfish+leg+extensor+muscle.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM+1-43+labeling+of+synaptic+vesicle+pools+at+the+Drosophila+neuromuscular+junction.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+methods+for+recording+synaptic+potentials+from+the+NMJ+of+Drosophila+larvae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+quantal+analysis+more+convenient,+fast,+and+accurate:+user-friendly+software+QUANTAN.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complexin+fusion+clamp+regulates+spontaneous+neurotransmitter+release+and+synaptic+growth.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+of+vesicle+pools,+rates+of+mobilization,+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant,+shibire.">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. 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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 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 synapti...

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 ...

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

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6.0K 次观看.  Wayne State University. The overall goal of this technique is to monitor synaptic activity of visualized synaptic boutons at the drosophila larval neuromuscular junction. An excellent model system readily amendable to genetic manipulations. This technique can address key questions in your science.Such as how mutations of synaptic proteins regulate synaptic transmission. The main advantage of this technique is that the activity of a limited number of release sites can be monitored and recorded currents of fast...

视频: 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...

recording synaptic currents at the drosophila larval neuromuscular junction

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...

Recording Synaptic Currents at the Drosophila Larval Neuromuscular Junction

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...

why quantification matters: characterization of phenotypes at the drosophila larval neuromuscular junction

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.

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

using drosophila larval neuromuscular junction and muscle cells to visualize microtubule network

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous...

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

detection of in situ protein-protein complexes at the drosophila larval neuromuscular junction using proximity ligation assay

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...

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

drosophila neuromuscular junction (nmj) quantification: a method to assess synaptic morphology and function

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...

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

dissection and imaging of active zones in the drosophila neuromuscular junction

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...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

channelrhodopsin2 mediated stimulation of synaptic potentials at drosophila neuromuscular junctions

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching...

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

whole-cell recordings of light evoked excitatory synaptic currents in the retinal slice

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

visualizing the live drosophila glial-neuromuscular junction with fluorescent dyes

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel  Inside-out  tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent...

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

dissecting and recording from the c. elegans neuromuscular junction

Dissecting and Recording from The C. Elegans Neuromuscular Junction

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...

in vivo visualization of synaptic vesicles within drosophila larval segmental axons

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...

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

electrophysiological methods for recording synaptic potentials from the nmj of drosophila larvae

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

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...

Viewing Microtubule Networks in &lt;em&gt;Drosophila&lt;/em&gt; Neuromuscular Junctions and Muscle Cells

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dissection and immunohistochemistry of the drosophila adult leg to detect changes at the neuromuscular junction for an identified motor neuron

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

two algorithms for high-throughput and multi-parametric quantification of drosophila neuromuscular junction morphology

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).

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

the neuromuscular junction: measuring synapse size, fragmentation and changes in synaptic protein density using confocal fluorescence microscopy

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

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...

in vitro neuromuscular junction induced from human induced pluripotent stem cells

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for...

3d particle tracking for noninvasive in vivo analysis of synaptic microtubule dynamics in dendrites and neuromuscular junctions of drosophila

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

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.

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 ...

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.

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 ...

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 ...

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.

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 (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. ...

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.

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 ...

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 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 ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.