I would like to offer great thanks to David Carlson, Professor of English at Matsumoto Dental University, for his kind support in writing this report. I would also like to thank Tadafumi Adachi, a seminarist in our laboratory, for his compliance as a subject in this investigation.

1. Preparation of EMG electrodes
 <ol>
 <li>Begin by preparing nine electrodes. Eight of them are used for recording signals from sites over the muscle, and one is a reference electrode. </li>
 <li>After connecting the recording and reference electrodes to the amplifier, fill the discs with electric-conductance paste. Every type is OK, but a low fluid type is better.</li>
 <li>Before attaching the electrodes to the skin, have the subject down firmly, and determine the approximate location of the masseter muscle on the face. </li>
 <li>The superior tendon of the masseter muscle attaches to the zygomatic arch, and its inferior tendon to the angle of mandible.</li>
	<li>Place the eight recording electrodes onto a long piece of adhesive tape in equal inter-electrode distance, and attach the array to the skin surface. The first recording electrode is placed on the zygomatic arch, and the last one on the angle of mandible.</li>
 	<li>Finally, attach the reference electrode to the tip of the nose.</li>
</ol>
2. EMG recording
<ol>
	<li>To begin recording, first set the parameters of the multi-channel analogue amplifier (MEG6100). Set the gain to x 500, the low pass filter to 0.5 Hz, and the high pass filter to 10 kHz.</li>
 	<li>Connect the eight outputs of the analogue amplifier to an Analogue-Digital converter (PCI-MIO-16E-4). The digital signal is processed with LabView software with a sampling rate of 20kHz.</li>
 	<li>When everything is connected, record EMGs during quick chewing-gum clenching on the ipsilateral side.</li>
 	<li>The traces from the eight channels are presented on the LabView display immediately after the recording process has been completed.</li>
</ol>
3. EPP extraction and observation of its features
<ol>
	<li>To extract the EPP component, first remove the action potential component from the raw EMGs using a high-cut digital filter (Butterworth filter).</li>
 	<li>Begin applying the filter with a cut-off frequency of 10 Hz. A slow wave that deflects in the positive or negative direction should appear. This wave corresponds to the EPP, and the difference in polarity reflects the sink-source relation of the end plate current.</li>
 	<li>When recording from the masseter muscle, the most negative deflection of the wave is expected in the trace from the most inferior recording site, and the polarity should shift around 3 sites away from that site.</li>
 	<li>The magnitude of the EPP component can be measured as a peak point of the deflection or an area during the deflection.</li>
 	<li>The polarity change of the EPP component can be determined by plotting the EPP wave from each channel against a reference channel that showed the most negative deflection.</li>
 	<li>Now by increasing the cut-off frequency, the slow wave will become oscillatory. The oscillation will increase with increasing cut-off frequency and will generally become obvious at a cut-off frequency of 30 Hz.</li>
 	<li>The phase of the wave will reverse across the trace in which the polarity of the 10Hz filtered EPP wave showed a reversal.</li>
 <li>The phase shift of the oscillation can be checked with the same graphs that were applied to the 10Hz-filtered EPP wave.</li>
 <li>From the interval between neighboring positive and/or negative peaks of the oscillation, we can get an approximation of the frequency of the oscillation</li>
</ol>
4. EMG recording results
<img src="/files/ftp_upload/1653/1653Fig1a.jpg" alt="Figure 1 a" /> Figure 1a: Shown here is a schematic of the eight sites along the masseter muscle that were recorded from.
<img src="/files/ftp_upload/1653/1653Fig1b.jpg" alt="Figure 1 b" /> Figure 1b: We see a sample of the monopolar EMGs recorded simultaneously from the sites over the masseter muscle in response to quick gum clenching on the ipsilateral side. The EPP component was extracted using a high-cut digital filter with a cut-off frequency of 10Hz, which is superimposed on each raw EMG. In this trial, the most negative deflection was observed in traces-5 and -6 and the polarity changed across trace-3.
<img src="/files/ftp_upload/1653/1653Fig1c.jpg" alt="Figure 1c" /> Figure 1c: When the cut-off frequency of the high-cut digital filter was set to 30Hz, the slow wave exhibited an oscillation. Its phase also shifted across trace 3, where the polarity of the 10Hz-filtered EPP wave changed. The interval between neighboring positive peaks of the oscillation was measured. In this subject, the average inter-peak interval was 31.7 &plusmn; 6.5 msec over 16 recordings.

<ol>
	<li>Eccles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The physiology of synapses. , (1964).</li><li>Mitzdorf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+source-density+method+and+application+in+cat+cerebral+cortex:+investigation+of+evoked+potentials+and+EEG+phenomena.">Current source-density method and application in cat cerebral cortex: investigation of evoked potentials and EEG phenomena.</a> Physiol. Rev. 65, 37-100 (1985).</li><li>Rall, W., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+reconstruction+of+field+potentials+and+dendrodendritic+synaptic+interactions+in+olfactory+bulb.">Theoretical reconstruction of field potentials and dendrodendritic synaptic interactions in olfactory bulb.</a> J. Neurophysiol. 31, 884-915 (1968).</li><li>Richardson, T. L., Turner, R. W., Miller, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action-potential+discharge+in+hippocampal+CA1+pyramidal+neurons:+current+source+density+analysis.">Action-potential discharge in hippocampal CA1 pyramidal neurons: current source density analysis.</a> J. Neurophysiol. 58, 981-996 (1987).</li><li>Kumai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+the+neuromuscular+junction+of+the+human+masseter+muscle+estimated+from+the+low+frequency+component+of+the+surface+electromyogram.">Location of the neuromuscular junction of the human masseter muscle estimated from the low frequency component of the surface electromyogram.</a> J. Jpn. Physiol. 55, 61-68 (2005).</li><li>Mito, K., Sakamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+muscle+fiber+conduction+velocity+of+m.+masseter+during+voluntary+isometric+contraction.">Distribution of muscle fiber conduction velocity of m. masseter during voluntary isometric contraction.</a> Electroencephalogr. Clin. Neurophysiol. 40, 275-285 (2000).</li><li>Tokunaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+configuration+of+the+myoneural+junctions+of+human+masticatory+muscle+detected+with+matrix+electrode.">Two-dimensional configuration of the myoneural junctions of human masticatory muscle detected with matrix electrode.</a> J. Oral Rehabili. 25, 329-334 (1998).</li><li>Rohen, J. H., Yokochi, C., L&#x0171;tjen-Drecoll, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Color atlas of anatomy. , (2006).</li><li>Wichmann, T., DeLong, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillation+in+the+basal+ganglia.">Oscillation in the basal ganglia.</a> Nature. 400, 621-622 (1999).</li></ol>

<ol><li>As the negative potential is considered to be formed by a flow-in of the endplate current, and the positive deflection, by its flow-out [1,2,3,4], the trace showing the most negative deflection must correspond to the site locating the neuromuscular junction [5]. Figure 1b indicates the neuromuscular junction of the masseter muscle locate in its inferior portion close to the angle of mandible, which is approximately the same as results obtained by another method utilizing the conduction manner of the motor unit action potential [6,7].</l...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Analogue filter</td>
<td>&nbsp;</td>
<td>Nihon Kohden</td>
<td>MEG6100</td>
<td>pre-,main-amplifier</td>
</tr>
<tr>
<td>A/D converter</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI-MIO-16E-4</td>
<td>PCI board for computer</td>
</tr>
<tr>
<td>Connection interface</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>BNC-2090</td>
<td>8ch BNC adaptor</td>
</tr>
<tr>
<td>Disc electrodes</td>
<td>&nbsp;</td>
<td>Nihon Kohden</td>
<td>NS-11</td>
<td>Ag/AgCl (&Phi;8mm)</td>
</tr>
<tr>
<td>Electrode past</td>
<td>&nbsp;</td>
<td>Sanshin</td>
<td>SA-5</td>
<td>Semi-fluid carbon past</td>
</tr>
<tr>
<td>LabVIEW (Digital filter)</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>V. 8.5</td>
<td>Programming language</td>
</tr>
<tr>
<td>Chewing gum</td>
<td>&nbsp;</td>
<td>Lotte</td>
<td>Green gum</td>
<td>Test food in clenching</td>
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extraction of the epp component from the surface emg

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component which can be extracted with an appropriate signal filter. Two factors are important: the EMG must be recorded in monopolar fashion, and the recording must be done so the low frequency signal corresponding the EPP is not eliminated. This report explains how to extract the EPP component from the EMG of the masseter muscle in a human subject. The surface EMG is recorded from eight sites using traditional disc electrodes aligned along over the muscle, with equal inter-electrode distance from the zygomatic arch to the angle of mandible in response to quick gum clenching. A reference electrode is placed on the tip of the nose. The EPP component is extracted from the raw EMGs by applying a high-cut digital filter (2nd dimension Butterworth filter) with a range of 10-35 Hz. When the filter is set to 10 Hz, the extracted EPP wave deflects either negative or positive depending on the recording site. The difference in the polarity reflects the sink-source relation of the end plate current, with the site showing the most negative deflection corresponding to the neuromuscular junction. In the case of the masseter muscle, the neuromuscular junction is estimated to be located in the inferior portion close to the angle of mandible. The EPP component exhibits an interesting oscillation when the cut-off frequency of the high-cut digital filter is set to 30 Hz. The EPP oscillation indicates that muscle contraction is adjusted in an intermittent manner. Abnormal tremors accompanying various sorts of diseases may be substantially due to this EPP oscillation, which becomes slower and is difficult to cease.

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Extraction of the EPP Component from the Surface EMG

The endplate potential (EPP) component can be extracted from the surface EMG using a digital filter. The extracted EPP shows oscillation with a frequency of about 30 Hz.

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component ...

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12.5K Views.  Matsumoto Dental University. The endplate potential (EPP) component can be extracted from the surface EMG using a digital filter. The extracted EPP shows oscillation with a frequency of about 30 Hz.

Video: Extraction of the EPP Component from the Surface EMG

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

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RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.

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Purification of Progenitors from Skeletal Muscle

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Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
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The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

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Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

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The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

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The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

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Extraction of the EPP Component from the Surface EMG

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