ありがとう博士マーク ・ m ・ リッチとダニエル ・ ミランダ編集コメント、アフマド ・ Khedraki (A.A.V. にスタートアップ資金) の金融支援この手法、およびライト州立大学の確立を助けるため。

<ol>
	<li>Angaut-Petit, D., Molgo, J., Connold, A. L., Faille, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+levator+auris+longus+muscle+of+the+mouse:+a+convenient+preparation+for+studies+of+short-+and+long-term+presynaptic+effects+of+drugs+or+toxins.">The levator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins.</a> Neurosci Lett. 82 (1), 83-88 (1987).</li><li>Erzen, I., Cvetko, E., Obreza, S., Angaut-Petit, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+types+in+the+mouse+levator+auris+longus+muscle:+a+convenient+preparation+to+study+muscle+and+nerve+plasticity.">Fiber types in the mouse levator auris longus muscle: a convenient preparation to study muscle and nerve plasticity.</a> J Neurosci Res. 59 (5), 692-697 (2000).</li><li>Bertone, N. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbonic+anhydrase+inhibitor+acetazolamide+shifts+synaptic+vesicle+recycling+to+a+fast+mode+at+the+mouse+neuromuscular+junction.">Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction.</a> Synapse. , (2017).</li><li>Garcia-Chacon, L. E., Nguyen, K. T., David, G., Barrett, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrusion+of+Ca2++from+mouse+motor+terminal+mitochondria+via+a+Na+-Ca2++exchanger+increases+post-tetanic+evoked+release.">Extrusion of Ca2+ from mouse motor terminal mitochondria via a Na+-Ca2+ exchanger increases post-tetanic evoked release.</a> J Physiol. 574 (Pt 3), 663-675 (2006).</li><li>Murray, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+vulnerability+of+motor+neurons+and+dissociation+of+pre-+and+post-synaptic+pathology+at+the+neuromuscular+junction+in+mouse+models+of+spinal+muscular+atrophy.">Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy.</a> Hum Mol Genet. 17 (7), 949-962 (2008).</li><li>Nadal, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+muscarinic+acetylcholine+autoreceptors+(M1,+M2+and+M4+subtypes),+adenosine+receptors+(A1+and+A2A)+and+tropomyosin-related+kinase+B+receptor+(TrkB)+modulate+the+developmental+synapse+elimination+process+at+the+neuromuscular+junction.">Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.</a> Mol Brain. 9 (1), 67 (2016).</li><li>Rousse, I., St-Amour, A., Darabid, H., Robitaille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse-glia+interactions+are+governed+by+synaptic+and+intrinsic+glial+properties.">Synapse-glia interactions are governed by synaptic and intrinsic glial properties.</a> Neuroscience. 167 (3), 621-632 (2010).</li><li>Rozas, J. L., Gomez-Sanchez, L., Tomas-Zapico, C., Lucas, J. J., Fernandez-Chacon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+neurotransmitter+release+at+the+neuromuscular+junction+in+a+mouse+model+of+polyglutamine+disease.">Increased neurotransmitter release at the neuromuscular junction in a mouse model of polyglutamine disease.</a> J Neurosci. 31 (3), 1106-1113 (2011).</li><li>Takeuchi, A., Takeuchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+analysis+of+relationship+between+end-plate+potential+and+end-plate+current.">Further analysis of relationship between end-plate potential and end-plate current.</a> J Neurophysiol. 23, 397-402 (1960).</li><li>McLachlan, E. M., Martin, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non+linear+summation+of+end+plate+potentials+in+the+frog+and+mouse.">Non linear summation of end plate potentials in the frog and mouse.</a> The Journal of Physiology. 311 (1), 307-324 (1981).</li><li>Obis, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+protein+kinase+C+epsilon+isoform+modulates+acetylcholine+release+in+the+rat+neuromuscular+junction.">The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction.</a> Mol Brain. 8 (1), 80 (2015).</li><li>Silveira, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ryanodine+and+inositol+triphosphate+receptors+modulate+facilitation+and+tetanic+depression+at+the+frog+neuromuscular+junction.">Ryanodine and inositol triphosphate receptors modulate facilitation and tetanic depression at the frog neuromuscular junction.</a> Muscle Nerve. 52 (4), 623-630 (2015).</li><li>Wood, S. J., Slater, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+factor+at+the+neuromuscular+junction.">Safety factor at the neuromuscular junction.</a> Prog Neurobiol. 64 (4), 393-429 (2001).</li><li>Miranda, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+Cl-+channel+defects+reveal+disrupted+skeletal+muscle+maturation+in+R6/2+Huntington's+mice.">Progressive Cl- channel defects reveal disrupted skeletal muscle maturation in R6/2 Huntington's mice.</a> J Gen Physiol. 149 (1), 55-74 (2017).</li><li>Waters, C. W., Varuzhanyan, G., Talmadge, R. J., Voss, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington+disease+skeletal+muscle+is+hyperexcitable+owing+to+chloride+and+potassium+channel+dysfunction.">Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (22), 9160-9165 (2013).</li><li>Khedraki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depressed+Synaptic+Transmission+and+Reduced+Vesicle+Release+Sites+in+Huntington's+Disease+Neuromuscular+Junctions.">Depressed Synaptic Transmission and Reduced Vesicle Release Sites in Huntington's Disease Neuromuscular Junctions.</a> Journal of Neuroscience. 37 (34), 8077-8091 (2017).</li><li>Greene, E. 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 anatomy of the rat. , (1955).</li><li>Magrassi, L., Purves, D., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+that+stain+living+nerve+terminals.">Fluorescent probes that stain living nerve terminals.</a> J Neurosci. 7 (4), 1207-1214 (1987).</li><li>Jack, J. J. B., Noble, D., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electric current flow in excitable cells. , (1983).</li><li>Voss, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+inhibits+chloride+channels+in+mature+mammalian+skeletal+muscle+by+activating+P2Y(1)+receptors.">Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y(1) receptors.</a> Journal of Physiology-London. 587 (23), 5739-5752 (2009).</li><li>Albuquerque, E. X., McIsaac, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+slow+mammalian+muscles+after+denervation.">Fast and slow mammalian muscles after denervation.</a> Experimental Neurology. 26 (1), 183-202 (1970).</li><li>Santafe, M. M., Urbano, F. J., Lanuza, M. A., Uchitel, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+types+of+calcium+channels+mediate+transmitter+release+during+functional+recovery+of+botulinum+toxin+type+A-poisoned+mouse+motor+nerve+terminals.">Multiple types of calcium channels mediate transmitter release during functional recovery of botulinum toxin type A-poisoned mouse motor nerve terminals.</a> Neuroscience. 95 (1), 227-234 (2000).</li><li>Gaffield, M. A., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+mobility+in+mouse+motor+nerve+terminals+with+and+without+synapsin.">Synaptic vesicle mobility in mouse motor nerve terminals with and without synapsin.</a> J Neurosci. 27 (50), 13691-13700 (2007).</li><li>Zhang, Z. S., Nguyen, K. T., Barrett, E. F., David, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+ATPase+Inserted+into+the+Plasma+Membrane+of+Motor+Terminals+by+Exocytosis+Alkalinizes+Cytosolic+pH+and+Facilitates+Endocytosis.">Vesicular ATPase Inserted into the Plasma Membrane of Motor Terminals by Exocytosis Alkalinizes Cytosolic pH and Facilitates Endocytosis.</a> Neuron. 68 (6), 1097-1108 (2010).</li></ol>

著者が明らかに何もありません。

準備および神経筋伝達電流または電圧クランプ条件下での測定のためマウス ラル筋肉の使用は、ここで説明。ラルを解剖するために考慮すべきいくつかの重要なポイントがあります。電極と電極串刺しの刑で筋肉エイズから過剰な結合組織のクリーニングは結合組織串刺しの刑にそれらを配置する場合ことができます暗礁。しかし、離れて撮影することができます結合組織を削除簡単に筋肉...

シナプス機能を検査するためのエクセレント モデルの動的神経や骨格筋のシステム関係に洞察力を提供するいますと、神経筋接合部のシナプス伝達を勉強します。オーリスの挙筋(ラル) は薄い筋肉、神経筋接合簡単に可視化することを可能にします。以前のレポートは、シナプスの薬物や毒素を調べるとラル1,2の骨格筋線維型の特性を特徴とした、ラルを使用しての利便性を説明しています。数多くの研究は、神経筋生理学3,4,5,6,7、8を調べる、ラルを使用しています。電気生理学、簡単にラルの神経筋接合部を観察する能力を運動終板に電極を正確に配置可能し、シナプス伝達を記録容量クランプの問題が大幅に減少します。電流クランプ記録膜時定数 (τm) 入力抵抗 (Rで) などの筋膜特性が容易に得られます。さらに、これらのプロパティは、同じの筋線維が筋膜特性にシナプス機能の直接比較を可能にする、神経筋伝達を記録するために使用から測定できます。これらのデータ分析は、多くの神経筋疾患と変更されたアクティビティの状態の物理的なメカニズムに重要な洞察を提供できます。
ここで説明する手法の重要な側面は、クランプ電圧の非線形電流クランプで検出された対象ではない、筋膜特性の独立してシナプスの録音用です。神経筋伝達を調べる現在のクランプではなく電圧クランプを使用する利点は、1950 年代の9での努力を開拓によって設立されました。電流クランプの下で 10-15 mV の振幅を超えるエップスは mEPP 振幅9の線形製品ではありません。たとえば、平均 mEPP は 1 mV、5 の EPP mV 5 mEPPs (5 の QC); の製品といえます一方、40 の EPP mV 以上 40 mEPPs の製品になります。この非直線性大きいエップスでは、膜電位と平衡電位のアセチルコリン受容体の違いは、EPP の原動力 (~-10 mV)、大きいエップスの間に大幅に減少します。電圧クランプ実験中に筋膜電位が変化しないために、この問題は電圧クランプ実験で避けます。欠点は、電圧クランプ実験電流クランプ記録よりも完了までに技術的により困難なことです。これを念頭において、マクラクランとマーティンは、エップス10電流クランプ記録の非線形性の簡単な数学的な補正を開発しました。修正は11,12,13をうまくが、重要なは、筋膜特性が乱れていないと仮定します。
筋膜特性、特に条件または筋肉を混乱させる病気の状態を勉強して場合を考慮する重要です。たとえば、ハンチントン病の R6/2 トランスジェニック モデルからの骨格筋は、安静時塩化カリウム電流14,15で進歩的な減少による脱です。結果として、mEPPs、エップスは R6/2 骨格筋で増幅されます。確かに、付加的な要因は、mEPPs とエップスを変更できます。変更は SNARE タンパク質8に関連しているように見えたエップス、ハンチントン病マウス (R6/1) の別のモデルで動作します。変更された神経筋伝達を引き起こすメカニズムを評価するためには、電圧クランプを使用して変更された筋膜特性の影響を除去するために有益となります。最近の研究では、ここで説明する手法を使用して両方の電流と電圧クランプ条件下で R6/2 神経筋伝達を調べた。モーターの常数全体だった電圧クランプと少ない 1% 誤差より終板16の長さの定数内で 2 つの電極を配置することによってそれはその電圧クランプと R6/2 筋の神経筋伝達の対照的な測定値が得られた電流クランプ レコードを修正.これは筋膜プロパティが変更された場合、非線形性のエップスを修正することができないことを強調表示し、筋膜特性の独立した電圧クランプ記録を得ることの利点を示しています。ここに提示されたプロトコルは、条件またはシナプスとシナプス後膜の特性に影響を与える病気の状態を調べることに最適です。

<table border="0" cellpadding="0" cellspacing="0" width="806">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus Compound Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">BX51WI</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x Objective</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">UMPLFLN10XW</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40x Objective</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">LUMPLFLN40XW</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:216px;">Borosilicate Glass</td>
			<td style="width:183px;">Sutter Instruments</td>
			<td style="width:119px;">BF150-86-7.5</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">CCD Camera</td>
			<td style="width:183px;">Santa Barbara Instruments Group</td>
			<td style="width:119px;">ST-7XMEI</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:216px;">Axoclamp 900A Amplifier</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:119px;">2500&#8208;0179&#160;&#160;</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mater-9 Pulse Generator</td>
			<td style="width:183px;">AMPI</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iso-flex Stimulus Isolator</td>
			<td style="width:183px;">AMPI</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pCLAMP 10 Data Acquisition and Analysis Software</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:119px;">1-2500-0180</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concentric Bipolar Electrode</td>
			<td style="width:183px;">FHC</td>
			<td style="width:119px;">CBDSH75</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ball-joint Manipulator</td>
			<td style="width:183px;">Narishige&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Non-metalic Syringes 34 Gauge</td>
			<td style="width:183px;">World Precision Instruments</td>
			<td style="width:119px;">MF34G-5</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nikon Stereomicroscope</td>
			<td style="width:183px;">Nikon</td>
			<td style="width:119px;">SMZ800N</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">No. 5 Forceps</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:216px;">Spring Scissors</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:119px;">15006-09</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">No. 2 Forceps</td>
			<td style="width:183px;">Roboz</td>
			<td style="width:119px;">RS-5Q41</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microdissecting Scissors</td>
			<td style="width:183px;">Roboz</td>
			<td style="width:119px;">RS-5912SC</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:183px;">Dow Corning</td>
			<td align="right" style="width:119px;">2404019862</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hair Removal Cream</td>
			<td style="width:183px;">Nair</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">Grass SD9 Stimulator</td>
			<td style="width:183px;">Grass Medical</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Model P-1000 Micropipette Puller</td>
			<td style="width:183px;">Sutter Instruments</td>
			<td style="width:119px;">P-1000</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">Axon Digidata 1550 Low-noise Data Acuisition System</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Low Pass Bessell Filter</td>
			<td style="width:183px;">Warner Instrument Corp.</td>
			<td style="width:119px;">LPF-8</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Left-handed Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641/45DL</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Right-handed Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641/45DR</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Single Motion Controler</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MC100e</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;width:216px;">Crossed Roller Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641R</td>
			<td style="width:288px;">This was added to the Z-axis of the Left and Right-handed micromanipulators to allow the z axis to be motorized. This custom set-up is cheaper and less bulky than buying a 4-axis motorized micromanipulator. It also allows us to control both micromanipulators with one controller</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:517px;">All chemicals were orded from Fisher except,</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BTS</td>
			<td style="width:183px;">Toronto Research Chemicals</td>
			<td style="width:119px;">B315190</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CTX</td>
			<td style="width:183px;">Alomone Labs</td>
			<td style="width:119px;">C-270</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-Di-2-Asp</td>
			<td style="width:183px;">Molecular Probes</td>
			<td style="width:119px;"></td>
			<td style="width:288px;">Molecular probes is no longer a company. Now ordered through Fisher</td>
		</tr>
	</tbody>
</table>

levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions

このプロトコルは、電流クランプとクランプ電圧条件下で神経筋接合部からレコードのシナプス伝達に手法を説明します。オーリスの挙筋(ラル) の前のヴィヴォ準備は、薄い筋肉の運動神経終板に電極を串刺し神経筋接合部の簡単な可視化を提供するために使用されます。このメソッドは、運動終板の膜特性と同様に自発ミニチュア終板電位と電流 (mEPPs と mEPCs)、神経誘発神経終板電位と電流 (エップスおよび Epc) の録音できます。このメソッドから得られた結果を含める素量 (QC)、小胞リリース サイト (n) の数、小胞の放出 (prel)、シナプス促通、うつ病と筋膜時定数 (τ の確率m) 入力抵抗とします。このテクニックの人間の病気のマウス ・ モデルへの適用は、病気の状態でキーの病態を強調表示し、新規治療戦略の特定できます。完全に電圧遮断単一シナプス、によってこのメソッドは、シナプス伝達の現在利用可能な最も詳細な分析の 1 つを提供します。

図 8は、12 週齢野生型 R6/2 マウスから電流パルス (図 8 a) と電流クランプの下の 1 つのラール繊維から電圧応答 (図 8 b) の例を示します。MEPPs の存在は、これらのレコードに運動終板から撮影されたことを示します。レコードは、通常の生理食塩液で得られました。これらの電流クランプのレコードは?...

Watch this Scientific Journal Video about Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions at JoVE.com

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

オーリスの挙筋哺乳類の神経筋伝達電圧クランプ条件下での試験のための準備

本稿で説明されたプロトコルは、神経筋接合部で自然を記録するマウス挙オーリス長(ラル) 筋とシナプス後電位の神経誘発 (電流クランプ) と電流 (電圧クランプ) を使用します。この手法の使用は、正常と病気の条件の下でのシナプス伝達のメカニズムに重要な洞察を提供できます。

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

levator-auris-longus-preparation-for-examination-mammalian

Research

JoVE Journal

Neuroscience

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

9.3K ビュー.  Wright State University. 本稿で説明されたプロトコルは、神経筋接合部で自然を記録するマウス挙オーリス長(ラル) 筋とシナプス後電位の神経誘発 (電流クランプ) と電流 (電圧クランプ) を使用します。この手法の使用は、正常と病気の条件の下でのシナプス伝達のメカニズムに重要な洞察を提供できます。

ビデオ: Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

皮下ムスカリン拮抗薬の管理およびマウスにおける挙筋オーリスの筋筋のトリプル免疫染色

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

神経科学

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

同時に電圧クランプとアンペロとドーパミン放出の単一細胞計測

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</sub亜ミトコンドリアの小胞膜のパッチクランプ記録を実行するための&gt; ATPアーゼ小胞の準備とテクニック

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

反応性アストロ サイトと増殖の in Vitro虚血のような条件下での監視

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

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.

神経筋接合部機能を測定

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

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.

全細胞電圧クランプ記録のための電気生理学的方法<em&gt;ショウジョウバエ</em&gt;感光体

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

平坦化された哺乳類の皮質皮質モジュールの可視化

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

膠様質ニューロンの細胞パッチク ランプ記録のため急性脊髄スライスの準備

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

神経筋解析のためのラット声帯の調製

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

生理学的条件下でのα-シヌクレイン構造ダイナミクスの研究のためのミリ秒水素/重水素交換質量分析

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...