この作品は、NIHのグラントEY17606（APS）によってサポートされていました。

1。準備電極<ol><li> 1M NaClに両方それらを浸漬し、それらの間に15Vの駆動〜20秒に1 Hzの正弦波を用いて、記録と参照電極を準備する</li><li>マイクロピペットプラー（サターインスツルメンツP - 97）を使用して、記録電極を行います。パッチピペットは、1.2mmの外径と0.69ミリメートル（サッタインスツルメンツ、カタログ番号B120 - 69 - 10）の内径とホウケイ酸ガラスを使用してください。電極の先端を通して直列抵抗を測定する。 MΩ〜12は、細胞体のためMΩ〜5〜直径の使用中の5μmの細胞体のための〜直径の使用中の20μm以下、ピペットの先端径を選択する標的細胞の大きさに基づいて。 </li><li>地上/参照電極を準備する。 1M NaCl中で、注射器を使用すると、（カタログ番号A7002、シグマ）3.5％寒天を含む1 mMのNaClを含む小さなセクションのポリエチレンチューブ（Intramedic、PE205）を記入してください。 Ag / AgCl参照電極上にこの寒天橋を置きます。 </li></ol> 2。準備ソリューション<ol><li>外部浴溶液、エイムズ&quot;メディアを含む重炭酸塩（1 L）：エイムズの1本&quot;培地（Sigma、カタログ番号A1420）、細菌を防ぐため、 炭酸水素ナトリウム1.9gの、ペニシリン-ストレプトマイシン（カタログ番号P0781、シグマ）の7 mLの成長。 〜280 mOsmに浸透圧を調整します。 </li><li>ソリューションをスライス、エイムズ&quot;pH緩衝HEPES（1 L）を含有する培地：エイムズの1本&quot;中、NaClの0.887グラム、HEPESの2.38グラム、ペニシリン - ストレプトマイシンの7 mLの。 1M NaOHおよび/または1M HClを用いて〜7.4にpHを調整する。 〜280 mOsmに浸透圧を調整します。 </li><li>カリウム-アスパラギン酸（K - ASP）ピペット内部の溶液（mMで）：125 K -アスパラギン酸、10のKCl、10 HEPES、5 NMG - HEDTA、0.5のCaCl 2、1 ATPとMg、0.2 GTPとMg、pHをするために調整されるNMG - OH、及び浸透圧を持つ〜7.3〜280 mOsmに調整されます。 </li><li>のdH 2 Oを150mlに溶けて、網膜を含む低ゲル化温度アガロース（5重量％）、寒天7.5gの（カタログ番号A7002、シグマ）を安定させるための寒天バックストップ</li></ol> 3。実験の日<ol><li>雪解け〜K - Aspを内部溶液と冷凍庫からお好みの蛍光体の1.5mLの。経験的に決定されるべき適切な濃度に蛍光体を希釈する。 </li><li>遮光貯蔵容器として水浴（30-32 ° C）に所定の場所にNaHCO 3を （〜200mL）を含むエイムズ&quot;メディアを注ぎ、5％CO 2 / 95％O 2ガスとの平衡化</li><li> 、HEPESを含むエイムズ&quot;メディアの〜110 mLのボトルをいっぱいに氷の上に位置し、100％O 2で平衡。 </li><li>ファラデーケージの上に温水タンクにNaHCO 3を含む残りのエイムズ&quot;メディアを置き、そして5％CO 2 / 95％O 2ガスを用いて平衡化します。パラフィルムは、溶液の上部空間にガスをトラップするために使用されることがあります。 </li><li> HEPESと〜115℃でアガロースを融解することで熱溶液とエイムズ&quot;メディアを25mLに、（カタログ番号A0701、シグマ）0.75gを添加することにより、低ゲル化温度アガロース（3％）の準備。気泡のない透明な溶液、及び水浴（〜42 ° C）にストアするまでかき混ぜる。 </li><li> K - Aspを内部溶液を記録ピペットを記入し、チップ抵抗を確認してください。 </li></ol> 4。実験を始める<ol><li>マウスを安楽死させるとすぐに湾曲したハサミを使って目を削除します。赤外線ゴーグルは、視覚的な色素の漂白を防ぐために、すべての手順を使用する必要があります。 </li><li>鉗子で目を押さえながら転がり、そして薄い両面刃を使用してからそれらを防ぐために、フィルター紙の上に目を置く、角膜にスリットを作る。 </li><li>すぐに眼球が破裂されると、エイムズ&quot;メディアで満たされた皿に、彼らの目を移す。 </li><li>エントリポイントとして角膜にスリットを使用して、小さなはさみはレンズと硝子体を除去することができる後に、角膜の残りの部分を切り取るために使用されます。網膜は、アイカップにアタッチしたままになって、そして32で闇の中にアイカップを保管° Cを5％CO 2 / 95％O 2で平衡化したエイムズ&quot;メディアにいることに注意してください。 </li></ol> 5。スライス<ol><li>アイカップのいずれかを二等分すると網膜色素上皮から網膜を解離する。組織が平らにできるように、網膜の辺を削除します。 </li><li>溶かした寒天と35ミリメートルのペトリ皿を埋めるためにラテックス電球と小さなガラスパスツールピペットを使用して、その一貫性をテストするために寒天を介して全体に鉗子をドラッグします。 </li><li>あなたは、寒天が固化し始めることができる場合： <ol class="lalpha"><li>寒天の上に網膜を転送します。 </li><li>網膜の周りに余分な溶液を吸収するキムワイプのねじれた部分を使用してください。 </li><li>直接網膜上に場所寒天の2〜3滴を、迅速にその周りに壁を形成するために、網膜上のプラスチック製のシリンダーを配置。寒天内で網膜を中心にしながらプラスチックの円筒の側面の下の追加溶かした寒天を滴らせる。 </li><li>ペトリ皿tを転送する冷却するために氷水浴をO。 </li></ol></li><li> 〜45秒後、氷水浴から組織を除去し、プラスチック製のシリンダーの外ブロック寒天を押し出すプランジャーを使用して、網膜から最も遠い側から押す。 </li><li>網膜を含む寒天の小さなブロックを切り取って試料ディスク上に寒天のバックスに対して所定の位置にブロックを瞬間接着剤に片側かみそりの刃を使用してください。 </li><li>振動ミクロトームのトレイに試料ディスクを転送し、網膜を持つブロックが完全に水没することができるようにトレイに氷冷エイムズのHEPESを注ぐ。 </li><li> 〜200μmの厚さと網膜のスライスは、最大ブレード振動速度で切断し、前進翼の速度は、各スライスで網膜を切って4〜5秒かかるように設定することができます。 </li><li>一度にそれぞれの使用可能なスライスのいずれかを削除する11号手術用メスの刃を使用してください</li><li>網膜を含む適切なスライスが録音室に配置されますと、それらを横断するナイロン糸とスライスのアンカーでダウン重み付けされています。ナイロン糸は、録画の遮るもののない網膜を残して、網膜の周囲の寒天を押したままにします。 </li><li>正立顕微鏡のステージに録音室を転送する、カーテンを閉じて、赤外カメラでスライスを表示するには、赤外光源をオンにします。 </li></ol> 6。録画<ol><li>一度良い網膜スライスは、（そのまま光受容体および適切な切削角を持つ）℃、5％で平衡化したCO 2 35 37に加熱される加熱されたエイムズ&quot;メディアと5 mL / minのより高い率で網膜を灌流開始識別されます。 / 95％O 2。 </li><li>いくつかの表面の損傷は、スライスのプロセスに起因する明らかな可能性があります。細胞のこの通常薄い層が壊れた先端にピペットを用いて除去し、そして穏やかに死んだ細胞体を離れて吸うことができます。これは一般的に生きている細胞を下に明らかになります。その位置関心の地層中に配置されている健康な細胞体を識別する。 </li><li> KAsp内部溶液をマイクロピペットを記入し、ピペットの先端部を介して正（外側）の圧力を適用し、ダウンマイクロマニピュレーターを用いて、セルのレベルに録音室にピペットの先端を下げると。 </li><li>それは細胞膜をディンプルようにピペットの先端を移動し、高抵抗のシールを作るために穏やかな負の（内側）の圧力を適用する。全細胞モードは細胞に侵入する電極に負圧を適用することによって達成することができます。 </li><li> LEDを使用して網膜組織を刺激するし、光に応答を呼び起こすことができる。 </li><li>録音がピペット内部溶液中でフルオロフォアからの蛍光を想起させることによって作らされたセルを撮影。 </li></ol> 7。代表的な結果<img src="/files/ftp_upload/2107/2107fig1.jpg" alt="図1" /> 図1。ここでは、増加する強さの光の点滅に暗順応網膜ニューロンの光誘発電位が示されている。 <img src="/files/ftp_upload/2107/2107fig2.jpg" alt="図2" /> 図2。細胞は蛍光体、ルシファーイエローで満たされている網膜のスライスからの蛍光画像。誘発蛍光は、パッチのレコーディングが行われたから、細胞の形態を示しています。

<ol>
	<li>Weblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+along+and+between+rods+in+the+tiger+salamander+retina.">Transmission along and between rods in the tiger salamander retina.</a> J Physiol. 280, 449-470 (1978).</li><li>Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+connections+among+retina+neurons+in+living+slices.">Synaptic connections among retina neurons in living slices.</a> J Neurosci Methods. 20, 139-149 (1987).</li><li>Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+contrast+adaptation+in+salamander+bipolar+cell.">Temporal contrast adaptation in salamander bipolar cell.</a> J Neurosci. 21, 9445-9454 (2001).</li><li>Armstrong-Gold, C., &amp;amp, R. i. e. k. 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=Bandpass+filtering+at+the+rod+to+second-order+cell+synapse+in+salamander+(Ambystoma+tigrinum)+retina.">Bandpass filtering at the rod to second-order cell synapse in salamander (Ambystoma tigrinum) retina.</a> J Neurosci. 23, 3796-3806 (2003).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat Neurosci. 4, 877-886 (2001).</li><li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li></ol>

利害の衝突は宣言されません。

脊椎動物の網膜からのスライスの記録は、何十年も1,2のために作られており、網膜回路が入射光をエンコードする方法のより深い理解を提供することにかなり成功している。かみそりの刃に直接振動ミクロトームで切断するのではなくの利点は、網膜のスライスが表面に少ないダメージを被ることがあります。吸引ピペットで、このダメージとターゲットだけ細胞のタイプのほとん?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>B120-69-10</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene tubing</td>
<td>&nbsp;</td>
<td>Intramedic</td>
<td>PE205</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7002</td>
<td>&nbsp;</td>
<tr>
<td>Low gelling temp agarose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0701</td>
<td>&nbsp;</td>
<tr>
<td>Ames' Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1420</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P0781</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

patch clamp recordings from mouse retinal neurons in a dark-adapted slice preparation

私たちの視覚経験は、私たちの網膜光受容体の視物質が光エネルギーの光子を吸収し、細胞膜中の環状ヌクレオチド依存性チャネルの閉鎖につながる細胞内イベントのカスケードを開始するときに開始されます。膜電位で生じる変化は、桿体と錐体シナプス端末の両方からの神経伝達物質の放出量の削減に順番につながる。光受容体の膜電位の光誘発変化が網膜の下流の活動につながるかを測定するために、科学者は数十年の1,2のための網膜スライス標本からの電気生理学的記録を行っている。過去にこれらのスライスは、ろ紙に接続されて網膜組織にカミソリの刃を使って手動でカットされている。近年でスライスの別の方法は、網膜組織が低ゲル化温度の寒天に埋め込まれているそれによって開発され、クールな解決策にスライスミクロトーム3,4を振動させる。この準備は少なく、表面の損傷と非常に堅牢な光誘発反応と網膜のスライスを生成します。ここでは、この手順を視覚的に色素を漂白避けるために、赤外光の下で実行する方法を文書化します。

Watch this Scientific Journal Video about Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation at JoVE.com

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

ここでは、電気生理学的記録のためのマウスの網膜の暗順応のスライスを生成するための手順を説明します。

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

patch-clamp-recordings-from-mouse-retinal-neurons-dark-adapted-slice

Research

JoVE Journal

Neuroscience

14.3K ビュー.  University of Southern California. ここでは、電気生理学的記録のためのマウスの網膜の暗順応のスライスを生成するための手順を説明します。

ビデオ: Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

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

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

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

モーターニューロンとターゲットゼブラフィッシュの骨格筋からの対のパッチクランプ記録

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

神経科学

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

内耳有毛細胞におけるパッチクランプ記録は、単離されたゼブラフィッシュから

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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 ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

両生類網膜スライス標本における感光体と2次ニューロンからの同時ホールセル録音

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

の感覚ニューロンの分離<em&gt;アメフラシカリフォルニ</em&gt;グルタミン酸作動電流のパッチクランプ記録のために

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

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

胚ゼブラフィッシュマウスナー細胞からパッチクランプ記録

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

パッチクランプ技術を用いたダーク適応、マウス網膜スライス標本におけるニューロンにおける記録光誘発シナプス後応答

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

脳スライスにおける全細胞パッチクランプ記録

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

黒質ドーパミンニューロンの細胞体樹状突起のドメインからの亜細胞パッチクランプ記録

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...