我々は感謝してNIH（HJCへNS047073）からのサポートを認める。我々はまた、我々のアプリケーションのためにそれを使用することをお勧めクイック實、彼女の初期の実験のためにキャサリンケールに感謝しています。

1。電極の作製<ol><li>電極を作成するには、約2フィートの長さのエナメルコーティングさ0.001インチ径のステンレス鋼線の一部をカットするはさみを使用してください。 、ワイヤーの両端にパテの小さなボールを取り付けます半分に線を折る、とツイストペアを作成するために両端を回転させ、差動電極と呼ばれる。 </li><li>解明を防ぐためにワイヤーのテープは、両端。識別用の各差動電極（図1A）のためのテープの異なる色を使用して、4つの差動電極を作る。 </li><li>一緒に差動電極のねじれ2つ、1つの端にねじられていない2インチを残し、もう一方の端に1インチ。差動電極の残りのペア（図1B）を繰り返します。 </li><li>両端がねじられていないままであることを確認して、2つの差動電極の両方のセットをひねります。電極接合体をホールドアップと両端が自由なまま、4つの差動電極の接合部で家庭用シリコーン接着剤を適用する。ハングと接着剤は、少なくとも4時間（図1C）のために乾燥させます。 </li><li>枝を絶縁するために、木のような部分はフラット、可動、表面に電極アセンブリを定める。両端を扇形に広げるとテープで固定します。ちょうどその支店の前に電極アセンブリを昇格し、そのテープで固定するプラスチックの小片を使用してください。 </li><li>解剖顕微鏡で、シリコン接着剤でコートして枝をピンセットを使用してください。 1インチ長い末端は動物の内側になりますので、接着剤の非常に薄い上着が必要です。接着剤は、少なくとも3時間乾燥することができます。 </li><li> 2センチメートル1つの差動電極の1インチ長い方の端を短くしてください。各ワイヤ上で、シリコンとエナメルの1cmを削除します。録音用フックに1つの裸線をカール。フックから他の線を上に曲げると離れて、これは、アース線として機能します。残りの3つの電極のために繰り返します。 </li><li> 2インチの端にある4つの電極のいずれかを選択します。 2線のそれぞれに裸のステンレス鋼線の1cmを公開、シリコンをこすり、オフエナメルに鉗子を使用してください。 2本のワイヤの各々に半田ゴールドコネクタ。端を固定し、地上と録音端子との間の分離を維持するために、ラボのテープの一部を使用してください。残りの3つの電極のために繰り返します。 </li><li>動物の中で電極を固定するために、ケーブルにフックの端から約1.5インチのシリコン接着剤の1〜2センチメートルボールを取り付けます。少なくとも1時間（図1D）を乾かせてください。 <img src="/files/ftp_upload/1791/1791fig1.jpg" alt="図1" /> 図1電極接合体の作製。 （A）、その両端で折り返しとツイストペア線で構成される単一の差動出力電極は、テープで固定。 （B）2つの差動電極が両端にねじられていないスペースを残して、一緒にツイスト。異なる色のテープまたは別のマーキングは、両端を識別するために使用されるべきである。 （C）すべての4つの差動電極がテープの異なる色でマークされたそれぞれの側の個別電極、の別の支店を持つ、単一のケーブルを形成する単一電極アセンブリに結合。電極アセンブリの（D）最終的なフォーム。左側には、各差動電極の録音とアース線のためのカールのフックがあります。右側には、金色のコネクタピンは、アンプに接続するための配線に半田付けされています。シリコン接着剤のアンカーボールは、アセンブリの左側に向かって配置されています。 </li></ol> 2。移植のための動物の準備<ol><li> 350〜450グラム重量は約健康な動物を選択してください。海藻の小さな断片に反応して、動物は5〜3の間の秒の間隔で刺されを生成する必要があります。電極の注入との完全な手術は約1時間かかります。 </li><li>動物を麻酔するために、30％w / vの等張MgCl 2で、それを注入する。血リンパとMgCl 2の損失を防ぐために、注射部位は、ニードルを取り外す前に、瞬間接着剤で被覆することができる。 <img src="/files/ftp_upload/1791/1791fig2.jpg" alt="図2" /> 動物は、電極の注入、および切開の場所（バーで示される）のために固定されている方法を示す図2。模式図。 </li><li>ダウンその足で解剖トレイに麻酔動物をレイアウト。動物の頭部を上直面して、動物が離れて傾いて、その側に部分的であることを、左のように鰓の領域をシフト。前触手（図2）を突き止める。 </li><li>解剖顕微鏡のステージ上にトレイを置きます。離れて切開から、頭を上げ、尾の血リンパの収集に約20度の角度でトレイを支える。 </li><li>解剖はさみを使用して、目玉模様の右手に少し切開を起動して1〜1.5センチメートル前方に延長する。切開開放（;切開を示す眼点の近くにノートバー図2）を保持するトレイの端に接続されているリトラクターを使用してください。 </li><li>頬質量を公開するには、下に透明な膜を持ち上げるために鉗子を使用して皮膚とメイン切開に一致する開口部をカットするはさみを使用してください。リトラクターへの膜の端を挟む。 </li></ol> 3。電極注入<ol><li>マイクロマニピュレータの電極アセンブリを配置し、動物の切開の上に移動します。 </li><li> radular神経は、アクセスするための最も困難で、最初のアプローチされる必要があります。 radular神経にアクセスするための2つのオプションがあります。 </li><li>好適である最初のオプションは、、BN1とBN2の間に頬神経節の下を見ることです。 radular神経の枝が表示されている場合は、先の細いピンセットを使ってブランチ（ラベル1神経節の下に図3、RNのループ）を持ち上げるために鉤状に曲げられる。 </li><li> radular神経の枝は筋肉上記にアクセスできない場合、二つ目のオプションは頬神経節（2と表示された筋肉の下に図3、RN）の基部にある結合組織の隣のI2の小さな切開を介してアクセスすることです。 I2の切開は、I2筋肉の二つの層を通過する必要があります。ストレートピンセットによる筋肉の切開の縁の上に持って、RNのブランチを引き出すためにフック鉗子を使用してください。 </li><li>有鉤ピンセットで神経を保持しながら、神経に電極のフックを操作する鉗子の異なるセットを使用してください。 </li><li>神経を離れて頬質量から、他の神経や組織の明確なリフトアップされるようにフックを高めるためにマイクロマニピュレーターを使用。神経の伸び過ぎてはいけません。 </li><li>注射器で余分な水分をオフまたは吸引ワイプ金を使って電極を乾かします。水の表面張力は、それ自体で崩壊する神経、または神経の下に形成する水のシールを引き起こす可能性があります。接着剤が適用される前に、どちらの場合でも、領域が完全に乾燥する必要があります。 </li><li>フックのベースは、神経の底部に接触している瞬間接着剤の小さい軽打を適用します。瞬間接着剤を設定するには、 アメフラシ生理食塩水または体液のドロップを適用します。接着剤が湿っているときに何もワイヤーと神経との間の接着剤は電気的接続をブロックするような取得のように移動するべきではありません。 </li><li>もう一度フックを乾かします。アイスパックをクイック實の接着剤を混ぜる。 </li><li>完全にフックの露出配線をカバーするクイック- Silの接着剤の少量を適用するためにピンの先端、ではなく、アース線を使用してください。 </li><li>接着剤は、それがフックのセクションから離れてくるとすれば接着剤を再調整する、約5分間に設定することができます。時（図4の最終構成を示しています）ピンに触れ、それはもはやスティック糊が設定されています。 </li><li> radular神経電極がセットされると、I2筋肉にアクセスすることができます。 BN1とBN2の間の領域では、フックにフック鉗子を使用し、軽く上向きにバンドを引っ張って、筋肉の残りの部分からI2筋肉の2バンドを分離する。同じ手順を使用して、電極を取り付けます。 </li><li>頬神経2と3は簡単にアクセスされ、電極は同じ手順で接続されています。 </li><li>一度、すべての電極が接続され、アップシリコンアンカーへの動物に電極アセンブリをスライドさせます。 </li><li>切開は、動物の内部にアンカーを保ち、突起電極のケーブルの周りに閉じて縫合。良好なシール性を確保するためにステッチを瞬間接着剤。 </li><li>発泡スチロールの小さなブロックに電極のもう一方の端にある金色のコネクタの先端を挿入します。一晩回復のための曝気分離容器のメインタンクに動物を返します。 </li></ol><img src="/files/ftp_upload/1791/1791fig3.jpg" alt="図3" /> 図3。頬質量として知られているアメフラシ供給装置の図。頬神経（BN1、BN2、BN3）、終了する頬神経節の下部には、その後、筋肉の下に急落することradular神経（RN）、および電極が接続されているI2筋肉上の場所が示されている。 <img src="/files/ftp_upload/1791/1791fig4.jpg" alt="図4" /> 図4。神経に接続されている（または筋肉）、所定の位置に接着された単一の電極の模式図。カールフックのデ - 絶縁領域は、瞬間接着剤で固定アンカー神経（または筋肉）、と直接接触している、と電気的にクイック- SILを用いて液体培地から絶縁することに注意してください。接地電極のdeinsulated先端が流体媒体に露出していることにも注意してください。 4。録画<ol><li>少なくとも1.5時間の録音機器に接続しながら通気コンテナ内の動物を順応。動物はすべての電極が機能していることと、ノイズレベルが低いことを確認するために慣れている間に信号を監視します。 </li><li>神経と筋肉の電極からの信号を記録するためにコンピュータを使用しながら摂食行動を記録するビデオカメラを使用してください。それは同時にAxoscopeのコンピュータ上に追加のチャネルとして記録している間、カメラビューのデジタルカウンタを制御する矩形波信号を使用してビデオと電極の録音を同期させます。 </li><li>摂食行動は、デ手法によって誘発されるモートンとチール1993年にスクライビング。 </li></ol>無脊椎動物では制度的動物の使用およびケア委員会による正式な承認を必要としないが、我々は、 アメフラシのすべての治療法が害を最小限にし、動物に苦しみ、そして動物は完全に麻酔のときにすべての手術手技が行われることを確保している。 代表的な結果<img src="/files/ftp_upload/1791/1791fig5.jpg" alt="図5" /> 嚥下応答中の筋肉（伸筋I2）から図5（）同時4チャンネルの録音、三頬神経（radular神経、RN、頬神経2、BN2、と頬神経3、BN3）から。 （B）は、ツバメの動きは、容易に可視化できるように白いバンドで0.5 cmの間隔でマークされているプレカット海藻のストリップを（幅0.5センチ）、など、自由に振る舞う動物の同時ビデオ録画から静止フレーム動物飼料。

<ol>
	<li>Lu, H., Chestek, C. A., Shaw, K. M., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extracellular+stimulation+of+individual+neurons+in+ganglia.">Selective extracellular stimulation of individual neurons in ganglia.</a> J. Neural Eng. 5, 287-309 (2008).</li><li>Morton, D. W., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+buccal+nerve+activity+that+distinguishes+ingestion+from+rejection+can+be+used+to+predict+behavioral+transitions+in+Aplysia.">In vivo buccal nerve activity that distinguishes ingestion from rejection can be used to predict behavioral transitions in Aplysia.</a> J Comp. Physiol. A. 172, 17-32 (1993).</li><li>Warman, E. N., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+chronic+single+extracellular+recording+in+freely+behaving+animals+using+pipette+electrodes.">A new technique for chronic single extracellular recording in freely behaving animals using pipette electrodes.</a> J. Neurosci. Methods. 57, 161-169 (1995).</li><li>Ye, H., Morton, D. W., Chiel, H. J., J, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nbsp;Neuromechanics+of+multifunctionality+during+rejection+in+Aplysia+californica.">nbsp;Neuromechanics of multifunctionality during rejection in Aplysia californica.</a> J. Neurosci. 26, 10743-10755 (2006).</li></ol>

これらの録音を成功に導いたしたキー技術革新は、一本のケーブルに電極を組み合わせています。二つの電極の記録は前に使用されますが、4つの個々の電極に拡大すると、多くの場合、通常の餌の動きを作ることから動物を防ぐ外科的癒着の形成をもたらしたされています。つのケーブルに電極を組み合わせることで順番に免疫応答を減少させる動物、内部電極の大部分を削減し、動物はよ...

<!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>Enamel coated stainless steel wire</td>
<td>&nbsp;</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>0.001D, coating h</td>
</tr>
<tr>
<td>Household Silicone II Glue</td>
<td>&nbsp;</td>
<td>GE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kwik-Sil Adhesive</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Duro Qwik-Gel superglue</td>
<td>&nbsp;</td>
<td>Henkel corp.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A-M Systems model 1700 amplifier</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>&nbsp;</td>
<td>Filter settings:100-1000Hz nerves,10-1000Hz I2 muscle</td>
</tr>
<tr>
<td>Axoscope 10.1</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gold Connector Pins</td>
<td>&nbsp;</td>
<td>Bulgin</td>
<td>SA3148/1</td>
<td>&nbsp;</td>
<tr>
<td>Gold Connector Sockets</td>
<td>&nbsp;</td>
<td>Bulgin</td>
<td>SA3149/1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrode fabrication and implantation in aplysia californica for multi-channel neural and muscular recordings in intact, freely behaving animals

の主要な神経や筋肉から録音する<em&gt;アメフラシ</em&gt;摂食行動の間に私たちは無傷の動物での神経制御のパターンを研究することができます。同時に複数の神経と筋肉からの記録は私達の神経活動のタイミングに関する正確な情報を提供します。以前の記録方法は2つの電極のために働いているが、追加の神経や筋肉の研究は、複数の動物の録音を組み合わせて平均化が必要、それが困難なタイミングと位相の細部を決定するために作られた、のため変動の応答への応答から、とから動物への動物。 4つの個別電極を注入すると、通常の送り運動を行うことから動物を防ぐ癒着の形成のために非常に低い成功率を持っています。我々は、通常の餌の動きを可能にする動物の内部電極の大部分を減少させる電極の製造の新しい方法を開発した。それは可能な限り週限り長期間記録すること、長持ちする電極の信頼性の高い絶縁で、電極の結果を添付する接着剤の組み合わせを使用する。我々が記述する製造技術は、いくつかの追加の電極を組み込むように拡張することができる、と動物を脊椎動物に適用できるだろう。

Watch this Scientific Journal Video about Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals at JoVE.com

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

テクニックは、4を注入するために記述されています<em&gt; in vivoで</em&gt;電極の摂食行動の神経筋制御を監視する<em&gt;アメフラシカリフォルニ</em&gt;。

の電極の作製と注入アメフラシカリフォルニ無傷、自由に振る舞う動物における多チャンネル神経と筋肉の録音のための

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

electrode-fabrication-implantation-aplysia-californica-for-multi

Research

JoVE Journal

Neuroscience

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

13.0K ビュー.  Case Western Reserve University. テクニックは、4を注入するために記述されています in vivoで電極の摂食行動の神経筋制御を監視するアメフラシカリフォルニ。

ビデオ: Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

ポリイミドベースのマイクロ電極を用いて自由に動くマウスの高密度脳波録音

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

神経科学

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

小動物の慢性神経録音用超軽量電動マイクロドライブの設計およびアセンブリ

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

アン<em&gt; in vitroで</em生理的動きと送りモータプログラムを引き出すと録音用&gt;準備<em&gt;アメフラシカリフォルニ</em

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

体外における筋モータープールのために運動ニューロンの識別<em&gt;アメフラシカリフォルニ</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

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 ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

動物を振る舞う、覚醒中のシングルユニット記録のためにマイクロワイヤの組織化された配列を移植するための手順

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

マウスの電気生理学的記録のための設計と超軽量重量の作製、調整可能なマルチ電極プローブ

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

誘発電位録画ドライの非侵襲的なマルチ チャネルを使用してマウスの頭皮脳波センサー

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

皮質局所電界電位、心電図、筋電図、呼吸リズム自由行動ラットからの同時録音

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

自由に行動するマウスで長期的な感覚の衝突

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...