我々は感謝マーダーラボのメンバーを認識する。私たちは、彼女の支援と激励のために、私達の顧問、博士イヴマーダーに感謝。この研究は、国立神経疾患研究所およびストローク助成NS - 17813とNS - 058110によってサポートされていました。

1。総解剖<ol><li>動物の鈍い感覚と運動システムへの30分間氷上でカニを置きます。氷と一緒にすべての側面にそれを囲む。カニを取り扱う際には手袋を着用してください。 </li><li>解剖のパンと、使用するツールをレイアウトすることで総解剖の準備。 Rongeurs、骨切りはさみ、小さなはさみ、歯鉗子、テーパーエッジとヘラ、ブラックSylgard -コーティングディッシュ、そして虫ピンが必要です。 </li><li>ピンチからそれを防止し、解剖のパンに配置すると後肢による氷のバケツからカニを削除します。 </li><li>それぞれが内側にねじれて最初の爪を外します。その後、同じねじれ運動を使用して、各脚を取り外します。 </li><li> rongeursを使用して、口を覆う上顎を削除します。また、ソフトmaxilluleを削除します。 </li><li>再びrongeursを使用して、甲の外側後端から開始し、それの端に正しい握り方をする。 rongeursで取り締まると甲羅のその部分を折る。ヘラは、開口部を入力できるように十分な甲羅のを削除してください。反対側の目とリピートして甲羅の端まで削除を続けます。 </li><li>ヘラのテーパーのサイドで、静かに背甲の屋根から柔らかい内側の組織を削る。 STNSの損傷を防ぐために、甲羅に接触してへらをしてください。組織から分離されている甲の部分を脱却するためrongeursを使用してください。また、甲羅に組織を接続する任意の内転筋を分​​離するために確実に腹甲羅から軟組織の一部を分離。 </li><li>甲羅から突き出ている二つの小さな耳小骨があると、心臓と同じくらい遠く後部背甲を離れて壊す。可能な限り前方にカット。背甲の最も前方の部分を脱却しながら、指一本でセファロンを（&quot;顔&quot;）安定化させる。 </li><li>甲羅の腹側では、それらに接続された口上突起から下顎を取り除くために、外側にねじれ、そしてそれを引いて、下顎にrongeursを固定して、一度に顎を引き抜く。適切に行われれば筋肉を持つ二つの長い耳小骨は、抽出された下顎骨に接続する必要があります。 </li><li>ヘラを使って目の近くセファロンの側面から組織を離れて区切ります。層によって可能層として小骨の尾根に近いとして組織横断小さな解剖鋏、と。 </li><li>解剖のパンの壁にカニを置き、（爪がこのために使用される）場所に固定し、それを支える。接続された任意の組織を残さないことを確実に離れて口上突起から唇を（&quot;上唇&quot;）カット。骨切りはさみを使用すると、Cephalon社を削除するには、腹甲に2つの斜めカットを行います。 </li><li>歯鉗子で唇を取ると離れて腹側の小骨から唇を（&quot;下唇&quot;）カット。その後、静かに残りの腹甲から唇を持ち上げます。甲羅の内側を覆う組織から胃の腹側部分を分離するために小型のはさみを使用してください。胃がカニから抽出されるまで、幽門膨大部が表示されている場合、その両側に離れて組織から胃を切る。 </li><li>それでも手のひらに対して胃を休ませ、口の中に生理食塩水注ぎ、鉗子で唇が胃を開催。これは、胃を拡大し、それが簡単に開いてカットできるようになります。ダウン膨大部の間に小骨を介して幽門右の間に口の開口部からカット。次に2つの噴門枝を通して斜めに削減。胃の内側に3本の歯の先端を切り取ります。 </li><li>ダウン胃の内側にある大規模な、黒、Sylgardコーティングした皿に胃を置きます。昆虫ピンと準備のファイブコーナーを突き止めると（図1参照）全体の準備を浸漬し、冷生理食塩水で皿を埋める。 </li></ol> 2。細かい解剖<ol><li> 、マイクロダイセクションのツールは、微細な解剖のために必要とされる。特に、鉗子と春のはさみは、タングステン針とピンホルダーとしてだけでなく、必要とされる。明確な、Sylgardコーティングした、ペトリ皿およびいくつかの細かい線のピンも用意されている必要があります。 </li><li>最も低い倍率で始まる、口の開口部に使用される唇の近くにフラップを下に固定することによって開始します。準備をピンと張るようにし、必要な他の領域を固定するピンの残りの部分を並べ替えます。 </li><li>慎重に準備の下の部分をカバーする斑点皮下組織を持ち上げ、つの白いドットで皮下組織のパッチを残して全体にトリミング。 </li><li>準備の下部にある黄色の、かすかな、膜組織アップピール。 mvnsが弱く、この組織の下側に取り付けられている。それがプルアップされているように慎重に黄色の組織からmvnsを分離。黄色の組織をフォローアップし、脳と一緒にカット。 </li><li>脳から出る太いプロセスに従うことを拡張laterallY.これらはcommisural神経です。交連神経の端に到達すると、離れて脳の側から切断されているスポンジ状、オフホワイトの組織の塊を取り除く。 </li><li> イオンと息子はコグを満たす場合には、 イオンと息子の長さを明らかにするために筋肉や組織を切り取る。 </li><li>斑点のある皮下組織のパッチに戻り、それを遮断。これはSTGを取り巻く動脈に開口部を残してください。脇腹STGを、2つの筋肉を分離するために開口部を介してカット。これらの筋肉の端を持ち上げて、動脈からそれらを分離し、alnsと活動銀河核を露出させるそれらの下に右に切る。 STN上の嘘軟骨限り、最大隣接筋肉を区切ります。 </li><li> 、どこmvns、DGNにSTGをダウン周囲動脈組織を切り取る とDVNは満たしています。 lvnsにDVNをダウン動作とlvnsをカバーする白く、繊細な組織をカット。 </li><li>準備の下部近くにある小骨を介してPSNを見つけます。 PSNはLVNとdlvn接合を見つけるために使用することができます。 </li><li>次pynとPDNを見つけるためにdlvnをダウンに従ってください。 pynは多くの場合開口部に幽門膨大部を介してラップします。 PDNはdlvnのフォークオフと幽門膨大部と有酸素運動幽門弁の筋肉の間です。準備が明確なSylgard皿に転送されるとき、それらが容易に混同される可能性があるので、これらの神経の1つに接続いくつかの筋肉を残す。 </li><li>一度にすべての神経のが発見されている、STNSと組織と胃の不必要な神経の間の任意の残りの接続を切断する。慎重にミスの接続を切断する、離れて胃の残りの部分からSTNSを移動する。 </li><li>条件Sylgardは、もはやあまりにも粘着や乾いた感じなくなるまでSylgardの表面の上にこすることによって胃の組織の残りの質量を持つ明確なSylgard皿。 Sylgardは疎水性であり、Sylgardコーティングしたディッシュがこのように条件付けされていない場合STNSはそれに強く付着する。 </li><li>皿にいくつかの冷生理食塩水を追加。鉗子による交連神経の上端をつかんで、明確なSylgard皿にSTNSをもたらす。 </li><li> SylgardにSTNSを突き止める。交連神経から脳を切断し、ダウン第一コグの4つの両端を固定するminutenピンを使用してください。準備はIVNが離れてSylgardから上を指していることを確認することにより右側アップしていることを確認してください。 STGは、右サイドアップのときにDGNは STGの下にわずかに終了する必要があります。神経の残りのピンは細いワイヤーピンを下に終わります。 </li><li> STNSから残りの筋肉や組織を離れて清掃してください。 </li><li> Desheathピンホルダーとわずかに引っ掛け、罰金、タングステン針を使用してSTGを。離れて細胞体からSTGシースの隅に小さな穴を確認し、シースの層の間に取得するにはその穴を使用してください。慎重にSTG神経網と細胞体を公開する分離されたシースのフラップをカット。 </li><li>細胞体へのアクセシビリティを高めるために、細胞内記録用STGを安定させるためにシースのいずれかのフラップを突き止める。各神経が張りつめているとよく細胞外記録法（図3参照）のために間隔をあけていることを確認STNSの残りを再固定します。 </li></ol> 3。結果<ol><li>理想的には、神経のすべては、特に切り傷や損傷、から記録されるものの自由でなければなりません。神経のどれが絡まっていないかのように改謬されてください。 STGは、神経網の周りひげの形成に配置された細胞のすべてで無傷でなければなりません。無傷STNSは左右対称であり、 脚 、腕や頭などの前端としてmvnsとしてlvnsとホムンクルスのように見えます。 </li></ol>略語： <table border="1"><tbody><tr><td> STNS </td><td>口と胃の神経系</td></tr><tr><td> STG </td><td>口と胃の神経節</td></tr><tr><td> COG </td><td>交連神経節</td></tr><tr><td> OG </td><td>食道神経節</td></tr><tr><td> MVN </td><td>中央値心室神経</td></tr><tr><td>イオン</td><td>劣って食道神経</td></tr><tr><td>息子</td><td>優れた食道神経</td></tr><tr><td> AGN </td><td>前方胃神経</td></tr><tr><td> AlNの</td><td>前方外側神経</td></tr><tr><td> DGN </td><td>背側胃神経</td></tr><tr><td> DVN </td><td>背心室神経</td></tr><tr><td> LVN </td><td>外側心室神経</td></tr><tr><td> PSN </td><td>後部胃神経</td></tr><tr><td> pyn </td><td>幽門神経</td></tr><tr><td> PDN </td><td>幽門拡張器の神経</td></tr><tr><td> dlvn </td><td>外側心室神経の背枝</td></tr><tr><td> IVN </td><td>劣った心室神経</td></tr></tbody></table><img src="/files/ftp_upload/1207/figure_1_90ppi.jpg" alt=&quot;図1&quot;/&gt; 図1：解剖する前に胃のSTNSのおおよその配置を示す図。 <img src="/files/ftp_upload/1207/figure_2_90ppi.jpg" alt="図2" /> 図2：ライトグリーンの胃とSTNSのオーバーレイの下の部分に様々な筋肉の図。 STG内の各セルのタイプ、それらが支​​配することが知られている筋肉を使って表示されています。特定のセル（ナディムラボの礼儀）からの信号を運ぶ神経を解剖するために計画する際にこのマップが便利です。 <img src="/files/ftp_upload/old/1207_2009_1_12_5/figure_3.jpg" alt="図3" /> 図3：細胞内電極と細胞外の井戸（マーダーとブッチャー、2007年から）とSTNSの模式図。

<ol>
	<li>Harris-Warrick, R. M., Marder, E., Selverston, A. I., Moulins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dynamic Biological Networks: The stomatogastric nervous system. , (1992).</li><li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+circuit+dynamics+using+the+stomatogastric+nervous+system+of+lobsters+and+crabs.">Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs.</a> Annu. Rev. Physiol. 69, 291-316 (2007).</li><li>Maynard, D. M., Dando, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+stomatogastric+neuromuscular+system+in+Callinectes+sapidus,+Homarus+Americanus+and+Panulirus+argus+(Decapoda+crustacean).">The structure of the stomatogastric neuromuscular system in Callinectes sapidus, Homarus Americanus and Panulirus argus (Decapoda crustacean).</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 268 (892), 161-220 (1974).</li><li>Selverston, A. I., Russell, D. F., Miller, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stomatogastric+Nervous+System:+Structure+and+function+of+a+small+neural+network.">The Stomatogastric Nervous System: Structure and function of a small neural network.</a> Prog. Neurobiology. 7, 215-290 (1976).</li></ol>

STNS郭清は、電気生理学や免疫組織化学の実験を行う際に、または細胞培養用細胞を得るための第一歩です。に関係なく、意図した実験の、総解剖は変更されません。しかし、微細な解剖は異なる場合があります。先にあなたから録音する細胞とその神経の時間の計画を立てることが重要です。 STGのほとんどのニューロンは、胃内の少なくとも1つの筋肉を支配するプロセスを持っている（図...

<!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>Black Sylgard 170</td>
<td>Other</td>
<td>Dow Corning</td>
<td>170</td>
<td>Gross Dissection</td>
</tr>
<tr>
<td>Fine Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14090-09</td>
<td>&nbsp;</td>
<tr>
<td>Insect Pins size 6</td>
<td>Surgery</td>
<td>FST</td>
<td>26000-65</td>
<td>&nbsp;</td>
<tr>
<td>Mayo Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14110-17</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Spatula</td>
<td>Surgery</td>
<td>Ward Sci.</td>
<td>15 V 4313</td>
<td>&nbsp;</td>
<tr>
<td>Rongeurs</td>
<td>Surgery</td>
<td>FST</td>
<td>16000-14</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Forceps</td>
<td>Surgery</td>
<td>FST</td>
<td>11021-12</td>
<td>&nbsp;</td>
<tr>
<td>Fine Tungsten Wire (0.001 in. diameter)</td>
<td>Surgery</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>Fine Dissection</td>
</tr>
<tr>
<td>Forceps #5</td>
<td>Surgery</td>
<td>FST</td>
<td>91150-20</td>
<td>&nbsp;</td>
<tr>
<td>Minuten Pins</td>
<td>Surgery</td>
<td>FST</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Moria Spring Scissors (7mm blades)</td>
<td>Surgery</td>
<td>FST</td>
<td>15370-52</td>
<td>&nbsp;</td>
<tr>
<td>Petri Dish (100x15mm)</td>
<td>Other</td>
<td>Fischer	</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Pin Holder</td>
<td>Surgery</td>
<td>FST</td>
<td>26018-17</td>
<td>&nbsp;</td>
<tr>
<td>Super fine forceps #5SF</td>
<td>Surgery</td>
<td>FST</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 182</td>
<td>Other</td>
<td>Dow Corning</td>
<td>182</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten Needles</td>
<td>Surgery</td>
<td>FST</td>
<td>10130-05</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Wild</td>
<td>MC3</td>
<td>Misc.</td>
</tr>
<tr>
<td>Jonah Crab (C. borealis)</td>
<td>Animal</td>
<td>Commercial Lobster</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
						
		</div>

cancer borealis stomatogastric nervous system dissection

それはよく特徴付けされているセルの数が比較的少ない（約25℃のborealisの）が含まれているため、口と胃の神経節（STG）は、携帯電話とネットワークの相互作用を研究するための優れたモデルです。 STGのセルは、出力の広い範囲を示し、胃の運動行動する責任があります。胃は3つの内歯と食べ物、そしてそれが中腸に到達する前に食べ物をフィルタリングする幽門を分解する胃工場が含まれています。 STGは、胃の工場と中枢パターン発生器（CPGは）として知られている幽門を制御する2つのリズミカルな出力を生成します。 STG内の各セルは、これらのリズムのいずれかまたは両方に参加することができます。これらのCPGはは、神経調節の研究、恒常性、携帯電話とネットワークの可変性、ネットワークの開発、およびネットワークの復旧を可能にする。 グロスと細かい解剖、ヨナのカニ（ がんのborealis）から口胃神経系の解剖（STNSは）2つの部分に分けて行われます。総解剖では胃全体はカニから解剖される。細かい解剖中にSTNSは、解剖顕微鏡と顕微解剖ツール（図1参照）を使用して胃から抽出されます。 STNSはSTG、食道神経節（OG）、および交連神経節（COG）と同様に胃の筋肉を支配する神経が含まれています。ここで、我々は細胞外記録用に使用されるSTGの細胞は細胞内および末梢神経から記録される電気生理学的実験の準備のためSTNSの完全な解剖を実行する方法を示します。目的の神経を見つけるための適切な手法がsomaの複数形と神経網を明らかにするために神経節をdesheathingの私たちの手法としてだけでなく、示されています。

Watch this Scientific Journal Video about Cancer Borealis Stomatogastric Nervous System Dissection at JoVE.com

Cancer Borealis Stomatogastric Nervous System Dissection

ヨナのカニの口胃神経系（STNS）（<em&gt; C.ボレアリス</em&gt;）電気生理学、免疫組織化学、および細胞培養の研究にも使用できます。グロスと細かい解剖：STNSの抽出は、2つの部分で行われます。

癌ボレアリス口胃神経系の解剖

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

cancer-borealis-stomatogastric-nervous-system-dissection

Research

JoVE Journal

Biology

Cancer Borealis Stomatogastric Nervous System Dissection

12.5K ビュー.  Brandeis. ヨナのカニの口胃神経系（STNS）（ C.ボレアリス）電気生理学、免疫組織化学、および細胞培養の研究にも使用できます。グロスと細かい解剖：STNSの抽出は、2つの部分で行われます。

ビデオ: Cancer Borealis Stomatogastric Nervous System Dissection

Dissection of Drosophila Ovaries

ショウジョウバエの卵巣の解剖

生物学

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPCのマウス胚の解剖

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. 
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

EAEモデルラットの中枢神経系から単核細胞の単離

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

ショウジョウバエ幼虫NMJの解剖

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

の解剖サッカロマイセスセレビシエ ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

ロブスター属Americanus口胃神経系の解剖

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

アダルトゼブラフィッシュからの臓器の解剖

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

大腸癌への進行中に視野欠損欠損PMS2、ERCC1、Ku86、CcOI

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

の解剖と染色ショウジョウバエ幼虫卵巣

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

受容体-リガンド相互作用を研究するための BW レポーター システム

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...