I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.

1原油Dauerフェロモン生産<ol><li> OP50 E.を育てる200rpmでLBブロス100mLに振とうしながら37℃でシングルコロニーO / Nから大腸菌 。注：このO / N培養物を、事前に作製し、4℃で保存することができる。 </li><li> N2 C.を育てる虫について15から20 OP50 E.を50μlを接種したNGM寒天（表1のレシピを参照してください）と6センチメートルペトリ皿大腸菌は菌までほとんど19を空乏化される。 </li><li> 1リットルのエルレンマイヤー中のS·メディアの5倍250ミリリットルを（表1のレシピを参照してください）makeは19フラスコ。注記：この溶液は、事前に行うことができる。 </li><li> OP50 Eの文化を育てるE.におけるOP50 1mlで接種することによりLBブロスOの4のL / Nが大腸菌大腸菌および16時間37℃で振とう。 </li><li> 4-（ステップ1.3で調製したSメディアとの4 1Lの三角フラスコのそれぞれに4-5プレートから1 S基底溶液（表1のレシピを参照）、転送線虫でプレート（ステップ1.2）から線虫を洗う5プレート各フラスコのための）19。 </li><li> 10分間650×gでステップ1.4からOP50を遠心し、上清を除去します。 S基底溶液10mlに各細菌ペレットを再懸濁。線虫の各フラスコに再懸濁した細菌の同量を転送します。 </li><li>解決策は、食品の枯渇を示す、クリアし始めるまで5日間 - （ - 25°Cの20）および100 - 4 150 rpmで室温でオービタルシェーカーフラスコを置きます。 </li><li>食料が枯渇したら、OP50 Eの別のO / N培養を育てるLBブロスの4倍1L中大腸菌 。 10分間650×gで、細菌を遠心分離し、線虫とSメディアフラスコのそれぞれに1フラスコからのペレットを追加します。食べ物が再びなくなるまでさらに3〜4日間、ステップ1.7のように、オービタルシェーカーにSメディアフラスコを返します。 </li><li>各フラスコからの溶液〜1ミリリットルのアリコートを取り、サブサンプリング、各1ミリリットルアリコートから1μLをし、線虫の数をカウントすることによって、線虫の濃度を推定。注：75,000線虫/ mlの - 線虫の人口50,000にする必要があります。この濃度での線虫の大部分はdauersある。 </li><li>いずれの汚染されたフラスコを捨てる。注：汚染が頻繁にステップ1.9の間に見られる真菌の菌糸として現れる。 </li><li> 4℃で10分間、4,000×gで遠心分離した線虫。線虫のペレットを捨て、上清を保持している。別の方法として、低効力フェロモンを作るためにこれらの動物を使用するための説明を参照してください。 </li><li> ＃1ワットマン濾紙で上澄みをろ過する。 NOTE：真空フラスコは、このプロセスを促進するであろう。 </li><li>さらに0.45μmの無菌真空濾過ユニットを通して上清をフィルタリングする。注：ろ液を4℃でO / N保たまたは1.14に進むことができます。 </li><li>攪拌棒を備えた2リットルのビーカーにろ液を追加します。加熱撹拌プレート上のドラフト内に置きます。攪拌しながら沸騰させ、約200mlに蒸発する。 500ミリリットルのビーカーにソリューションを転送します。約50ミリリットルがルになるまで沸騰続行フィート注：ソリューションがオーバー沸騰することは可能であるように注意がこのステップの間に注意する必要があります。最後の50ミリリットルの色はダークブラウンです。調理プロセス中の任意の時点で、加熱を停止することができ、ろ液をRTまたは4℃でO / Nを記憶する。 </li><li>クール1,000×gで5分間遠心分離してみましょう。 100ミリリットルのビーカーに上清を注ぎ、ペレットを捨てる。 </li><li>茶色の皮に、上清を煮詰める。暑さから削除し、へらで地殻を破る。地殻をカバーするのに十分な200プルーフのエタノールを追加します。パラフィルムでビーカーをカバーし、それは、室温でO / Nを座らせて。きれいなビーカーにエタノールを捨て、横に置く。地殻をカバーする新たなエタノールを追加します。 O / Nの抽出を3回繰り返します。抽出物をプール。 </li><li>エタノール抽出を乾燥させるために、真空ポンプを使用してください。真空ポンプが使用できない場合はドラフト内で攪拌しながら、50℃にきれいなビーカーと暖かいにエタノール抽出物を追加します。注：エタノールをlの粘着性の残留物を蒸発した後IGHTブラウン色がままにしてください。加熱が使用される場合、注意が長すぎるために加熱して溶液を燃焼しないように注意すべきである。 </li><li>滅菌蒸留水10mlに残渣を溶解。フィルターは1mlアリコートにおいて-20℃で0.22μmのシリンジフィルターとストアこの溶液を滅菌する。 NOTE：1mlの水は、元の液体線虫培養物100ml毎ための残渣を溶解するために使用される。液体培養フラスコの一つは、汚染に起因して破棄された場合、次に残留物を溶解するために使用される水の量を調整する。 </li></ol>フェロモン用量反応の2の決定<ol><li>ペプトンことなく、ノーブル寒天でNGM培地の6倍5ミリリットルを行うことで、dauerフェロモンを準備し、および50μg/ ml硫酸ストレプトマイシンとdauerフェロモンの範囲（表1のレシピを参照してください）4、19を補充した。13x100 mm単位で成分を混合ガラス培養管は、ブンゼンバーナーの上に沸騰してから、3センチメートルペトリ皿に注ぐ。 </li><li>午前の重量を測定icrocentrifugeチューブ。 EのO / N培養の1ミリリットルを追加します。マイクロチューブ内〜17000×gでチューブと遠心分離機に大腸菌 OP50。上清のすべてを削除します。再びチューブを計量し、細菌ペレットの重量を決定する。 4％の最終濃度（表1の処方を参照）M9緩衝液でペレットを再懸濁（w / v）の 。一定分量の各フェロモン板4の中央部への細菌懸濁液10μl。 RTで保管プレートO / N。 </li><li> 10-15妊娠N2野生型の雌雄同体のフェロモン板4の上に大人になって脱皮した後、ある日を選択してください。 4時間 - 3、25℃でプレートを保管してください。 </li><li>大人の雌雄同体のすべてをオフに選んでください。各プレートについての産卵数を記録します。パラフィルムでプレートを密封し、3日間17 25℃に戻ります。 </li><li> dauersのパーセンテージをカウントし（ 図2）。注：両方の方法のためには、dauers（およびより少ない元に留意することが重要である10トン非dauers）が壁を登るとペトリ皿の蓋の上になります。これは、線虫のための料理のすべての表面を調べることが重要である。 </li><li>用量反応曲線にデータをフィットし、 図3に示すように、（dauersを形成するために、動物の90％を誘導するのに十分なフェロモン）フェロモンのEC 90濃度を推定注：私たちは、典型的にはEC 90を推定するためにロジスティック回帰モデルを使用ししかし、異なる統計ソフトウェアパッケージは、同様の推定値を生成する別の方法を有することができる。 </li></ol> Dauer IL2ニューロンの3。ライブイメージング<ol><li>ステップ2.6で決定されたEC 90値を使用して、プロトコル2のようにdauerフェロモンプレートを準備します。 </li><li>統合されたIL2レポーター導入遺伝子を有する株（myIs13 [PのKLP-6 :: GFP]、myIs14 [PのKLP-6 :: GFP]、myIs16 [Pで、プロトコル2の方法を使用してdauersを誘導する<eM&gt; KLP-6 :: tdTomato]またはqIs56 [P ラグ-2 :: GFP]）12、20、21注：Q Is56を使用する場合には、GFPの発現は、中に数時間までIL2Qsに表示されませんdauer脱皮し、初期のリモデリング事象の一部が表示されません。 P ラグ-2 :: GFP導入遺伝子からの蛍光シグナルは、P KLP-6蛍光レポーターよりdauer中にIL2sにおける究極的に明るくなっている間に加えて、P ラグ-2は :: GFP導入遺伝子はまた、頭部の筋肉に弱く発現する細かいIL2の枝をマスクすることができます。 </li><li>産卵の後、大人の雌雄同体を削除するパラフィルムでプレートを密封し、25℃17で34時間インキュベートする。注：dauerの脱皮までの時間の長さは、個体間でわずかに異なります。 dauer脱皮の開始後5時間（39 - - デンドライト樹枝状分岐の中で最も急速な期間は4を開始する44時間後に産卵25℃で）12。 </li><li>イメージング前日は、EC 90濃度のdauerフェロモンとM9緩衝液中の10％（w / v）アガロースの溶液を作る。アリコートをアガロースの約100μlの顕微鏡スライド上に、迅速に、かつ慎重に、平らなアガロース面を作成するために上に別のスライドを置く。アガロースが固化した後、湿度室（ウェットペーパータオルでピペットチップボックス）にサランラップや店舗でスライドを包む。 </li><li> 34時間のインキュベーション期間の後、咽頭のポンピングを停止した線虫のためのフェロモンプレートを調べます。つのスライドの片面のみがそれをアガロース有するように、別個のスライド。 10％のアガロースパッドと0.1ミクロンのポリスチレンビーズ22の1μlの上に1つ以上の線虫を選択してください。注：よく同期人口、プレート上の線虫の大部分はL2dをからdauerに脱皮を締結されます。しかし、私の中で変​​動​​があるかもしれないndividuals。それは、動物はdauer脱皮内であった時間（例えば代表的な結果を参照してください）​​の量を推定するための追加機能（ストーマ閉塞、咽頭リモデリング）を注意することが重要である。 </li><li>線虫の向き（左/右、背側/腹側）を決定します。可能な限り低い蛍光強度100倍、15分毎にIL2QデンドライトのZスタック画像を取り込むか、特定の実験の必要に応じ。線虫はL2dのキューティクルおよび固定化から分離するまで、画像を撮影続行動物はdauerから回復した場合（咽頭のポンピングを開始する）ことは不可能であるか。積極的にイメージングしない場合には、湿度チャンバ内のスライドを置く。注：私たちは、電動ステージと微分干渉コントラスト（DIC）光学系と落射蛍光顕微鏡を使用しています。 </li></ol>

<ol>
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The authors have nothing to disclose

dauer特有の神経可塑性およびその他のdauer関連形態学的変化を調べたところ、遺伝子変異（ すなわち、DAF-C変異体）を介して、またはフェロモンに野生型動物の曝露のいずれかによってdauersの制御された、信頼性の高い形成する必要がある。 dauersを誘導する遺伝子変異の使用が便利であるが、それは、結果を混乱させることがある。したがってDAF-cの変異を有する収集されたデ?...

発達や行動の可塑性を含む表現型の可塑性は、不利な環境条件に適応するために重要であると進化1の原動力であると考えられる。C.虫が頻繁に開発·神経生物学における研究のために使用されるモデル生物である。理想的な条件下で、C.エレガンスは、成人の生殖段階に入る前に4幼虫の段階を経て開発しています。しかし、低食品の可用性と高い人口密度、Cの条件の下でエレガンスは、発達スイッチを受け、長い寿命とストレス耐性のdauerステージ2に入ることができる。 Dauersはおそらく、このストレス耐性を媒介する非dauersからのいくつかの形態学的および行動の相違を示す。 dauerエントリを規制する環境的合図と遺伝的経路は広範囲に3、4、5、6、7について説明したが、分子機構をdを発生する個別の表現型の変化を制御するuringのdauer形成はあまりよく理解されている。 dauer固有の表現型の検査はdauer動物の生産を必要とします。これは、3つの方法で達成することができる。1）Cの飢えた培養物からピッキング虫 、dauer形成構成的（DAF-c）の変異体の2）を使用し、精製されたフェロモンを通じてdauer形成の3）誘導。これは、Cと標準のNGMプレートからdauersを選ぶことは比較的簡単であるその細菌の食料供給を使い果たした虫個体群。私達の手の中に、標準的なNGMプレートがもともと3日間成長させ、その後3を接種したOP50 大腸菌 50μlを、を播種4 -成体雌雄同体は、一週間以内に食糧供給を排気して数千を生成し、25℃に保っさらに3日以内にdauers（多数の飢餓状態の非dauersに加えて）。しかし、この方法は、dの中に脱皮中に発生するプロセスを調べるため不十分であるアウアー。これは、典型的な飢餓プレート上の動物はdauerに入力している数千のかを判断することは困難である。さらに、飢餓状態のプレートの使用は、同期のための可能性を提供していません。 DAF-cの変異体の使用は、同期とdauersの信頼性の高い生成を可能にする。しかし、特定のDAF-C変異は、単独で、またはさらなる変異と組み合わせて他のdauer固有の表現型に影響を与えないという保証はありません。 dauerフェロモンの存在が最初Cassadaとラッセルによって暗黙以降ゴールデンとリドルによって実証された。フェロモンは、以来、化学的に2、図8、図9、図10に特徴付けられている。精製されたフェロモンは異なる形で両方の行動や発達過程9を規制ascarosidesの複雑なブレンド、11で構成されています。使用した粗dauerフェロモン抽出物dauerを可能にする野生型のバックグラウンドで同期されたdauersの信頼性が制御さ誘導。 </P&gt; 神経系および関連するグリア細胞がdauer幼虫形成が12、13、14時のリモデリングを受けています。これらの変更のいくつかは、そのようなdauer 13、14時のグリア細胞のリモデリングとして、不可逆的であるが、他のリモデリング事象が良好に戻った際に可逆的である環境条件。デンドライト樹枝状分岐、multidendriticニューロンと軸索リモデリング〜12の単一樹状からスイッチ：たとえば、4 IL2Qニューロンの集合が含むdauer形成時の迅速かつ可逆的神経細胞リモデリングを受ける。本明細書の方法は、モデル生物におけるストレス誘発神経可塑性の信頼性のin vivoイメージングを可能にします。生産、粗dauerフェロモンの試験のために提示される方法は、以前にいくつかの情報源4、8、15、16、17、18から説明され、コンパイルされている。

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">N2 C. elegans Bristol strain</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td style="width:212px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2660 myIs13[Pklp-6::gfp] III</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2762 myIs14[Pklp-6::gfp] V</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">JK2868 qIs56[Plag-2::gfp] V</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">OP50 E. coli</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes</td>
			<td style="width:152px;">Tritech Research</td>
			<td>60 mm T3308, 35 mm T3501</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;">NGM agar</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">3 g NaCl, 17 g agar, 2.5 g peptone in 975 mL H2O autoclaved cooled to 55&#176;C followed by 1 mL of 5 mg/ml cholesterol in ethanol, 25 mL of 1 M KPO4 buffer (pH 6.0), 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4</td>
		</tr>
		<tr height="356">
			<td height="356" style="height:356px;">S Media</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">S media consists of S basal solution (1.46 g NaCl, 0.25g K2HPO4, 1.5 g KH2PO4, 0.25 ml of 5 mg/ml cholesterol in ethanol and H2O to 250 mL. Autoclaved.) To the S basal solution add 2.5 mL of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate, H2O to 100 mL, autoclaved), 2.5 mL of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO4&#160;&#8226;7 H2O, 0.2 g MnCl2&#8226;4 H2O, 0.29 g ZnSO4&#160;&#8226;7 H2O, 0.025 g CuSO4&#160;&#8226;5 H2O, H2O to 1 liter. Autoclaved and stored in the dark), 0.75 mL of 1 M CaCl2, and 0.75 mL of 1 M MgSO4.</td>
		</tr>
		<tr height="392">
			<td height="392" style="height:392px;width:259px;">Dauer pheromone plates for dose-response assay</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 0.085 g Noble agar, 0.5 mL of 0.513 M NaCl and 5 &#181;L of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 mL culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H2O to bring the volume to 4.725 mL.&#160; Boil over a Bunsen burner until the agar is dissolved and then cool to 55 &#176;C. Add 125 &#181;L 1 M KPO4 buffer (pH 6.0), 50 &#181;l 0.1 M CaCl2, 50 &#181;L 0.1 M MgSO4 and 50 &#181;l of 5 mg/ml streptomycin sulfate to each tube.&#160; Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsphere beads 0.1 micron</td>
			<td style="width:152px;">Polysciences Inc.</td>
			<td>00876-15</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">M9 buffer</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 liter. Sterilize by autoclaving</td>
		</tr>
	</tbody>
</table>

in vivo imaging of dauer-specific neuronal remodeling in c. elegans

動物でストレス誘発表現型の可塑性を制御するメカニズムは、生体内で勉強することがしばしば複雑で困難である。ストレス誘発性可塑性の典型的な例はCのdauer段階である虫 。 Dauers、細菌食品やdauerフェロモン高濃度、低濃度の条件下で形成された代替案の発達幼虫期である。 Dauersは、広範な発達や行動の可塑性を表示します。例えば、4つの内側陰唇象限（IL2Q）ニューロンのセットはdauer形成中に豊富な可逆的リモデリングを受ける。 dauerエントリを調節するよく知られた環境の経路を利用して、大量の液体線虫培養物からの粗dauerフェロモンの製造のための以前に確立された方法が示されている。この方法では、50,000濃度 - 液体培養の線虫75,000 / mlを非常に強力な粗dauerフェロモンを生成するのに十分である。原油フェロモン効力はDETEです用量反応バイオアッセイによりrmined。最後に、dauer形成中IL2Qs のin vivoタイムラプスイメージングのために使用される方法が記載されている。

暑さと寒さに安定でもある黄色の液体中の粗dauerフェロモン結果（ 図1）の産生。原油フェロモンのアリコートの活性の明らかな損失で無期限に-20℃で保存されている。フェロモンの生産に続き、単用量反応バイオアッセイは、効力を概算するのに十分である。 3回、各実験からの平均を取る - しかし、ほとんどの実験のためにはバイオアッセイ2を繰り返す必要がある。 2フェロ?...

Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

CのDauer固有の神経リモデリングのin vivoイメージングエレガンス

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

in-vivo-imaging-of-dauer-specific-neuronal-remodeling-in-c-elegans

Research

JoVE Journal

Neuroscience

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

11.1K ビュー.  University of Illinois Urbana-Champaign. Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer. 

ビデオ: In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

神経科学

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

Cの生体レーザー軸索切断にエレガンス

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

シナプス蛍光の自動定量<em&gt; C.エレガンス</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Cの生体内神経カルシウムイメージングでエレガンス

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

神経高密度コア小胞の超解像イメージング

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

軸索分岐と神経形態形成中に形質膜配信の役割を定義するために組み合わせる方法論を活用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

適応マイクロ嗅覚を用いた男性c. の Elegans頭ニューロンのフェロモン応答における神経活動のイメージング

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.