我々は、ステージ上インキュベーターの設計と製造の間に彼の技術的な専門知識と優れた技術を貢献するためにドナルドハットソンに感謝します。私たちは、彼女の優秀な技術支援のためにもAyda Dashtaeiに感謝しています。すべての作業は、神経科学研究財団によってサポートされていました。

<ol>
	<li>Ligon, L. A., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+of+mitochondria+in+the+axons+and+dendrites+of+cultured+hippocampal+neurons.">Movement of mitochondria in the axons and dendrites of cultured hippocampal neurons.</a> J Comp Neurol. 427, 340-350 (2000).</li><li>Miller, K. E., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+coherent+low+velocity+axonal+transport+of+mitochondria.">Direct evidence for coherent low velocity axonal transport of mitochondria.</a> J Cell Biol. 173, 373-381 (2006).</li><li>Macaskill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miro1+is+a+calcium+sensor+for+glutamate+receptor-dependent+localization+of+mitochondria+at+synapses.">Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses.</a> Neuron. 61, 541-555 (2009).</li><li>Morris, R. L., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+mitochondria+along+microtubules+and+F-actin+in+living+vertebrate+neurons.">Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons.</a> J Cell Biol. 131, 1315-1326 (1995).</li><li>Rintoul, G. L., Filiano, A. J., Brocard, J. B., Kress, G. J., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+decreases+mitochondrial+size+and+movement+in+primary+forebrain+neurons.">Glutamate decreases mitochondrial size and movement in primary forebrain neurons.</a> J Neurosci. 23, 7881-7888 (2003).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in neuroscience. , (1999).</li><li>Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protocols for neural cell culture. , (2001).</li><li>Chen, S., Owens, G. C., Crossin, K. L., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+stimulates+mitochondrial+transport+in+hippocampal+neurons.">Serotonin stimulates mitochondrial transport in hippocampal neurons.</a> Mol Cell Neurosci. 36, 472-483 (2007).</li><li>Chen, S., Owens, G. C., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+inhibits+mitochondrial+motility+in+hippocampal+neurons.">Dopamine inhibits mitochondrial motility in hippocampal neurons.</a> PLoS One. 3, e2804-e2804 (2008).</li><li>Chen, S., Owens, G. C., Makarenkova, H., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC6+regulates+mitochondrial+transport+in+hippocampal+neurons.">HDAC6 regulates mitochondrial transport in hippocampal neurons.</a> PLoS One. , e10848-e10848 (2010).</li><li>Curran, M. A., Kaiser, S. M., Achacoso, P. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transduction+of+nondividing+cells+by+optimized+feline+immunodeficiency+virus+vectors.">Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.</a> Mol Ther. 1, 31-38 (2000).</li></ol>

利害の衝突は宣言されません。

レンチウイルスを介した感染培養神経細胞のミトコンドリアをターゲットと蛍光タンパク質の発現と延長期間のための生細胞のイメージングを可能にする安価なラボ構築ステージトップインキュベーターを採用し、我々は、ミトコンドリアの動きと神経調節の信号間のリンクを調査することができた、セロトニン（5 - HT）、ドーパミン（DA）、及びアセチルコリン（ACH）など。神経機能の心臓部である - 私たちの研究は、その、初めて、リンクなどの5 - HTとDAのような神経伝達物質によって変調されたニューロンの活動の変化にミトコンドリアの人身売買シグナル伝達経路を解明するために役立っています。我々は、標的蛍光タンパク質の使用は、より多くの生理学的に関連する重要な染料を使用して可能なはるかに短い期間よりも可能性が長期間にわたって培養神経細胞を生体内でミトコンドリアというラベルの観察を可能にすることがわかります。さらに、蛍光タンパク質のシグナルの強度は、光退色や光毒性の可能性を最小限に抑え、私たちは画像取得時の露光時間を短くすることができます。最後に、シンプルで安価なステージトップインキュベーターメディアの蒸発を最小限に抑えながら、周囲温度、湿度、およびCO 2のレベルを維持し、私たちはさらに数時間または数日にわたってニューロンを生体内でミトコンドリアの動きをたどることができます。ライブニューロンのミトコンドリアの長期的な観測のためにステージトップインキュベーターを作製する研究者が使用する材料の特性（例えば、ガス透過性膜媒体の蒸発を避けるために提供されて、私たちのデザインの正確な詳細に従う必要はありません）と（例えば、温度、湿度コントロール、浸透圧のpHは、メンテナンスのバッファリング）適用される原則は、一般的にこのプロトコールに記載されているものと一致している。

<table border="1"><tbody><tr><th>数量</th><th>説明（と図1の位置） </th></tr><tr><td> 1 </td><td>組織培養インキュベーター（H） </td></tr><tr><td> 1 </td><td>耐熱性プラスチックの筐体（デルリンまたは同等の）（） </td></tr><tr><td> 1 </td><td> 35ミリメートルGBMのふた用プラスチックフレーム（ペトリ皿に膜を貼るための）（M） </td></tr><tr><td> 1月2日</td><td>リニアフィートクリアPFTE（テフロン）膜材料</td></tr><tr><td> 4 </td><td>真鍮のつまみねじ</td></tr><tr><td> 2 </td><td>小さな10kOhmヒートシンク抵抗（ステージトップインキュベーターのエンクロージャ内部に発熱体として使用される） </td></tr><tr><td> 1 </td><td>変圧器（抵抗に9Vの電流の供給） </td></tr><tr><td> 1 </td><td>テラリウムの温度調節器とプローブ（トランスを経由してヒートシンクの抵抗への電源の恒温調節） </td></tr><tr><td> 1 </td><td> 2000ミリリットルの三角フラスコ（ステージトップインキュベーターの前にホースの振動抑制用マフラー）（K） </td></tr><tr><td> 1 </td><td> 1 / 8&quot;sorbothaneシート（ステージトップインキュベーターのベース用ガスケットの材料） </td></tr><tr><td> 1 </td><td>気密ABS（または同等の）プラスチック製のボックス（水槽ポンプ用ハウジング）（J） </td></tr><tr><td> 1 </td><td>水槽のポンプ（I） </td></tr><tr><td> 1 </td><td>小さなコンピュータの冷却ファン</td></tr><tr><td> 1 </td><td>冷却ファン用のプラスチック製筐体（L） </td></tr><tr><td> 1 </td><td>コンピュータの冷却ファン用9Vトランス</td></tr><tr><td> 20〜30フィート</td><td> Nalgeneまたはシリコーンのホース（組織培養とステージトップインキュベーター間の接続は、水分のトラップ）（F、G、N） </td></tr><tr><td> 2 </td><td>プラスチック製のストップコック（ステージトップインキュベーターの前と後の空気の流れを開いたり閉じたりする）（O） </td></tr><tr><td> 4 </td><td>とげのある真鍮のホースコネクタ（ステージトップインキュベーターと水槽のポンプのエンクロージャへの/からのホース接続用） </td></tr><tr><td> 2 </td><td>真鍮クイックカプラー（ホース接続用まで/ステージトップインキュベーターから） </td></tr><tr><td> 2 </td><td>真鍮クイックカプラー（ホース接続用まで/ステージトップインキュベーターから） </td></tr></tbody></table> 表1。ステージトップインキュベーターの部品： <table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>ポリ- D -リジン</td><td>シグマアルドリッチ</td><td> P7280 - 5mgの</td></tr><tr><td>ラミニン</td><td>ロシュダイアグノスティックス</td><td> 11243217001 </td></tr><tr><td> 35ミリメートルのガラスボトムディッシュ</td><td>マテック</td><td> P35GC - 0 - 14 - C </td></tr><tr><td> DMEM </td><td>ライフテクノロジーズ</td><td> 10567 </td></tr><tr><td> B27 </td><td>ライフテクノロジーズ</td><td> 17504-044 </td></tr><tr><td>グルタミン</td><td>ライフテクノロジーズ</td><td> 35050 </td></tr><tr><td>脂質が豊富なBSA </td><td>ライフテクノロジーズ</td><td> 11020-021 </td></tr><tr><td> L -アスパラギン</td><td>シグマアルドリッチ</td><td> P0380 - 100G </td></tr><tr><td> L -プロリン</td><td>シグマアルドリッチ</td><td> A8381 - 100G </td></tr><tr><td>ビタミンB - 12 </td><td>シグマアルドリッチ</td><td> V2876 - 100mgの</td></tr><tr><td> 5 - HT </td><td>シグマアルドリッチ</td><td> H9523 - 25mgを</td></tr><tr><td> 8 - OH - DPAT </td><td>シグマアルドリッチ</td><td> H8520 - 25mgを</td></tr><tr><td>ドーパミン</td><td>シグマアルドリッチ</td><td> H8502 - 5G </td></tr><tr><td>ブロモクリプチン</td><td>シグマアルドリッチ</td><td> B2134 - 25mgを</td></tr><tr><td> SKF38393 </td><td>シグマアルドリッチ</td><td> D047 - 100mgの</td></tr></tbody></table>

neuromodulation and mitochondrial transport: live imaging in hippocampal neurons over long durations

ミトコンドリア輸送と神経機能の関係を理解するために、それは1-3拡張された期間のためのライブ培養神経細胞におけるミトコンドリアの挙動を観察することが重要です。これは生きている細胞の細胞骨格成分、細胞小器官、および他の構造を標識することができるとの重要な染料と蛍光タンパク質を使用することで可能になりましたし、動的な蛍光顕微鏡を介して可視化した。例えば、胚性ニワトリ交感神経ニューロンにおいて、ミトコンドリアの動きは生体染色色素のローダミン123 4を用いて特徴付けられた。別の研究で、ミトコンドリアはミトコンドリア標的EYFP 5のトランスフェクションにより、ラットの前脳の神経細胞で可視化した。しかし、一次分以上のニューロン、時間、または数日間の撮影は、多くの問題を提示。何よりもこれらの間にある：1）そのような温度、湿度、および長時間の撮像セッション中に、pHなどの培養条件の維持、画像解析時に信号強度の取得画像の品質と正確な測定の両方を確保するため2）強い、安定した蛍光シグナル;および3）退色最小限に抑え、光毒性を避けるために、画像取得時の露光時間を制限する。 ここでは、観察、可視化、および高時間分解能で、最適な生命維持の条件下で培養海馬神経細胞におけるミトコンドリアの動きの分析を可能にするプロトコルを記述する。我々は、良好な温度調節と雰囲気ガスの流れを提供する手頃なステージトップインキュベーターを建設し、また安定したpHと浸透圧を確保し、メディアの蒸発の程度を制限している。このインキュベータは、一定の湿度レベルを提供する標準組織培養インキュベーター、5〜10％のCO 2 /空気の雰囲気に、入口、出口ホースを経由して、接続されている。この設計は、必ずしも多くの時間あるいは数日にわたって細胞の生存を保証していないはるかに高価な顕微鏡のインキュベーターに代わるコスト効率を提供しています。ミトコンドリア可視化するために、我々は、ミトコンドリアを対象としている赤色蛍光蛋白質をコードするレンチウイルスで細胞を感染させる。これは安定キセノン光源の使用と併せて、私たちは画像取得時の露光時間を制限することを可能にし、すべてが光退色と光毒性排除する、強力かつ持続的な信号を、保証します。ステージトップインキュベーターの上部にある2つの注入ポートは、意図した神経伝達物質や他の試薬の急性投与は、ミトコンドリアの動きを調節することができます。オルガネラを標的と赤色蛍光タンパク質と私たちのステージトップインキュベーター、従来の倒立蛍光顕微鏡、CCDカメラ、およびキセノン光源の組み合わせの和、レンチウイルスを介した表現で私たちがでミトコンドリア輸送のタイムラプス画像を取得することができます従来の重要な染料と既製の生命維持システムを導入する研究の可能性よりも長い期間にわたって生きているニューロン。

Watch this Scientific Journal Video about Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations at JoVE.com

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

我々は、長時間にわたって蛍光顕微鏡を介して神経細胞を生体内でミトコンドリアのイメージングを可能にするプロトコルを記述する。長期間にわたるイメージングは​​、我々の研究室で設計し、構築されたミトコンドリア標的蛍光蛋白質と安価なステージトップインキュベーターの使用のレンチウイルスを介した発現を介して実行されます。

ニューロモデュレーションとミトコンドリア輸送：長時間にわたって海馬ニューロンのライブイメージング

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

neuromodulation-mitochondrial-transport-live-imaging-hippocampal

Research

JoVE Journal

Neuroscience

15.8K ビュー.  The Neurosciences Institute. 我々は、長時間にわたって蛍光顕微鏡を介して神経細胞を生体内でミトコンドリアのイメージングを可能にするプロトコルを記述する。長期間にわたるイメージングは​​、我々の研究室で設計し、構築されたミトコンドリア標的蛍光蛋白質と安価なステージトップインキュベーターの使用のレンチウイルスを介した発現を介して実行されます。

ビデオ: Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Sf9細胞でとアメフラシ感覚ニューロンにおけるPKC転座のライブイメージング

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

神経科学

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

ライブラット皮質ニューロンのミトコンドリア膜電位と活性酸素種の定量

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

一次ニューロンにおける量子ドットで標識されたBDNFの軸索輸送のリアルタイムイメージング

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

ニコチン誘導性カルシウムシグナリングおよび腹側海馬軸索に沿って神経伝達物質放出のライブイメージング

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

細胞内のCaの蛍光と生物発光イメージング<sup&gt; 2+</sup&gt;高齢者における海馬ニューロン

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

培養海馬神経細胞のシナプス小胞エンドサイトーシスを測定

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

遺伝性痙性対麻痺におけるヒト人工多能性幹細胞由来ニューロンにおけるミトコンドリア輸送と形態学の解析

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

遺伝子組み換えCa2+ 指標(GEC)を用いたアストロサイトとニューロンのイメージングミトコンドリアCa2+ 取り込み

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

Live-imaging of Mitochondrial System in Cultured Astrocytes

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and function in astrocytes are implicated in cognition. This article describes the method for time-lapse imaging of astrocyte cultures equipped with a mitochondrial biosensor: MitoTimer. MitoTimer is a powerful and unique tool to assess mitochondrial dynamics, mobility, morphology, biogenesis, and redox state. Here, the different procedures for culture, image acquisitions, and subsequent mitochondrial analysis are presented.

This article describes the method for mitochondrial time-lapse imaging of astrocyte cultures equipped with MitoTimer biosensor and the resulting multiparametric analysis of mitochondrial dynamics, mobility, morphology, biogenesis, redox state, and turnover.

培養アストロサイトにおけるミトコンドリア系の生画像化

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and ...

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

レシオメトリック指標を用いた一次ニューロンにおけるミトコンドリアグルタチオンレドックス状態の生画像化

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...