我们要感谢他在舞台上方的孵化器的设计和制造技术的专业知识和高超的技巧贡献唐纳德赫特森。我们也感谢Ayda Dashtaei为她出色的技术援助。所有的工作是研究基金会的神经科学的支持。

<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>

采用有针对性地在感染培养的神经元线粒体荧光蛋白的慢病毒介导的表达和廉价的实验室建阶段顶的孵化器，允许延长工期的活细胞成像，我们已经能够进行调查线粒体运动和neuromodulatory信号之间的联系5 - 羟色胺（5 - HT），多巴胺（DA），乙酰胆碱（ACH）等。我们的研究有助于阐明信号通路，第一次，链接线粒体贩运活动的神经递质，如5 - HT和DA神经元调制的变化 - 心脏神经功能。我们发现，使用有针对性的荧光蛋白许可证标记长时间，可能会比更短的工期可以使用重要的染料有关的生理，生活培养的神经元的线粒体观察。此外，荧光蛋白信号的强度，使我们能够保持在图像采集的曝光时间短，最大限度地减少漂白或光毒性的可能性。最后，一个简单和廉价的阶段顶部的孵化器，保持环境的温度，湿度，和二氧化碳水平，同时尽量减少媒体的蒸发，使我们能够按照居住超过几小时甚至几天的神经细胞线粒体运动。想编造一个阶段顶部的“孵化器”在活的神经元线粒体的长期观测的研究人员需要按照我们的设计提供精确的细节，使用（例如，透氧膜，以避免媒体蒸发的材料特性）和应用（例如，温度和湿度控制，pH值，渗透压维护的缓冲）的原则一般是在本议定书中所描述的一致。

<table border="1"><tbody><tr><th>数量</th><th>说明（在图1的位置） </th></tr><tr><td> 1 </td><td>组织文化的孵化器（H） </td></tr><tr><td> 1 </td><td>耐热塑料外壳（聚甲醛或类似）（一） </td></tr><tr><td> 1 </td><td>为35mm GBM的盖子的塑料架（贴上膜培养皿）（男） </td></tr><tr><td> 1-2 </td><td>线性英尺清除PFTE（聚四氟乙烯）膜材料</td></tr><tr><td> 4 </td><td>铜拇指螺丝</td></tr><tr><td> 2 </td><td>小10kOhm散热片电阻（用于舞台上的孵化器外壳内的加热元件） </td></tr><tr><td> 1 </td><td>变压器（9V电流供应电阻） </td></tr><tr><td> 1 </td><td>水晶球温度控制器和探测器（恒温调节的电源通过变压器散热片电阻） </td></tr><tr><td> 1 </td><td> 2000毫升锥形瓶中（软管振动抑制孵化器前阶段顶部的消声器）（K） </td></tr><tr><td> 1 </td><td> 1 / 8“sorbothane表（顶部阶段，孵化器基地的垫片材料） </td></tr><tr><td> 1 </td><td>气密性ABS（或类似）塑料盒（水族水泵房）（J） </td></tr><tr><td> 1 </td><td>水族馆泵（一） </td></tr><tr><td> 1 </td><td>小型计算机冷却风扇</td></tr><tr><td> 1 </td><td>塑料外壳的散热风扇（L） </td></tr><tr><td> 1 </td><td>计算机9V变压器冷却风扇</td></tr><tr><td> 20 -30英尺</td><td> NALGENE或硅胶管（组织培养和阶段的孵化器之间的连接;水分陷阱）（F，G，N） </td></tr><tr><td> 2 </td><td>塑料活塞（打开和关闭阶段顶部孵化器前后空气流通）（海外） </td></tr><tr><td> 4 </td><td>刺黄铜软管接头（软管连接到/从舞台上的孵化器和水族馆泵的外壳） </td></tr><tr><td> 2 </td><td>黄铜快速接头（软管连接到/从舞台上的孵化器） </td></tr><tr><td> 2 </td><td>黄铜快速接头（软管连接到/从舞台上的孵化器） </td></tr></tbody></table> 表1。舞台上的孵化器部分 ： <table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>聚- D -赖氨酸</td><td> Sigma - Aldrich公司</td><td> P7280 - 5MG </td></tr><tr><td>层粘连蛋白</td><td>罗氏应用科学部</td><td> 11243217001 </td></tr><tr><td> 35毫米的玻璃底菜</td><td> MatTek </td><td> P35GC - 0 - 14 - C </td></tr><tr><td> DMEM培养液</td><td> Life Technologies公司</td><td> 10567 </td></tr><tr><td> B27 </td><td> Life Technologies公司</td><td> 17504-044 </td></tr><tr><td> Glutamax </td><td> Life Technologies公司</td><td> 35050 </td></tr><tr><td>脂质丰富的牛血清白蛋白</td><td> Life Technologies公司</td><td> 11020-021 </td></tr><tr><td> L -天冬酰胺</td><td> Sigma - Aldrich公司</td><td> P0380 - 100G </td></tr><tr><td> L -脯氨酸</td><td> Sigma - Aldrich公司</td><td> A8381 - 100G </td></tr><tr><td>维生素B - 12 </td><td> Sigma - Aldrich公司</td><td> V2876 - 100MG </td></tr><tr><td> 5 - 羟色胺</td><td> Sigma - Aldrich公司</td><td> H9523 - 25毫克</td></tr><tr><td> 8 - OH - DPAT </td><td> Sigma - Aldrich公司</td><td> H8520 - 25毫克</td></tr><tr><td>多巴胺</td><td> Sigma - Aldrich公司</td><td> H8502 - 5G </td></tr><tr><td>溴隐亭</td><td> Sigma - Aldrich公司</td><td> B2134 - 25毫克</td></tr><tr><td> SKF38393 </td><td> Sigma - Aldrich公司</td><td> D047 - 100MG </td></tr></tbody></table>

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

要了解线粒体运输和神经功能之间的关系，关键是观察 1-3延长工期的生活培养的神经元线粒体的行为。这是现在可以通过使用重要的染料和荧光蛋白与细胞骨架，细胞器，并在活细胞中的其他结构可以被标记，然后通过动态的荧光显微镜，可视化。例如，在鸡胚交感神经元，线粒体运动的特点，使用的重要染料罗丹明123 4 。在另一项研究中，线粒体mitochondrially针对性EYFP 5转染大鼠前脑神经元的可视化。然而，初级神经元，超过分钟，小时，甚至天的成像提出了一些问题。其中最重要的是：1）维护的培养条件，如温度，湿度，pH值在长成像会话; 2）一个强大，稳定的荧光信号，以确保获得的图像质量和信号强度的精确测量，在图像分析; 3）限制在图像采集的曝光时间，以尽量减少漂白，避免光毒性。 在这里，我们描述了一个协议，允许观察，可视化和高时间分辨率和最佳的生活支持条件下培养的海马神经元线粒体运动分析。我们已经构建了一个负担得起的阶段顶孵化器提供了良好的温度调节和大气中的气体流量，也限制了媒体蒸发的程度，以确保稳定的pH值和渗透压。这个孵化器连接，通过进口和出口软管，一个标准的组织培养孵化器，它提供恒定的湿度水平和 5-10％的CO 2 /空气气氛。这种设计提供了一个成本效益的替代昂贵得多显微镜孵化器不一定保证了许多数小时甚至数天的细胞活力。为了形象化线粒体，我们与编码，有针对性地线粒体红色荧光蛋白的慢病毒感染的细胞。这保证了强大的和持久的的信号，配合稳定的氙气光源的使用，使我们能够限制在图像采集的曝光时间，但排除漂白和光毒性。舞台上方的孵化器顶部的两个注射口允许的神经递质和其他试剂的急性政府调节线粒体运动。总之，表达慢病毒介导的细胞器有针对性的红色荧光蛋白和我们的舞台上方的孵化器，传统的倒置荧光显微镜，CCD摄像头，以及氙气光源的结合使我们能够获得线粒体运输时间推移图像生活过较长的持续时间可能比那些在研究部署常规重要的染料和关闭的，现成的生命支持系统的神经元。

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

我们描述了一个协议，允许在较长的时间，通过荧光显微镜观察生活中的神经元线粒体成像。过长时间的成像是通过一个mitochondrially有针对性的荧光蛋白，并利用一种廉价的舞台上方的孵化器，在我们的实验室设计和内置的慢病毒介导的表达。

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

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 次观看.  The Neurosciences Institute. 我们描述了一个协议，允许在较长的时间，通过荧光显微镜观察生活中的神经元线粒体成像。过长时间的成像是通过一个mitochondrially有针对性的荧光蛋白，并利用一种廉价的舞台上方的孵化器，在我们的实验室设计和内置的慢病毒介导的表达。

视频: 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.

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

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 ...

科研

神经科学

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.

活大鼠皮层神经元线粒体膜电位和活性氧的测定

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.

初级量子神经元轴突运输的实时成像点标记的BDNF

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.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

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.

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

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.

海马神经元中突触囊泡吞的测量

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.

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

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.

使用遗传编码的Ca2 + 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca2 + 摄取进行成像

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.

培养的星形胶质细胞中线粒体系统的实时成像

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.

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

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