这项工作是由美国国立卫生研究院（K22NS050137 JCB卡）的支持。

1。细胞培养<ol><li>使用前面描述的技术和培养皿镀玻璃底部涂上聚- D -赖氨酸和层粘连蛋白1（MatTek公司，亚什兰，MA），孤立和皮层神经元生长。 </li></ol> 2。准备的荧光探针TMRM和H 2 DCF - DA的股票解决方案<ol><li>准备10毫米的TMRM原液5.0毫克的TMRM溶解在1 mL无水二甲基亚砜。涡它为1分钟。然后，分装和储存在-20 ° C，避光，并使用1个月内。 </li><li>下一步，准备通过溶解在1毫升无水二甲基亚砜4.87毫克的H 2 DCF - DA的H 2 DCF - DA 10毫米的原液。同样，涡旋1分钟。然后，分装和储存在-20 ° C，避光，并使用一个星期内。 </li></ol> 3。载入TMRM和H 2 DCF - DA大鼠皮层神经元 TMRM是一个电位，细胞渗透性的荧光指示剂，在线粒体内部高度带负电荷的的积累。重要的是要使用低浓度的TMRM（10-50纳米范围内），以避免自动淬火线粒体TMRM。然后，TMRM荧光信号可直接合作关系到整个内线粒体膜ΔΨm。一个原因TMRM泄漏造成损失的荧光强度的线粒体ΔΨm损失。 H 2 DCF - DA的是细胞渗透探头转换成DCF - DA的细胞内酯酶，其氧化荧光的DCF结果。终浓度的H 2 DCF - DA 2-10μM和它之间的范围应在来自不同脑区的神经元测试经验，因为高负荷的浓度可能会导致在DCF的荧光，即使在没有对H 2 O 2的饱和。任何内源性或外源性氧化剂（例如，一氧化氮，过氧化氢）的存在会增加DFC的荧光强度。下面，我们提供了一个用于装载TMRM和H 2 DCF - DA大鼠皮层神经元的协议。 <ol><li>要加载的与TMRM大鼠皮层神经元，洗的培养神经元的台氏缓冲（文字叠加3次：结核病：145 mM的氯化钠，5 mM的氯化钾，10 mM的葡萄糖，1.5毫米氯化钙 ，1 mM的氯化镁2，和10 mM的HEPES;用NaOH调整pH至7.4）。然后，准备1 / 1000倍稀释10毫米TMRM股票在结核病的20纳米的TMRM，然后添加2μL稀释TMRM每1毫升的结核病。与在室温避光45分钟TMRM孵育的神经元。 45分钟后，安装在显微镜的阶段的培养皿中，并开始成像。 </li><li>负载与H 2 DCF - DA大鼠皮层神经元，洗培养的神经元有结核病的3倍。下一步，准备在结核病稀释H 2 DCF - DA的股票的1 / 10倍10毫米的H 2 DCF - DA 2微米，然后加入2μL 稀释 H 2 DCF - DA的每1毫升的结核病。然后，孵育45 min，室温在黑暗与H 2 DCF - DA神经元。 45分钟后，洗净的神经元与之前获得的图像，删除多余的荧光指示剂的结核病4倍。 </li></ol> 4。实时成像TMRM培养的神经元，以确定ΔΨm <ol><li>为了执行实时成像TMRM，共聚焦激光扫描显微镜（文字叠加：LSM 510，卡尔蔡司公司）孵育的神经元，与生活时间序列程序的应用程序，用于。适用于低分辨率和激光功率衰减（文本叠加：低解析度：256 × 256;激光功率：1％），以最大限度地减少获取图像，并避免漂白需要的时间。 </li><li> 。其次，调整TMRM加载使用反射光的神经元装的焦点。检查TMRM荧光照度在514纳米和570 nm处检测。设置一个摄像头略低于饱和度检测增益。 </li><li>一旦所有参数，包括分辨率，激光电源，检测相机的增益，和间隔时间推移获得的图像设置，不改变这些设置之间的实验。接下来，更改字段。开始采集图像。 </li><li>为了测试在ΔΨm的变化，如FCCP或2微克/毫升的寡，1微米的刺激可以被应用，这将大大去极化或超极化的线粒体膜电位，分别为。这些变化将反映在TMRM荧光强度，与基线荧光强度FCCP的情况下，或在TMRM寡的情况下的荧光强度的增加相比减少。 </li></ol> 5。与H 2 DCF - DA的培养，以确定活性氧的神经元的实时成像<ol><li>要与H 2 DCF - DA，首先培养的神经细胞进行实时成像，安装在显微镜的阶段培养皿中。调整我们细胞的焦点ING的反射光。检查DCF的荧光激发波长为488和515 nm处的排放。 </li><li>其次，调整激光功率的5-7％，探测器增益，分辨率为256 × 256。不要更改这些设置之间的实验。然后，将获得使用时间系列节目的现场图像的频率。 </li><li>选择一个新的领域，并开始获取图像。为了检测ROS水平的变化，治疗的H 2 O 2与100-200微米的细胞。这将体现在DCF的荧光强度的增加，与基线水平相比。 </li></ol> 6。数据分析<ol><li>使用感兴趣区域（文本叠加：投资回报率）从LSM程序的工具，选择的地区。然后，测量TMRM或ROS的荧光强度。选择从线粒体地区或整个细胞体成像的细胞来衡量TMRM或ROS的荧光强度，分别从投资回报率的投资回报。 </li><li>从所有的每个细胞TMRM或全细胞ROS的所有细胞的每个时间点的成像机构的投资回报计算的平均荧光强度。选择地区旁细胞的背景荧光强度计算。采取多次测量和计算的平均背景强度。 </li><li>从投资回报率，每次使用Microsoft Excel在每个单元的平均荧光强度减去平均背景荧光强度。减去背景强度后，正常化TMRM或DCF荧光强度，使用这个公式的基线荧光（文本叠加：ΔF= FF O / F O X 100，其中F =在任何时间点的荧光强度=基准荧光） 。然后，使用Sigma的剧情程序生成的波形图，显示随着时间的推移的荧光强度的变化。 </li></ol> 7。代表性的成果图1A显示了TMRM培养的大鼠皮层神经元的荧光图像。 FCCP此外，线粒体的解偶联剂，导致线粒体的去极化和亏损TMRM荧光强度（图1B）。此外FCCP（第350秒;图1C）前基线TMRM荧光水平保持稳定。随着时间的推移TMRM荧光变化的定量分析显示后FCCP（图1C）除了在TMRM荧光显着减少。 图1D显示与DCF装载的大鼠皮层神经元的的荧光图像。 H 2 O 2的结果除了增加在细胞体DCF的荧光强度（图1E）。基线DCF荧光水平不变（第120秒）前的H 2 O 2的应用程序。 DCF的荧光定时测量显示其稳定的水平，增加H 2 O 2处理（图1F）后。 <img src="/files/ftp_upload/2704/2704fig1.jpg" alt="图1"/> 图1。线粒体膜电位和生活大鼠皮层神经元的活性氧水平的评估。 （一）代表皮层神经元的荧光图像加载TMRM。扫描基线TMRM荧光后，神经细胞治疗与protonophore FCCP（1μM）。右边是一个伪与明亮的黄色和黑色代表最高和最低强度，分别TMRM荧光强度栏。从线粒体地区TMRM荧光损失表示的ΔΨm崩溃后FCCP治疗（B组）。 TMRM荧光强度的变化在不同时间点的定量表示FCCP治疗前后是显示面板C.（四）加载H2DCF - DA大鼠皮层神经元的荧光图像。确定基准DCF的荧光后，细胞治疗与200微米H 2 O 2，DCF荧光的变化进行了评估。在DCF荧光增加反映后，H 2 O 2处理（五）增加ROS水平。定量分析的DCF荧光的变化，前后H 2 O 2处理，显示面板F.比例尺= 10微米 Video.7.1 - labmedia 2704_Joshi.avi 活细胞成像的皮层神经元TMRM前后FCCP此外，使用40X目标。伪强度显示最大（FCCP除了前明亮的黄色）和下降（红色，后FCCP此外）TMRM后的荧光强度FCCP此外<a href="http://www.jove.com/files/ftp_upload/2704/supplemental_video1.avi">，点击这里查看</a>视频 视频。 7.5 - labmedia 2704_Joshi.avi H 2 O 2除了使用40X目标之前和之后，现场皮层神经元细胞DCF的成像。基线DCF的荧光在细胞体浅绿色和过氧化氢除了增加了DC明亮的绿色荧光强度<a href="http://www.jove.com/files/ftp_upload/2704/supplemental_video2.avi">F点击这里观看</a>视频

<ol>
	<li>Hilgenberg, L. G., Smith, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+dissociated+mouse+cortical+neuron+cultures.">Preparation of dissociated mouse cortical neuron cultures.</a> J. Vis. Exp. , (2007).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+membrane+potential+using+fluorescent+rhodamine+derivatives.">Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.</a> Biophys. J. 76, 469-477 (1999).</li><li>Ward, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+precursor+protein+intracellular+domain+(AICD)+disrupts+actin+dynamics+and+mitochondrial+bioenergetics.">The amyloid precursor protein intracellular domain (AICD) disrupts actin dynamics and mitochondrial bioenergetics.</a> J. Neurochem. 113, 275-284 (2010).</li><li>Gunasekar, P. G., Kanthasamy, A. G., Borowitz, J. L., Isom, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor+activation+produces+concurrent+generation+of+nitric+oxide+and+reactive+oxygen+species:+implication+for+cell+death.">NMDA receptor activation produces concurrent generation of nitric oxide and reactive oxygen species: implication for cell death.</a> J. Neurochem. 65, 2016-2021 (1995).</li></ol>

我们提出了一个一步一步的过程，描述如何确定在使用荧光灯指标TMRM 和 H 2 DCF - DA，分别的大鼠皮层神经元的ΔΨm和ROS。对于其他类型的细胞，是很重要的经验确定最终的TMRM或H 2 DCF - DA的浓度和加载时间。在一般情况下，TMRM浓度范围从20-200纳米，并与TMRM细胞孵育时间从20到60分钟不等。终浓度的H 2 DCF - DA的范围2-10μM，并装载包含这个指标的解决方案中的细胞孵育30-45分钟不等。 重要的是要优化激光功率和扫描速度的图像，同时避免光毒性细胞，并在没有任何刺激的情况下的荧光强度的变化（例如TMRM荧光闪烁）。优化的光学设置应在荧光信号，这不是在刺激的情况下，超过或低于饱和（阈值）的结果。取得的最佳条件，在选择一个特定的激光功率和扫描速度的领域收集的图像时，有探头的荧光强度在没有任何刺激10-15分钟的实时成像的情况下没有变化。 其他荧光探针来确定ΔΨm包括罗丹明123和四甲基若丹明乙酯（TMRE）。然而，他们发现，在孤立的线粒体 2抑制呼吸过程。更重要的是，TMRM没有线粒体呼吸的影响，并已在低浓度低的光毒性和漂白与其他探测器相比。 H 2 DCF - DA是一个很好的指标为活性氧的细胞，因为它是在保留和认识的几个氧化剂品种，如过氧化物，超氧化物，一氧化氮 4 。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>玻璃底培养皿</td><td> MatTek公司</td><td> P35G - 1.5 - 14 - C </td></tr><tr><td> NbActive4 </td><td> BrainBits </td><td> NbActive4 </td></tr><tr><td> TMRM </td><td> Invitrogen公司</td><td> T668 </td></tr><tr><td> H 2 DCF - DA </td><td> Invitrogen公司</td><td> C400 </td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S6191 </td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P3911 </td></tr><tr><td>氯化钙2•2H 2 O </td><td>西格玛</td><td> C3306 </td></tr><tr><td>氯化镁2•6H 2 O </td><td>西格玛</td><td> M2670 </td></tr><tr><td> D -葡萄糖</td><td>西格玛</td><td> G6152 </td></tr><tr><td> HEPES </td><td> Invitrogen公司</td><td> 15630 </td></tr></tbody></table>

determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons

线粒体膜电位（ΔΨm）是维持生理功能的呼吸链产生ATP的关键。的ΔΨm的一个重大损失，使细胞的能量耗尽，随后死亡。活性氧（ROS）是重要的信号分子，但他们在病理条件下的积累会导致氧化应激。在细胞内ROS的两个主要来源是环境中的毒素和氧化磷酸化的过程中。线粒体功能障碍和氧化应激有牵连的许多疾病的病理生理;因此，ΔΨm和ROS的能力，以确定可以提供有关的细胞和线粒体功能的生理状态的重要线索。 几个荧光探针（TMRM，罗丹明123，TMRE，JC - 1）可以用来确定在多种细胞类型Δψm，许多荧光指标（H 2 DCF - DA）可以用来确定活性氧，Dihydrorhodamine 123 Dihydroethidium 。几乎所有可用荧光探针用于评估ΔΨm或ROS是单一波长的指标，增加或减少其荧光强度成正比，增加或减少ΔΨm或ROS水平的刺激。因此，当务之急是在基准水平和应用特定的刺激后，这些探针的荧光强度来衡量。这允许一个确定的基线水平和刺激之间的荧光强度的变化的百分比。这种荧光强度的变化反映在ΔΨm或ROS相对水平的变化。在这段视频中，我们展示了如何应用在大鼠皮层神经元的荧光指标，TMRM，以确定在TMRM之间的基线水平和应用FCCP，线粒体解偶联剂后的荧光强度的百分比变化。 FCCP治疗造成下级TMRM荧光反映线粒体膜电位去极化。我们还展示了如何应用的荧光探针 H 2 DCF - DA的，以评估在皮层神经元的ROS水平，首先在基线和应用H 2 O 2后，然后。此协议（稍作修改），也可以用来确定在ΔΨm和ROS的变化，在不同类型的细胞和神经元在大脑的其他区域隔离。

Watch this Scientific Journal Video about Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons at JoVE.com

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

我们证明在皮层神经元的应用荧光指标，TMRM TMRM荧光强度的相对变化，以确定应用程序之前和之后的一个特定的刺激。我们也应用荧光探针H<sub&gt; 2</sub&gt; DCF - DA评估皮层神经元的活性氧物种的相对水平。

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 ...

determination-mitochondrial-membrane-potential-reactive-oxygen

Research

JoVE Journal

Neuroscience

64.7K 次观看.  Loyola University Chicago. 我们证明在皮层神经元的应用荧光指标，TMRM TMRM荧光强度的相对变化，以确定应用程序之前和之后的一个特定的刺激。我们也应用荧光探针H 2 DCF - DA评估皮层神经元的活性氧物种的相对水平。

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

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

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

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 ...

科研

神经科学

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

刺激特定的皮质视觉引发潜在形态模式

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...