Name: monitoring neuronal survival via longitudinal fluorescence microscopy
Uploaded: 2019-01-19T15:00:00
Description: Watch this Scientific Journal Video about Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy at JoVE.com

我们感谢 steve finkbeiner 和 finkbeiner 实验室的成员, 感谢他们的开创性机器人显微镜。我们还感谢 dan peisach 构建了图像处理和自动生存分析所需的初始软件。这项工作是由国家神经疾病和中风研究所 (ninsds) r01-ns097542, 密歇根大学蛋白质折叠疾病倡议, 安阿伯积极反对 als 资助。

密歇根大学动物使用和护理委员会批准了所有脊椎动物工作 (pro00007096 号议定书)。实验是经过精心策划的, 以尽量减少牺牲的动物数量。怀孕的女性野生类型 (wt), 非转基因长埃文斯大鼠 (北拉图斯) 单独被安置在配备了环境丰富的房间, 并由密歇根大学实验动物医学 (ulam) 单位 (ulam) 照顾,根据 nih 支持的实验动物护理和使用指南。根据美国兽医协会和密歇根大学安乐死方法的《安乐死准则》的...

<ol>
	<li>Lockshin, R. A., Zakeri, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+in+health+and+disease.">Cell death in health and disease.</a> Journal of Cellular and Molecular Medicine. 11, 1214-1224 (2007).</li><li>Kepp, O., Galluzzi, L., Lipinski, M., Yuan, J., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+assays+for+drug+discovery.">Cell death assays for drug discovery.</a> Nature Reviews Drug Discovery. 10, 221-237 (2011).</li><li>Lemasters, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+permeability+transition+in+cell+death:+a+common+mechanism+in+necrosis,+apoptosis+and+autophagy.">The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy.</a> Biochimica et Biophysica Acta Bioenergetics. , 177-196 (1998).</li><li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labelled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.</a> Journal of Immunological Methods. 184, 39-51 (1995).</li><li>Chien, K. R., Abrams, J., Serroni, A., Martin, J. T., Farber, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+phospholipid+degradation+and+associated+membrane+dysfunction+in+irreversible,+ischemic+liver+cell+injury.">Accelerated phospholipid degradation and associated membrane dysfunction in irreversible, ischemic liver cell injury.</a> Journal of Biological Chemistry. 253, 4809-4817 (1978).</li><li>Zwaal, R. F. A., Comfurius, P., Bevers, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+exposure+of+phosphatidylserine+in+pathological+cells.">Surface exposure of phosphatidylserine in pathological cells.</a> Cell and Molecular Life Sciences. 62, 971-988 (2005).</li><li>Mitchell, D. B., Santone, K. S., Acosta, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cytotoxicity+in+cultured+cells+by+enzyme+leakage.">Evaluation of cytotoxicity in cultured cells by enzyme leakage.</a> Journal of Tissue Culture Methods. 6, 113-116 (1980).</li><li>Moran, J. H., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+beta-NADH-linked+fluorescence+assay+for+lactate+dehydrogenase+in+cellular+death.">A rapid beta-NADH-linked fluorescence assay for lactate dehydrogenase in cellular death.</a> Journal of Pharmacological and Toxicological Methods. 36, 41-44 (1996).</li><li>Mossman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cellular+growth+and+survival:+Application+to+proliferation+and+cytotoxicity+assays.">Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.</a> Journal of Immunological Methods. 65 (1-2), 55-63 (1983).</li><li>Guerzoni, L. P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Modulation+of+TrkB+Receptor+Signaling+upon+Sequential+Delivery+of+Curcumin-DHA+Loaded+Carriers+Towards+Promoting+Neuronal+Survival.">In Vitro Modulation of TrkB Receptor Signaling upon Sequential Delivery of Curcumin-DHA Loaded Carriers Towards Promoting Neuronal Survival.</a> Pharmaceutical Research. 34 (2), 492-505 (2017).</li><li>Arrasate, M., Finkbeiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+microscope+system+for+determining+factors+that+predict+neuronal+fate.">Automated microscope system for determining factors that predict neuronal fate.</a> Proceedings of the National Academy of Science of the United States of America. 102, 3840-3845 (2005).</li><li>Christensen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivariate+survival+analysis+using+Cox&#8217;s+regression+model.">Multivariate survival analysis using Cox&#8217;s regression model.</a> Hepatology. 7, 1346-1358 (1987).</li><li>Barmada, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+induction+enhances+TDP43+turnover+and+survival+in+neuronal+ALS+models.">Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models.</a> Nature Chemical Biology. 10, 677-685 (2014).</li><li>Barmada, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+toxicity+in+neuronal+models+of+amyotrophic+lateral+sclerosis+by+hUPF1.">Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1.</a> Proceedings of the National Academy of Science of the United States of America. 112, 7821-7826 (2015).</li><li>Malik, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrin+3-dependent+neurotoxicity+is+modified+by+nucleic+acid+binding+and+nucleocytoplasmic+localization.">Matrin 3-dependent neurotoxicity is modified by nucleic acid binding and nucleocytoplasmic localization.</a> Elife. 7, (2018).</li><li>Archbold, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP43+nuclear+export+and+neurodegeneration+in+models+of+amyotrophic+lateral+sclerosis+and+frontotemporal+dementia.">TDP43 nuclear export and neurodegeneration in models of amyotrophic lateral sclerosis and frontotemporal dementia.</a> Scientific Reports. 8, 4606 (2018).</li><li>Green, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAN+translation+at+C9orf72-associated+repeat+expansions+is+selectively+enhanced+by+the+integrated+stress+response.">RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response.</a> Nature Communications. 8, 2005 (2017).</li><li>Park, S. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+essential+Sis1+chaperone+reduces+TDP-43+effects+on+toxicity+and+proteolysis.">Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis.</a> PLOS Genetics. 13, e1006805 (2017).</li><li>Gupta, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Proline/Arginine+Dipeptide+from+Hexanucleotide+Repeat+Expanded+C9ORF72+Inhibits+the+Proteasome.">The Proline/Arginine Dipeptide from Hexanucleotide Repeat Expanded C9ORF72 Inhibits the Proteasome.</a> eNeuro. 4, (2017).</li><li>Angelov, B., Angelova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+clustering+of+the+neurotrophin+receptor+TrkB+revealed+by+super-resolution+STED+microscopy.">Nanoscale clustering of the neurotrophin receptor TrkB revealed by super-resolution STED microscopy.</a> Nanoscale. 9 (28), 9797-9804 (2017).</li></ol>

本文提出了在单细胞基础上直接监测神经元存活的方法。与传统的细胞死亡检测仅限于离散时间点和整个细胞群相比, 这种方法允许对多种因素进行持续评估, 如细胞形态、蛋白质表达或定位, 以及可以确定每个因素如何以一种前瞻性的方式影响细胞的存活。
这种方法可以修改, 以适应广泛的实验需求。成像的频率和持续时间可以很容易地调整, 任何感兴趣的蛋白质都可以与荧?...

异常细胞死亡是许多疾病的驱动因素, 包括癌症、神经退行性变和中风1。对细胞死亡进行可靠和敏感的检测对于这些疾病的描述以及扩大或降低细胞存活率的治疗策略的发展至关重要。目前有几十种技术可以直接或通过代理标记2测量细胞死亡。例如, 可以通过选择性染色死细胞或活细胞3的重要染料, 或通过监测血浆膜 4,5, 6上特定磷脂的外观, 从视觉上评估细胞死亡情况..细胞内成分或细胞代谢物在细胞溶解时释放到培养基中的测量也可用作细胞死亡的代名词 7,8。或者, 细胞活力可以通过评估代谢活动9,10近似。尽管这些方法提供了评估细胞存活的快速方法, 但它们并非没有警告。每种技术都将培养作为一个单一的群体来观察, 因此不可能区分单个细胞及其独特的存活率。此外, 这种基于人群的检测无法衡量可能对细胞死亡很重要的因素, 包括细胞形态、蛋白质表达或定位。在许多情况下, 这些检测仅限于离散的时间点, 不允许随着时间的推移对细胞进行连续观察。
相反, 纵向荧光显微镜是一种高度灵活的方法, 可在单细胞基础上直接和持续地监测死亡风险11。简单地说, 纵向荧光显微镜可以对数千个单个细胞进行长时间的跟踪, 从而精确地确定细胞死亡和增强或抑制细胞死亡的因素。在其基础上, 该方法涉及瞬态转染或转导细胞与矢量编码荧光蛋白。然后建立一个独特的信托, 每个转染细胞相对于这个地标的位置允许用户在几个小时、几天或几周的时间里对单个细胞进行成像和跟踪。当按顺序查看这些图像时, 细胞死亡的特征变化是细胞体的荧光、形态和碎片, 从而能够为每个细胞分配死亡时间。计算出的死亡率, 由危险函数确定, 然后可以定量比较条件之间, 或有关选择细胞特征使用单变量或多变量 cox 比例危害分析12。这些方法结合在一起, 可以准确和客观地识别细胞种群中的细胞死亡率, 并识别显著预测细胞死亡和存活的变量 (图 1)。
虽然这种方法可用于监测任何有丝分裂后细胞类型的存活在各种电镀格式, 该协议将描述条件转染和成像大鼠皮质神经元培养在96孔板。

<table>
	<tbody>
		<tr>
			<td>Neurobasal Medium</td>
			<td>GIBCO</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>CompactPrep Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12863</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride Hexahydrate</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Kynurenic Acid Hydrate</td>
			<td>TCI</td>
			<td>H0303</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly D-Lysine</td>
			<td>Millipore</td>
			<td>A-003-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>52887</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Fisher</td>
			<td>A3582801</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>GIBCO</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>TPP</td>
			<td>0876</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring neuronal survival via longitudinal fluorescence microscopy

标准的细胞毒性检测, 需要收集裂解液或固定细胞在多个时间点, 有有限的敏感性和能力来评估影响神经元的命运因素。这些检测需要观察离散时间点的单独细胞群。因此, 随着时间的推移, 单个细胞不能前瞻性地跟踪, 严重限制了区分亚细胞事件 (如点状形成或蛋白质定位不当) 是否为疾病的致病性驱动因素、稳态反应的能力,或仅仅是巧合的现象。单细胞纵向显微镜克服了这些限制, 使研究人员能够确定种群之间的生存差异, 并以更高的灵敏度得出因果关系。本视频指南将概述一个具有代表性的工作流程, 用于测量表达荧光蛋白标记的大鼠原代皮质神经元单细胞存活情况的实验。观众将学习如何实现高效的转化, 收集和处理图像, 使单个细胞的前瞻性跟踪, 并使用 cox 比例危害分析比较神经元群的相对生存。

利用本文所述的转染过程, div4 大鼠皮质神经元被编码为荧光蛋白 mapple 的质粒转染。从转染后24小时开始, 细胞连续10天每24小时用荧光显微镜成像。生成的图像如图 2所示进行了组织, 然后对细胞死亡进行拼接、堆叠和评分 (图 1)。图 3显示了3个有代表性神经元的时间过程, 其中两个神经元在实验过程中死亡...

Watch this Scientific Journal Video about Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy at JoVE.com

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy

在这里, 我们提出了一个协议, 在单细胞的基础上监测生存情况, 并确定显著预测细胞死亡的变量。

通过纵向荧光显微镜监测新生儿生存

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to ...

该协议使研究人员能够监测单细胞的存活率，并确定显著预测细胞死亡或存活的变量。传统的细胞毒性测定评估细胞群，缺乏区分单个细胞的能力。纵向显微镜允许为种群中每个细胞分配死亡时间。该技术非常适合评估神经退行性疾病和其他疾病的新疗法。这种方法已被证明对探索神经退行性疾病中的疾病机制无价，并可以同样有效地应用于其他细胞死亡感兴趣的疾病。研究人员可能难以达到足够的转染效率，而不会产生不适当的毒性。使用多通道移液器并熟悉协议，随着时间的推移应减少这些困难。显微镜是一种固有的视觉方法。因此，除了阅读之外，通过观看可能更容易理解这种技术。首先，将适当数量的减少血清介质和转染试剂组合在一个管中，并在单独的管中减少血清介质和DNA。在室温下孵育五分钟。然后，将两管的内装物混合，在室温下孵育20分钟。使用多通道移液器和消毒塑料槽两次洗皮质神经元，每井用100微升神经基底介质清洗。将有条件介质和其他剩余介质储存在 37 摄氏度。每井更换 100 微升以前准备的 NBKY 溶液的 NBM。20分钟后，移液50微升的转染试剂和DNA混合物滴入每个井。在37摄氏度下孵育细胞20分钟。使用 NBKY 溶液冲洗细胞两次。然后添加100微升的有条件介质和100微升的NBC解决方案到井。要开始对细胞进行成像，请将96井板放在荧光显微镜上。使用每个板唯一的受托标记作为未来对齐的参考，并保存图像供将来参考。然后，导航到感兴趣区域并记录相对于标记的 x 和 y 坐标。使用每个板唯一的受托标记作为未来对齐的参考，并保存图像供将来参考。最后，关注表达荧光标签的转染细胞。在适当的荧光通道中以定期间隔拍摄多个图像。要从图像处理开始，请双击斐济图标。然后，单击图像下划线处理宏到斐济栏上以打开斐济内的宏。根据文本协议调整图像下划线处理宏的 2 行到 7 行。然后，单击拼接，然后网格/集合拼接。调整下拉菜单、类型和顺序中的设置，直到生成精确拼接的图像。根据这些选择调整网格类型和在第 8 行和第 9 行中缝合顺序变量。然后，调整第 10 行以指定每井的图像数，如有必要，将第 14 行的背景减法选项设置为 true。调整第 15 行以根据文本协议设置滚动球半径，然后单击"运行"。最后，打开输出图像堆栈，根据文本协议手动识别死神经元。将数据记录在电子表格文件中。要执行统计分析，请双击图标以求生存。R 脚本。突出显示第二行并单击运行按钮以加载生存库。然后，更改第 5 行中的路径以调用输入电子表格，然后单击运行按钮。突出显示第 8 行和第 9 行，然后单击运行按钮以执行 Cox 比例危险分析。突出显示第 12 行到 16 行和 19 行到 24 行。单击运行按钮，将累积死亡风险和生存数据绘制为卡普兰-迈尔曲线。在这项研究中，大鼠皮质神经元在电镀后四天内被转染，质粒编码为荧光蛋白M苹果。转染后24小时，神经元连续10天每24小时用荧光显微镜成像。在图像采集后，图像得到处理，并按顺序检查，以标记细胞死亡。每个细胞的死亡时间由细胞体荧光、形态和碎片的变化决定。对突变神经元的生存数据进行考克斯比例危害分析，导致危险比为2.2。这一结果表明，与野生型种群相比，突变神经元的死亡率更快。尝试此过程时，要记住的最重要的事情是小心移液。最大限度地减少空气暴露和避免气泡对于成功转染至关重要。转染后，除了细胞存活率外，纵向荧光显微镜还可用于跟踪可能导致细胞死亡的各种因素，包括蛋白质定位、周转和表达水平。在神经退化领域，纵向显微镜的动态性质揭示了疾病相关蛋白质的聚集是否代表有毒或保护性事件。这些试剂或仪器均无危险。

Research

JoVE Journal

Neuroscience

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

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

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

科研

神经科学

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

神经肌肉接头处：测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

器官细胞生存在器官细胞切片培养

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy

使用动脉内血压监测和通气通过气管切除术的小鼠脑死亡诱导

While both living donation and donation after circulatory death provide alternative opportunities for organ transplantation, donation after donor brain ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

在行为和刺激反应期间为整个大脑成像准备成人德罗索菲拉褪黑麦片加斯特

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...