这项工作是支持由寿险公司“AG2R - LA - MONDIALE”，巴黎高等DES神经科学（ENP），法新社国立德拉Recherche“的ANR - 09 - NEUR - 004”框架“的ERA - NET神经“第七框​​架计划由欧盟委员会和巴斯德基金会。塞巴斯蒂安瓦格纳Letten基金会的支持。

1。光学校正：测量LED电源<ol><li>将一个LED阵列积极冷却风扇，并加盖该LED /散热器aparatus一个准直透镜的散热片。 </li><li>在明照明用LED /散热片/风扇/镜头仪器的更换灯泡。本设备必须仔细定位，使平行的LED光束对聚光镜的光沿直线路径传播。确保散热片/风扇是正确的接地系统的共同点。 </li><li>电源，可以给当前的快速和方波驱动LED阵列。该电源可控制脉冲发生器产生一个5V TTL脉冲。 </li><li>中心沿之间的视场光阑和冷凝器的镜头定义光路的准直光束。理想的情况下，LED光束会稍微过量的全面开放的视场光阑。通常情况下，更紧密平行的LED光束会少填补这个光圈，会产生铁道部E电源牺牲的统一性。在我们的设置中，我们增加了选择准直透镜，预计其在consenser膜片的共轭平面略有扩大图像的LED阵列光均匀。 </li><li>实现由注重冷凝器，使视场光阑（最靠近光源的隔膜）的形象是集中对片室（ 图1）科勒照明。一个镜头纸薄组织可以作为投影屏幕，可视化集中在其他深度的视场光阑的图像。 </li><li>钻一系列已知直径在一个不透明的材料的针孔。放置的光学功率计传感器，这些小针孔。将功率计对试样阶段，中心的电能表集中的视场光阑，通过简单的移动功率计，直到它提供了最大读数的形象。张贴在这个位置上的功率计。 </li><li>完全打开光圈（aperature隔膜和视场光阑）。系统地将附片室/功率计，相对光学光路计算照射范围内的光功率的均匀性。构建你的系统的统一性情节。如果在显微镜正确设置与科勒照明目标的重点为中心，最大功率应直接下的目标，这一重点之外的地区，现在应该收到一个已知金额按均匀情节权力。 </li><li>对于每一个针孔大小，建立标准曲线的光功率与针孔表面积。通过调整输入电流LED阵列，产生这条曲线在多种功率级别，从每条曲线，计算出每平方毫米的电源。如果是用于修补光学LED阵列，确保引进修补必要的光学元件（冷凝器，针孔和过滤器）知道多少光线下修补照明传输。 </li><li>翻转功率计面对目标，在470nm的光照强度计算汞灯开启时。 </li><li>添加一个活切片室，重建标准曲线，以确定通过散射脑组织传输的光功率。 </li></ol> 2。程序切片和电 A部分：切片制备<ol><li>麻醉（60毫克/公斤氯氨酮和甲苯噻嗪剂2mg/kg）和杀头的鼠标。解剖脑人工脑脊髓液（学联，MM：124 1.3 3氯化钾，氯化钠， 硫酸镁 ，26碳酸氢钠，1.25 NaHPO 4，20葡萄糖，氯化钙2〜310mOsm，pH值7.4时的混合物起泡95％O2和5％CO 2 5,1），注意不要损坏嗅球。琼脂分开的两个半球和地方，甚至一个边缘（水平段）的腹面。 </li><li>胶琼脂和每个皮质半球的背水面的vibratome夹头，慢慢填补，用冰冷学联的洗澡。腹面切片在300微米的部分，传输每个加热（34-36 ° C）节和含氧学联，使他们能够恢复30-45分钟。 </li></ol> B部分：松散的门槛补丁测量秒杀<ol start="3"><li>带来切片室温30分钟后，轻轻放置在显微镜下不断灌注含氧学联室录音室中分得一杯羹。 EYFP - ChR2存在过分刺激ChR2感染神经元的风险，可以确认下epifluorescence（图2A） </li><li>拉吸管拉马（萨特的P - 97）玻璃电极。该电极与学联填充。当学联浴尖阻力应介于7-10兆欧。 </li><li>在荧光灯照明，定位片与成熟形态健康ChR2 EYFP的神经元。同时定位下神经元的修补光学SOMA。 </li><li>随着光积极通过电极尖端的压力，降低对确定的荧光神经元的补丁电极。膜接触时，迅速释放出积极的压力，并申请通过尖端的吸量小而短暂的。一个千兆欧姆密封应质膜和补丁电极的墙壁之间。 </li><li> GIGA密封即使没有形成，如果电极是足够接近一个荧光的神经元扣球活动应该产生一个可衡量的的局部领域的潜力。不同剂量的光闪烁激活ChR2在这个神经元。由于光剂量是两个LED电源和持续时间的函数，计算光线的多少，是必要的，以唤起动作电位在多个权力和持续时间（图2b）。此外观察扣球汞灯照明下发生多少。 </li></ol> 3。代表性的成果： 在我们的显微镜（奥林巴斯BX51WI），我们的LED是我N线与2孔和一个聚光镜，从而保持了原有工厂安装的弧光灯的光路。通过关闭两个视场光阑和aperature隔膜，我们可以实现明对比足够的膜片钳记录（ 图1b）。所有隔膜完全打开，我们揭露切片channelrhodopsin激活（ 图1A）的最大光功率。在我们的显微镜，这个补丁配置产生的光密度约三个数量级，比最高的满场密度较低（4.1μW/ 2毫米与6.88毫瓦/平方毫米）。 一个成人出生的单ChR2 EYFP的表达粒细胞表明，我们看到强大的迁移在延髓的迁徙流（图2a）一个宽松的补丁录音神经母细胞的慢病毒感染后的标签成人出生的嗅球颗粒和periglomerular神经元周5毫秒的刺激在格言UM功率（6.88毫瓦/平方毫米 ）足以唤起扣球（ 图2b）。由于细胞之间的表达水平的变化，通过的门槛秒杀的光量会有所不同，应为每个感兴趣的细胞类型的统计学描述。 <img src="/files/ftp_upload/3379/3379fig1.jpg" alt="图1" /> 图1。满场photostimulation和膜片钳切片电LED阵列设置。要激活channelrhodopsin（ChR2），我们通过打开背面的光圈和冷凝器光学（一）项目一个准直光束。此配置可完全关闭的视场光阑和调节视场光阑的宽度（b）改为高对比度的修补光学。缩写：A.，B.散热器风扇，C. LED阵列，D.准直透镜，E.镜，F.场光阑，G.光圈，H.聚光镜， 一 SAmple阶段，J.目标。 <img src="/files/ftp_upload/3379/3379fig2.jpg" alt="图2" /> 图2。膜片钳和整场photostimulation嗅球（一）一个300μm的水平切片图像。可以看出，从嗅球的核心辐射Lentivirally感染的成人出生的颗粒细胞表达ChR2 - EYFP。光剂量需要引起扣球可以通过增加LED闪光灯的持续时间（二）发现。这粒细胞的阈值是5ms的完整的LED亮度（2.43mW/mm 2 ）。 （一）规模= 500微米，（B）= 50ms的规模。

<ol>
	<li>Nissant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+promotes+synaptic+plasticity+in+the+olfactory+bulb.">Adult neurogenesis promotes synaptic plasticity in the olfactory bulb.</a> Nature Neuroscience. 12, 728-730 (2009).</li><li>Apicella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+Cells+in+Piriform+Cortex+Receive+Convergent+Input+from+Distinct+Olfactory+Bulb+Glomeruli.">Pyramidal Cells in Piriform Cortex Receive Convergent Input from Distinct Olfactory Bulb Glomeruli.</a> Journal of Neuroscience. 30, 14255-14260 (2010).</li><li>Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=genetically+targeted+optical+control+of+neural+activity.">genetically targeted optical control of neural activity.</a> Nature. 8, 1263-1263 (2005).</li><li>Bardy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=where+new+inhibitory+neurons+release+neurotransmitters+in+the+adult+olfactory+bulb.">where new inhibitory neurons release neurotransmitters in the adult olfactory bulb.</a> The Journal of Neuroscience. 30, 17023-17034 (2010).</li><li>Grubb, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+of+the+first+synapse+in+olfaction:+development+and+adult+neurogenesis.">Functional maturation of the first synapse in olfaction: development and adult neurogenesis.</a> The Journal of neuroscience. 28, 2919-2932 (2008).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit-breakers:+optical+technologies+for+probing+neural+signals+and+systems.">Circuit-breakers: optical technologies for probing neural signals and systems.</a> Nature reviews. Neuroscience. 8, 577-581 (2007).</li><li>Grossman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-site+optical+excitation+using+ChR2+and+micro-LED+array.">Multi-site optical excitation using ChR2 and micro-LED array.</a> Journal of neural engineering. 7, 16004-16004 (2010).</li><li>Dhawale, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-redundant+odor+coding+by+sister+mitral+cells+revealed+by+light+addressable+glomeruli+in+the+mouse.">Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse.</a> Nature neuroscience. 13, 1404-1412 (2010).</li><li>Weick, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+control+of+transplantable+human+ESC-derived+neurons+via+optogenetic+targeting.">Functional control of transplantable human ESC-derived neurons via optogenetic targeting.</a> Stem cells. 28, 2008-2016 (2010).</li><li>Toni, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+born+in+the+adult+dentate+gyrus+form+functional+synapses+with+target+cells.">Neurons born in the adult dentate gyrus form functional synapses with target cells.</a> Nature Neuroscience. 11, 901-907 (2008).</li><li>Grubb, M. S., Burrone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2+Localised+to+the+Axon+Initial+Segment.">Channelrhodopsin-2 Localised to the Axon Initial Segment.</a> PLoS ONE. 5, e13761-e13761 .</li><li>Tye, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amygdala+circuitry+mediating+reversible+and+bidirectional+control+of+anxiety.">Amygdala circuitry mediating reversible and bidirectional control of anxiety.</a> Nature. 471, 358-362 (2011).</li></ol>

没有利益冲突的声明。

近年来，人们在神经科学的研究6 optogenetic工具的普及爆炸。因此，它正变得越来越重要，希望开始使用这些新工具的实验室，以降低进入障碍。在这里，我们描述了如何进行一个简单和低成本的改造和传统的膜片钳钻机的校准，所以它可以做满场channelrhodopsin表达神经元光刺激。特别是，我们嗅球成年神经发生专门控制，新出生的神经元的研究应用此技术。 我们演示?...

<table border="1">
	<tbody>
		<tr>
			<td>Material</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue No.</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>100 mg/ml</td>
			<td>Imalg&egrave;ne 1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>2%</td>
			<td>Rompun</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbps;</td>
			<td>Sigma </td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>P5405</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>M1880</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5761</td>
		</tr>
		<tr>
			<td>NaHPO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5011</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>G7021</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>C7902</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>A9539</td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>P-97</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>1.5 mm O.D./1.17 mm I.D.</td>
			<td>Harvard Apparatus</td>
			<td>GC150T-10</td>
		</tr>
		<tr>
			<td>LED array</td>
			<td>&nbsp;</td>
			<td>Bridgelux</td>
			<td>BXRA-C2000</td>
		</tr>
		<tr>
			<td>Collimating lens</td>
			<td>40 mm beam diameter</td>
			<td>Thor Labs</td>
			<td>LEDC1</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>2.8 amp</td>
			<td>A1W Electronik</td>
			<td>HKO2800</td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>&nbsp;</td>
			<td>Thor Labs</td>
			<td>PM 100</td>
		</tr>
		<tr>
			<td>Heatsink </td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Fan</td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>&nbsp;</td>
			<td>Leica</td>
			<td>VT1200S</td>
		</tr>
	</tbody>
</table>

using affordable led arrays for photo-stimulation of neurons

标准片电已使研究人员能够探测单个细胞的电反应记录在电器或药理操纵 1,2神经电路的各个组成部分。随着发明的方法，以光学控制基因有针对性的神经元（光遗传学），研究人员现在有一个前所未有的水平超过特定群体的神经元控制在标准片的准备。特别是，感光channelrhodopsin - 2（ChR2），使研究人员能够激活神经元，光3,4。通过结合基于LED的标准切片电ChR2 photostimulation小心校准，我们能够更详细地探测成人出生interneurons的嗅球，嗅觉系统的第一个中央继电器的作用。使用病毒表达ChR2 - YFP专门在成人出生的神经元，我们可以选择性地控制在旧环境的年轻成年出生的神经元ð成熟的神经元。我们的光控，使用一个简单而廉价的LED系统，我们展示了如何该系统可以进行校准，以了解需要多少光线唤起扣球在单个神经元的活动。因此，简短的闪烁的蓝灯可以远程控制ChR2转新生细胞的放电模式。

Watch this Scientific Journal Video about Using Affordable LED Arrays for Photo-Stimulation of Neurons at JoVE.com

Using Affordable LED Arrays for Photo-Stimulation of Neurons

成人出生的神经元表达ChR2可以操纵片电生理的准备，以考察他们对嗅觉神经回路的功能的贡献。

使用光刺激神经元的负担得起的LED阵列

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

using-affordable-led-arrays-for-photo-stimulation-of-neurons

Research

JoVE Journal

Neuroscience

18.5K 次观看.  Institut Pasteur and Centre National de la Recherche Scientifique (CNRS). 成人出生的神经元表达ChR2可以操纵片电生理的准备，以考察他们对嗅觉神经回路的功能的贡献。

视频: Using Affordable LED Arrays for Photo-Stimulation of Neurons

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

使用电动和一维和二维文化磁场神经元的外部激励

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

利用生命病毒介导的人神经元转导研究头塔聚集的体外模型

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

多柔性聚合物电极阵列的慢性植入

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).