作者要感谢毕业后的学者计划办公室和诺华医学研究所作为一个整体为这项研究提供资金。

1. 细胞的制备
注意: 这个协议, 包括数据生成, 使用 HEK293 细胞转基因与杆状病毒编码 NR1 和 NR2A 细胞。
<ol><li>种子适当数量的 HEK293 细胞, 并添加 NR1 和/或 NR2A 病毒在适当的最终浓度 (1.00 µL 每个)。对于一个384井板, 使用1万细胞/以及在最后的体积30µL。</li>
	<li>或者, 种子 HEK293-NR1-NR2A 细胞在适当的细胞数量和容量 (为384井板材, 使用1万个细胞/井在最后容量30µL)。添加四环素在2µg/毫升的浓度, 以诱导 NR2A 表达, 并添加保护化合物 (100 µM MDL105,519 或5µM CGP07066724)。</li>
	<li>注意: 杆状病毒编码 NR1 和 NR2 应该有一个近似的效价之间的 5 x 109 -10 x 109感染单位每毫升24。</li>
</ol>2. NMDA 受体活性的测定
<ol><li>在任何保护性化合物的存在下, 用 HEK293 细胞转基因与 NR1/NR2 或 HEK293-NR1-NR2A 细胞一起制备384井板。使用透明底, 黑色框架, 聚 d-赖氨酸涂层板。 注: 保护与100µM MDL105,519 是用来赋予对甘氨酸的敏感性, 而5µM CGP070667 用于赋予谷氨酸的敏感性。</li>
	<li>在37摄氏度和 5% CO2上孵育盘子16小时 (隔夜)。</li>
	<li>将盘子从孵化器中取出, 并通过减慢翻转板在兼容的 biowaste 容器上轻轻地丢弃细胞介质。在纸巾上轻轻地敲击盘子, 将介质从盘子边缘清除, 并排出剩余的介质。</li>
	<li>在10毫升的孵化缓冲器 (HBSS pH 7.5、20 mm HEPES、1 mm 氯化镁2、1 mm 丙磺舒) 中, 将一小瓶 calcium6 染料 (1 片尺寸) calcium6-dye。在37摄氏度和 5% CO2上孵化2小时的板材。</li>
	<li>从孵化器中取出盘子, 让细胞调整到室温10分钟。</li>
	<li>轻轻地倒出 calcium6-dye, 如步骤2.3 所述, 并添加30µL 的化验缓冲器 (HBSS pH 7.5, 20 毫米 HEPES, 1.8 毫米 CaCl2, 1 毫米丙磺舒)。</li>
	<li>重复步骤 2.6, 共3次洗涤。最后一次清洗后, 在细胞上留下30µL 的化验缓冲器。</li>
	<li>让细胞在室温下休息5分钟。</li>
	<li>制备配体板, 使4倍的库存400µM 甘氨酸和400µM 谷氨酸 (最终浓度100µM; 配位的最佳浓度应确定如下所述)。将至少25µL 的配体料转移到 V 底384井板中。</li>
	<li>将配体板和细胞板装入 FDSS (功能性药物筛选系统)。</li>
	<li>测量电池板的基线荧光 (Fo) 30 秒。用 FDSS 配药功能将配体的10µL 转移到细胞板中, 通过记录 calcium6-dye 荧光量5分钟来测量钙流量。</li>
	<li>通过将基线荧光 (f最大/fo) 获得的最大荧光划分, 计算最大荧光比。</li>
</ol>3. NMDAR 表达水平的优化
<ol><li>要确定使用的杆状病毒的最佳数量, 执行 NR1 和 NR2A 病毒的滴定。准备一个有 HEK293 细胞的384井板 (推荐1万个细胞/好, 30 µL 介质), 并包括步骤2.1 所述的保护化合物。</li>
	<li>在板块的不同行中添加 NR1 病毒的数量变化 (例如: 分别添加0µL、2µL、1µL、0.5 µL、0.25 µL)。为数据准确性添加重复项。</li>
	<li>在板块的不同列中添加不同数量的 NR2A 病毒 (例如: 分别添加0µL、2µL、1µL、0.5 µL、0.25 µL)。为数据准确性添加重复项。</li>
	<li>在37摄氏度和 5% CO2上孵育盘子16小时 (隔夜)。</li>
	<li>通过在 FDSS 上执行钙通量测量, 确定 NR1 和 NR2A 病毒的最佳比率和数量 (见上文)。</li>
</ol>4. 配体滴定法
<ol><li>按照步骤2.3 中的说明准备单元格。</li>
	<li>在其他配体的饱和浓度 (100 µM) 的存在下, 制备每个配体的稀释, 作为4倍的库存溶液。例如: 对于甘氨酸的10µM 最终测试浓度, 制备40µM 的甘氨酸溶液, 包括400µM 谷氨酸。</li>
	<li>按照步骤2.11 中的说明进行 FDSS 测量。</li>
</ol>5. 复合测试
<ol><li>按照步骤1中的说明准备单元格。</li>
	<li>在检测缓冲器中制备配体板, 其最终配体浓度为5倍。</li>
	<li>在检测缓冲器中, 用所需的最后浓度的4倍的库存准备化合物板。对于最终的化合物浓度为10µM 在化验缓冲器, 准备一个40µM 的库存解决方案。<ol>
<li>将二甲基亚砜 (亚砜) 常数的浓度保持在所有样品中, 方法是在亚砜中制备预稀释的化合物, 然后再稀释成测定缓冲器。</li>
		<li>对于化合物在3至30µM 的最终测定中的剂量反应, 在亚砜中制备化合物的预稀释, 从1到10毫米不等。稀释那些在化验缓冲器到4倍的存货浓度。亚砜的最终浓度不应超过0.5%。 注意: 建议在 triplicates 中测试每个样本。</li>
	</ol>
</li>
	<li>将配体、复合物和细胞板装入 FDSS。</li>
	<li>测量基线荧光30秒。</li>
	<li>添加10µL 的复合库存, 并测量荧光5分钟。</li>
	<li>添加10µL 的配体股票, 并测量荧光5分钟。</li>
	<li>在测量的最后5分钟内, 通过计算荧光比曲线下的面积来确定荧光比的积分。 注: 另外, 在配体添加后, 荧光的最大比值可用于分析。</li>
</ol>

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作者没有利益冲突, 也没有什么可透露的。

这种化验的成功主要取决于使用的 HEK 细胞的健康状况。需要使用指数增长和低通道数的细胞。这种分析涉及许多转移和补充的解决方案, 所以使用谨慎将确保更高的准确性, 其结果。化合物和所有其他试剂的浓度也应进行交叉检查, 以尽量减少错误。当用测定缓冲液取代细胞培养基进行钙通量测定时, 应轻轻倾斜板, 使细胞不分离。最后, 在本协议中使用聚 d-赖氨酸涂层板增加了细胞对板的附着。</...

随着目前医学的进步, 预期寿命显著增加;然而, 与年龄相关的疾病的流行率也是如此。中枢神经系统疾病 (CNS), 如精神分裂症, 肌萎缩侧索硬化症, 阿尔茨海默氏病, 帕金森病等, 也不例外, 并预计将增加在未来十年1,2,3. 被称为 n-甲基-d-天门冬氨酸受体 (NMDAR) 的离子型谷氨酸受体的故障与阿尔茨海默病、精神分裂症、外伤性脑损伤、中风、糖尿病和青光眼等有联系, 这就保证了需要研究他们的生物学为发展有效, 疾病修改疗法4,5,6,7。
NMDARs 由四单体或亚基4,8,9组成。NMDAR 的结构构成在脑子7,10显示发展和区域可变性。NMDARs 参与突触可塑性, 认知和节奏的世代为呼吸和运动11,12,13。作为电压门控通道, 它主要是在静止膜电位 (-70 mV) 不导电, 并被镁堵塞, 以防止离子进一步渗透。该通道是由两个配体, 谷氨酸和甘氨酸/d-丝的结合激活, 并在突触膜 AMPA 受体介导的, 另一子类离子型谷氨酸受体的同时去极化。退极化去除 NMDAR 镁堵塞, 使阳离子的汇集, 特别是钙14,15,16。尽管激活 NMDAR 是细胞存活的必要条件, 但过度活化可能导致细胞死亡17,18,19通过兴奋。这, 除了受体的复杂性质, 使其具有挑战性的研究开发有效的治疗所必需的。
对 NMDAR 的研究开发了不同的方法。然而, 每一个都有相应的警告。例如, 一种广泛使用的技术是一种基于荧光的检测方法, 用于测量在四环素诱导启动子 (NMDAR) 控制下稳定细胞系内细胞内钙的变化.然而, 在这个系统中, 所需配体的 supramaximal 浓度和 NMDAR 抑制剂在测量过程中存在的要求, 几乎不可能检测到竞争对手的活动。在其他类似的系统中, 功能受体的表达导致毒性, 要求通道阻滞剂, 如氯胺酮21,22保存细胞培养。这些通道阻滞剂坐在受体的核心, 很难洗出来, 尤其是以平板为基础的格式, 所以他们干扰的功能研究的受体。最后, 在电生理测量, 如补丁夹紧, 有有限的吞吐量, 和大规模的研究是非常昂贵的23。尽管如此, 上述系统对甘氨酸刺激不敏感;因此, 研究 NMDAR 的甘氨酸依赖性活动成为一项挑战。
在这里, 我们描述了一个新的方法来研究 NMDAR, 克服了讨论的局限性。我们的技术是利用杆状病毒表达系统来表达受体在功能水平上的最佳比例的亚基在16小时。此外, 杆状病毒的使用允许一种简单的组合方法, 它提供了一个广泛的描述不同的重组 NMDAR 亚型。与其他化验不同, 该协议不需要通道阻滞剂, 因为使用弱拮抗剂。该方法的最大优势是, 在弱拮抗剂的冲刷后, 受体对个体甘氨酸和谷氨酸结合部位的调制敏感, 除了甘氨酸/d-丝氨酰胺和谷氨酸配体结合的双重调制网站。该检测概括已知的 NMDAR 受体药理作用及其已知的阳性和阴性调节剂的影响。最后, 这种体外细胞检测的产生克服了过量钙流入引起的细胞毒性, 允许高通量的受体功能研究, 从而加速 NMDAR 调制器的发现。在疾病状态。

<table>
	<tbody>
		<tr>
			<td>HEK-293</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Human NMDA (NR1/NR2A) Receptor Cell Line</td>
			<td>ChanTest Corporation</td>
			<td>CT6120</td>
			<td></td>
		</tr>
		<tr>
			<td>pFastBac Dual Expression Vector</td>
			<td>ThermoFisher Scientific</td>
			<td>10712-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well Clear Flat Bottom Microplate</td>
			<td>Corning Life Sciences</td>
			<td>3844</td>
			<td></td>
		</tr>
		<tr>
			<td>FLIPR Calcium 6-QF Assay Kit</td>
			<td>Molecular Devices</td>
			<td>R8192</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>49621</td>
			<td></td>
		</tr>
		<tr>
			<td>D-serine</td>
			<td>Sigma-Aldrich</td>
			<td>S4250</td>
			<td></td>
		</tr>
		<tr>
			<td>L701,324</td>
			<td>Tocris</td>
			<td>907</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Boston Bio Product</td>
			<td>BB-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>E506</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher Scientific</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>ThermoFisher Scientific</td>
			<td>P36400</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX media</td>
			<td>ThermoFisher Scientific</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>MDL105,519</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>NVP-AAM077</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>CGP070667</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate

n-甲基-d-天门冬氨酸 (NMDA) 受体 (NMDAR) 被归类为离子型谷氨酸受体, 在学习和记忆中具有重要作用。NMDAR 的故障, 表现为过度或活动不足造成的突变, 改变表达, 贩运, 或本地化, 可能导致许多疾病, 特别是在中枢神经系统。因此, 了解受体的生物学以及促进发现化合物和小分子, 对于目前防治神经系统疾病的努力至关重要。目前研究该受体的方法有限制, 包括低吞吐量, 高成本, 并无法研究其功能能力, 由于必要存在的渠道阻滞剂, 以防止 NMDAR 介导的兴奋。此外, 现有的检测系统只对谷氨酸的刺激很敏感, 对 NMDAR 的另一种共配体甘氨酸的刺激缺乏敏感性。在这里, 我们提出了第一个以板块为基础的高通量的检测方法, 研究一种对同配体、谷氨酸和 d-丝氨酸/甘氨酸有敏感性的 NMDA 受体。这种方法允许研究不同的 NMDAR 亚基组成, 并允许功能研究的受体在甘氨酸和/或谷氨酸敏感模式。此外, 该方法不需要在测量过程中存在抑制剂。通过本试验可以检测到正、负构调节剂的作用, 并在本系统中复制了 NMDAR 的已知药理学。该技术克服了现有方法的局限性, 具有很高的成本效益。我们相信, 这项新的技术将加快发现治疗 NMDAR 介导的病理。

在测试小分子的影响之前, 你必须确定 NMDARs 的最佳表达水平以及最佳配体浓度。如所述, 在384井板中, HEK293 细胞每井1万个细胞, 在5µM CGP060667, 然后转基因的 NR1 和 NR2A 病毒数量不等。在一夜之间孵化后, 配体诱导的钙通量被测量 (图 1)。这些实验的结果显示了每一个亚基使用的病毒的最佳数量和比率 (图 1, 黄色盒)。
<p class="jo...

Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

本议定书的目的是促进对 NMDA 受体 (NMDAR) 的研究, 使之更大规模地进行, 并允许检查小分子的调节作用及其治疗应用。

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

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Research

JoVE Journal

Neuroscience

9.1K 次观看.  Novartis Institutes for BioMedical Research. 本议定书的目的是促进对 NMDA 受体 (NMDAR) 的研究, 使之更大规模地进行, 并允许检查小分子的调节作用及其治疗应用。

视频: A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

科研

神经科学

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

光偏好的检测，以研究的先天和昼夜调节感光特性<em&gt;果蝇</em&gt;幼虫

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

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

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

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 ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

高通量检测，以检查个体产蛋首<em&gt;果蝇</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

Correlative Light and Electron Microscopy to Study Microglial Interactions with &amp;#946;-Amyloid Plaques

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).