This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

本文所述的所有实验均按照英国动物（科学程序）1986年法进行的。 1.准备仪器，文化传媒，和菜名<ol><li>准备两个不锈钢实验室解剖剪刀和两个不锈钢镊子实验室。高压灭菌器的所有仪器和地方文化引擎盖O / N在紫外光（UV）光灭菌。 </li><li>使500毫升最小必需培养基（MEM）培养基（10％胎牛血清[FBS]，20mM的氯化钾，25mM的碳酸氢钠 ，30mM的D-葡萄糖，2mM的L-谷氨酰胺，100U / ml青霉素，100 100μg/ ml链霉素和6微克/毫升氨苄青霉素），并过滤杀菌。储存在4℃数月。 </li><li>准备包含14个毫米，1号盖玻片厚度24孔培养板。高压灭菌的盖玻片和外衣在100毫克/升聚d-赖氨酸（PDL）如前所述3。紫外线消毒O / N。 </li></ol> 2.准备CGC培养液<ol><li>制备“溶液A”（100毫升）的高压灭菌，处理过的UV光，无菌的dh 2 O含有250mg葡萄糖，300毫克牛血清白蛋白[BSA]的，955毫克磷酸盐缓冲盐水[PBS]（ 钙离子和毫克+游离）和1毫升3.8％ 硫酸镁 ·7H 2 O. </li><li>制备含1毫升5毫克/毫升胰蛋白酶稀释于9ml溶液A的&#39;溶液B&#39;（10毫升）中</li><li>准备“溶液C”（10毫升）含1000单位的DNA酶，0.5毫克的大豆胰蛋白酶抑制剂，100微升3.8％硫酸镁4·7H 2 O，稀释至10毫升溶液A. </li><li>制备，含3.2 ml溶液的C，稀释至20ml用溶液A&#39;溶液D&#39;（20毫升）中</li><li>制备的BSA / Earle氏平衡盐溶液（EBSS）含有EBSS，25mM的碳酸氢钠 ，3mM的硫酸镁溶液·7H 2 O，和4％BSA，pH 7.4中，先前所描述的3。 </li><li>制备LME通过加入纯LME到5毫升的MEM培养基，得到150毫LME的终浓度，使用台式pH计返回溶液至pH 7.4，并过滤灭菌使用28mm的聚醚砜（PES）为0.2μm注射器过滤器。 </li></ol> 3.培养信用担保公司<ol><li>收集4-7日龄的幼鼠小脑如前所述3。含冰5毫升解决方案的培养皿立即到位。 </li><li>倒出从小脑和地方组织在培养皿盖过量的溶液。 </li><li>切碎组织，在至少三个不同方向的一个良好的火烧刀片。 </li><li>加入切碎的组织到溶液B（冲洗培养皿以溶液，以确保有限的组织丢失）并置于水浴中，在37℃下进行5分钟。轻轻摇动每两分钟。 </li><li>添加溶液D（20毫升）到管中以中和胰蛋白酶，抖动和占rifuge在65×g离心5分钟。 </li><li>倒掉上清。 </li><li>重悬在4毫升的溶液C.磨碎很好地减小直径，直到悬浮液是尽可能均匀的三个火烧玻璃吸管（约10每次）。 </li><li>慢慢地，轻轻地对BSA / EBSS顶部加几滴这种匀浆。如果它开始通过BSA / EBSS下沉，更磨碎和/或补充一点的解决方案C.将匀浆应该坐在一个层上的BSA / EBSS之上。不要摇晃。旋转，在100×g离心5分钟。 </li><li>除去上清，重悬沉淀（软）在1ml MEM培养基。 </li><li>计数使用血球和盘细胞为80万细胞/盖玻片在500μlMEM培养基。发生在一个培养箱中在37℃下用6％的CO 2。 </li><li>第二天（预习后24-36小时）改变介质。使含有10μM的细胞周期抑制剂阿糖呋喃胞苷（阿糖胞苷）的MEM培养基中的溶液。 <ol><li>对于每一个盖玻片，保留250微升旧培养基的新鲜24孔板，吸去休息，加入250微升正常的MEM培养基中，并吸出此以洗涤细胞，然后加入250μl的旧培养基的回细胞和顶部向上用250微升含阿糖胞苷介质中。 注：细胞可以来自6天内使用体外 （DIV）。 </li></ol></li></ol> 4.选择性地耗尽小胶质细胞（在演出6格） <ol><li>添加纯LME至5ml MEM培养基的单独的等分试样，得到150毫LME的终浓度。 </li><li>返回该溶液至pH 7.4，并过滤灭菌使用28mm的聚醚砜（PES）为0.2μm针筒过滤器。 </li><li>随着对于对照培养正常的MEM培养基稀释溶液至50mM的（2×LME）的MEM培养基中并置于水浴中的浓度在37℃下10分钟。 </li><li>除去一半（250微升）从CGC培养的MEM培养基中，并保持在37℃。 </li><li>处理细胞用含有2×LME（250微升）的MEM培养基， 即 1:1稀释得到的25mM的阱浓度，或与对照培养物预热MEM培养基而不LME（250微升）。 </li><li>孵育细胞在37℃下在湿润的气氛中，用6％CO 2下1小时。 </li><li>洗涤细胞在新鲜预热的MEM培养基两次以除去含LME介质，然后更换保留培养基和新鲜预热的MEM培养基等体积的相应孔中。 </li><li>返回培养至孵化器（加湿用6％的CO 2，37℃）和在任何进一步处理离开休息24小时。 </li></ol>

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The authors declare that they have no competing financial interests.

最重要的步骤，以确保成功选择性消除小胶质细胞来自CGC和/或混合培养物有：1）保持无菌，健康的文化CGC; 2）过滤灭菌的含LME介质并返回该溶液至pH 7.4; 3）保持保留CGC媒体和LME-含37媒体 °C，这样可以避免热冲击;和4）迅速工作，以减少单元保持在培养箱外的时间。 我们使用25毫米LME从我们的CGC文化耗尽胶质 - 以前在我们的实验室进行了优化的浓度。尽管如此，一些?...

人类大脑包含估计为85十亿神经元和另一个85十亿非神经元细胞，包括神经胶质1。对于过去的100年神经科学家主要集中在神经元细胞群的大部分时间里，相信神经胶质细胞比被动的支持细胞为神经元提供结构支持更小 - “神经胶质细胞”翻译成英语的，因此希腊词源“胶水”。然而，最近它已成为日益明显的是，神经细胞 - 胶质细胞的相互作用可能是更为基本的神经生物学，神经生理学和许多神经变性疾病的发生和进展的基本方面。小脑颗粒细胞（信用担保公司），最丰富的神经元的同质人口在人的大脑，支配小脑和弥补90％以上，其细胞成分。因此，这些细胞已被广泛用于在体外作为吨的模型系统他研究神经发育，功能和病理2-6。 不过，CGC文化还含有小胶质细胞和神经胶质细胞等在争议显著的比例。其结果是，CGC数据推定显示直接神经元对不同的细胞治疗可能实际上出现 - 在部分或全部 - 从在培养相邻神经胶质的间接二次响应。为了评估这一点，我们选择的除去从CGC神经元培养小胶质用的L-亮氨酸甲酯（LME）的助剂。 LME是一个lysomotropic剂原本用于选择性地破坏巨噬细胞7，并一直使用也选择性地从神经元，星形胶质细胞和神经胶质混合培养8,9,10消耗小胶质细胞。 LME由巨噬细胞和小胶质细胞内化，其中它引起溶酶体破裂和随后的细胞凋亡13,14。巨噬细胞和小胶质细胞是典型丰富的溶酶体，使他们成为particula暴露于LME治疗RLY脆弱。该协议提供了一个功能强大，简单易用的方式，以确定小胶质细胞在利用CGC和其他神经元/胶质细胞培养系统实验的贡献。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>The curved end facilitates removal of the cerebellum&#160;</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146</td>
			<td>Straight, sharp point facilitates rodent P4-7 dissection&#160;</td>
		</tr>
		<tr>
			<td>L-leucine methyl ester hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>7517-19-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E7510-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>27964-99-4</td>
			<td>Coat coverslips 1 day before use&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ED1 antibody&#160;</td>
			<td>Abcam</td>
			<td>ab31630</td>
			<td></td>
		</tr>
	</tbody>
</table>

selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

这种技术的以选择性地消除从CGC和/或混合培养物的小胶质细胞的能力依赖于研究者的准确识别，并从它们的周围细胞分化的小胶质细胞的后续能力。这可以使用一个小胶质特异性细胞制造者，如植物凝集素B4中实现， 如图1所示。作为表现出在图2中，没有观察到改变星形细胞和神经元密度和形貌进行记录，对于各主要的治疗组。重要的是，观察到的神经元或星形细胞形态...

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

小胶质细胞的小脑颗粒细胞使用L-亮氨酸甲酯选择性耗尽

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

Research

JoVE Journal

Neuroscience

8.6K 次观看.  University of Washington. Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. 

视频: Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

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 ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

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

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

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

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

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 ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

应用 ImageJ 对免疫组化制备组织显微照片小胶质细胞形态学的定量研究

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

髓鞘有机小脑片培养的制备及免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...