这项研究是FMMU新发现的基础和部分美国国立卫生研究院拨款的支持。

1。胶质细胞培养来自不同地区的中枢神经系统神经胶质细胞可以培养。整个过程是在过程图所示。 <img src="/files/ftp_upload/2111/2111process.jpg" alt="过程1" />第1天涂层培养板和盖玻片<ol><li>干燥灭菌的玻璃显微镜下一轮盖玻片高压灭菌器。 </li><li>板到灭菌的24孔培养板消毒盖玻片。 </li><li>外套与多聚赖氨酸和根的温度下2小时孵化的盖玻片。 </li><li>大衣6步骤3相同的方式培养板。 </li><li>两次洗净的盖玻片和6孔培养板，用蒸馏水和空气干燥文化引擎盖。 </li><li>在6孔板，每孔在24孔板和2毫升，每孔加200μLDMEM培养基（含10％胎牛血清）和37度下，将它们放入孵化器5 ％的CO 2 </li></ol>第2天的隔离皮层和神经胶质细胞培养<ol><li>消毒正流夹层罩。 </li><li>紫外线灯开启20分钟。 </li><li>喷雾用70％乙醇的所有表面和使用前等待15分钟。 </li><li>改变紧张：由低温麻醉幼鼠（P2 - P6），并在framen使用操作系统剪刀的万能基地斩首。 </li><li>打开头盖骨沿矢状使用虹膜剪和关闭颅骨剥离缝合。 </li><li>删除前脑和他们放进冷冻L15中等，体视显微镜下，仔细，清洁血管硬膜和软膜，隔离皮层和L15的中型洗几次。 </li><li>显微剪刀切成小块的皮质。 </li><li>添加0.125％胰蛋白酶EDTA制备成皮质件，并培育与L15的介质，在37度15分。 </li><li>组织块转移到20ML的DMEM培养基含20％FBS，于50ml试管中，脱氧核糖核酸酶原液加入终浓度为10ug/mL，吸组织解决方案，并与火抛光的玻璃Pasteure吸管的20倍，收集的单细胞悬浮液。 </li><li>洗细胞悬液一次DMEM培养基，重新暂停含10％胎牛血清的DMEM细胞，计数细胞与血球，种子细胞，在显微镜下5000-10000/cm 2 。细胞保持在37度5％的孵化器，改变介质的每周2次的一半。 </li></ol> 2。背根神经节神经元的分离，培养和纯化第1天准备文化的物质<ol><li>外套与多聚赖氨酸，洗盖玻片消毒盖玻片两次提取水和空气干燥，放入24孔板。 </li><li> 100μLNeurobasal培养基添加2％B27与2.5S神经生长因子（50毫微克/毫升），放入37度5％的CO 2板。 </li></ol>第2天，从胚胎分离的背根节<ol><li>消毒胶质细胞培养相同的方式流罩。 </li><li>过量的CO 2，由70％乙醇消毒腹部安乐死一个孕鼠（E15），胚胎从子宫中分离到L15的冷冻介质。 </li><li>隔离脊髓背根神经节在体视显微镜和转让下连接与L15的冷冻介质为35mm菜。 </li><li>收集单细胞悬液和用NBF培养基（Neurobasal培养基含2％B27与2.5S神经生长因子（50毫微克/毫升））。 </li></ol>第3天净化DRG神经元<ol><li> 18H - 24H，神经元的播种后，添加1毫米的集中FUDR股票解决方案的神经元文化的终浓度为20微米和200μL的最终体积。 </li><li> 72小时后，更换Neurobasal含2％B27和2.5S神经生长因子（50ng/mL）的培养基没有FUDR中期的一半。之后，改变培养基隔日。 </li></ol> 3。共培养背根神经节神经元与神经胶质细胞<ol><li>当神经胶质细胞培养达到合流（播种后20天左右），他们准备与神经元共培养。 </li><li> 24小时前加入纯化的神经元，神经胶质细胞的培养基改变Neurobasal培养基中含有2％B27与2.5S神经生长因子（50毫微克/毫升）。 </li><li>纯净的神经元是从文化的井，单细胞悬浮液获得通过火抛光Pasteure吸管机械传递。 </li><li>计数后，接种于融合的神经胶质细胞神经元在Neurobasal培养基含2％B27与2.5S神经生长因子（50ng/mL）500/cm 2 。 </li><li>神经细胞粘附和神经胶质细胞神经​​突起的生长，可以在不同时间点记录和图像分析软件和使用的细胞类型特异性抗体的免疫细胞化学分析。 </li></ol> 4。代表性的成果<ol><li> 胶质细胞培养：播种后，胶质细胞将成为融合为20天左右。在第一阶段对比显微OPE，这是很容易识别，不同形态的神经胶质细胞亚群形成了不同的增长模式的子结构和GFAP阳性星形胶质细胞占90％以上immuocytochemisty技术（图1，图2）所示。 </li><li> DRG神经元的文化 ：在我们的方法，FUDR治疗72小时后，DRG神经元的纯度将达到6天之内高达99％高，DRG神经元表现出其独特的形态和高密度突起的生长（图3，图4）。 </li><li> 胶质细胞和神经元共培养 DRG细胞的粘附和轴突生长发生在播种后4小时内的神经胶质细胞容易，仔细观察发现，神经细胞的粘附和突起的生长是由神经胶质细胞亚群，形成了特殊的经济增长模式的子结构的影响，这可以很容易地确定相差显微镜下，免疫细胞化学法（图5，图6）。 </li></ol><img src="/files/ftp_upload/2111/2111fig1.jpg" alt="图1" /> 图1。融合的神经胶质细胞的形态。皮质神经胶质细胞，接种于多聚赖氨酸涂布盖玻片培养20天，请注意不同的经济增长模式的神经胶质细胞，细胞辐射的方式排列在左侧。 <img src="/files/ftp_upload/2111/2111fig2.jpg" alt="图2" /> 图2。聚合胶质细胞胶质纤维酸性蛋白（GFAP）抗体标记注意GFAP（红色），在右侧的阳性细胞的辐射安排。 <img src="/files/ftp_upload/2111/2111fig3.jpg" alt="图3" /> 图3。背根神经节神经元生长在体外无FUDR治疗 。污染的细胞形成背根神经节神经元“的背景。 <img src="/files/ftp_upload/2111/2111fig4.jpg" alt="图4" /> 图4。背根神经节神经元增长FUDR治疗后在体外 。背景污染的细胞已经完全消除。 <img src="/files/ftp_upload/2111/2111fig5.jpg" alt="图5" /> 图5。背根神经节神经胶质细胞生长的神经元的神经细胞粘附和突起的生长受到抑制，并在右侧的神经胶质细胞对辐射的细胞排列有限。 <img src="/files/ftp_upload/2111/2111fig6.jpg" alt="图6" /> 图6。背根神经节神经元的神经丝蛋白抗体标记的神经胶质细胞生长 。突起（绿色）是抑制辐射安排左侧的神经胶质细胞，并在右侧的限制，所有的神经胶质细胞标记GFAP抗体（红色）。

<ol>
	<li>Rossignol, S., Schwab, M., Schwartz, M., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+time+to+move?.">Spinal cord injury: time to move?.</a> J Neurosci. 27 (44), 11782-11792 (2007).</li><li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat Rev Neurosci. 7 (8), 617-627 (2006).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Matyash, V., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+astrocyte+morphology+and+physiology.">Heterogeneity in astrocyte morphology and physiology.</a> Brain Res Rev. , (2009).</li><li>Wanner, I. B., Deik, A., Torres, M., Rosendahl, A., Neary, J. T., Lemmon, V. P., Bixby, 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=A+new+in+vitro+model+of+the+glial+scar+inhibits+axon+growth.+Glia.">A new in vitro model of the glial scar inhibits axon growth. Glia.</a> 56 (15), 1691-1709 (2008).</li><li>Kuffler, D. P., Sosa, I. J., Reyes, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+chondroitin+sulfate+proteoglycan+inhibits+dorsal+root+ganglion+neuron+neurite+outgrowth+and+substrate+specificity+via+a+soma+and+not+a+growth+cone+mechanism.">Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism.</a> J Neurosci Res. 87 (13), 2863-2871 (2009).</li><li>Kleitman, N., Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+method+for+the+study+of+myelination.">Tissue culture method for the study of myelination.</a> Culturing nerve cells. , P559-P559 (1998).</li></ol>

这个实验协议的目的是要达到两个目标，以研究神经胶质细胞，神经细胞粘附和突起的生长，特别是星形胶质细胞的异质性的影响。第一个目标是保持星形胶质细胞的异质性，尽可能在本实验中，胶质细胞的融合文化，丰富的星形胶质细胞混合小学文化，无任何化学处理和消化的传播，这可能会进一步破坏细胞，并诱导星形胶质细胞的损伤反应。最后的星形胶质细胞纯度为90％以上，GFAP阳性污染?...

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<td>DMEM(high glucose)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-039</td>
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<td>L15 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11415-114</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437-077</td>
<td>&nbsp;</td>
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<td>Neurobasal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>&nbsp;</td>
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<td>poly-lysine</td>
<td>&nbsp;</td>
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<td>P4832</td>
<td>culture grade</td>
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<td>NGF(2.5S)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13257-019</td>
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<td>FUDR</td>
<td>&nbsp;</td>
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<td>F0503</td>
<td>&nbsp;</td>
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<td>neurofilament antibody</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab24575</td>
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study glial cell heterogeneity influence on axon growth using a new coculture method

在中央的所有哺乳动物的中枢神经系统，切断损伤后的轴突无法再生到原来的目标和功能恢复是非常差的1。轴突再生的失败是几个因素，包括敌对的神经胶质细胞环境，抑制髓鞘相关分子和减少内在的神经元的再生能力2的综合作用的结果。星形胶质细胞是最突出的中枢神经系统的神经胶质细胞类型，并在轴突功能的生理和病理条件3下发挥重要作用。对比同源少突胶质细胞，星形胶质细胞是一个由不同的星形胶质细胞亚群具有不同的形态和基因表达的 4组成的异质细胞群。这种异质性的功能意义，如对轴突的生长影响，在很大程度上是未知的。 为了研究神经胶质细胞，尤其是在星形胶质细胞神经​​元行为的异质性的功能，我们建立了合作培养高纯度的背根与从大鼠皮层获得的神经胶质细胞神经​​节神经元的一种新方法。通过这项技术，我们能够直接比较不同的星形胶质细胞亚群在相同条件下神经细胞的粘附和轴突的生长。 在这份报告中，我们给这种方法的星形胶质细胞的分离和文化，背根神经节神经元的分离和提纯，和背根神经节神经元与星形胶质细胞共培养的详细协议。这种方法也可以扩展到其他脑区，研究细胞或地区的特定神经元和神经胶质细胞之间的相互作用。

Watch this Scientific Journal Video about Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method at JoVE.com

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

在这个协议中，我们描述了一个新的方法来研究与轴突生长的神经胶质细胞的异质性的影响<em&gt;在体外</em&gt;共培养体系。大鼠皮层神经胶质细胞培养汇合和高度纯化的大鼠背根神经节神经元共培养。在相同的文化，不同的神经胶质细胞对神经细胞的粘附和轴突的生长影响比较直接。这种方法提供了一种新的方式直接研究胶质细胞对神经细胞粘附和轴突生长的异质性影响。

研究胶质细胞轴突的生长异质性的影响，使用一个新的共培养法

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

study-glial-cell-heterogeneity-influence-on-axon-growth-using-new

Research

JoVE Journal

Neuroscience

17.2K 次观看.  Cedars Sinai Medical Center, UCLA. 在这个协议中，我们描述了一个新的方法来研究与轴突生长的神经胶质细胞的异质性的影响在体外共培养体系。大鼠皮层神经胶质细胞培养汇合和高度纯化的大鼠背根神经节神经元共培养。在相同的文化，不同的神经胶质细胞对神经细胞的粘附和轴突的生长影响比较直接。这种方法提供了一种新的方式直接研究胶质细胞对神经细胞粘附和轴突生长?...

视频: Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

科研

神经科学

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

研究风味编码的新方法

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

胶质细胞和神经细胞的三维重建与虚拟现实分析方法

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...