洗澡的录音技术最早是由皮尔斯（1984）8和北奥等。 （1990）9，测量尾翻转时产生的电场。该技术后来又修改和完善唐纳德爱德华兹（美国乔治亚州立大学）博士在他的前研究生博士发地答：伊萨和他的前博士后副延Herberholz博士的实验室。已作出进一步的改进，并在马里兰大学的新研究中的应用已经在实验室的博士延Herberholz的测试。我想感谢给我帮助与实验中使用他的高速视频系统和我的研究助理大卫Rotstein和威廉利登，我的同事大卫耶格尔博士。

第1部分：录音室<ol><li>录音室是长方形的薄壁玻璃。商会尺寸为8.5厘米× 2厘米× 5厘米（长x宽x高）动物为2.5 - 3.5厘米（从主席台到尾节）总长度。 见图。例如在我们的实验中使用的一腔1。 </li><li>或者可以从其他材料（例如，无毒的透明塑料），录音室。钱伯斯的大小可能会有所不同根据实验过程和商会应为每一个实验系列定制。为了达到最佳效果，室尺寸应尽可能小，而不限制在他们的自然行为的动物。作为一个经验法则，室的长度和宽度不应动物的大小的三倍以上。 </li></ol>第2部分：浴电极和地线<ol><li>一对记录电极和接地电极。电极是由绝缘铜线（0.25毫米绝缘AWG 26）。洗澡电极的一端连接到细胞外放大器（AM系统1700，见下文）和0.5 - 1.0 mm的绝缘层剥离的另一端。接地线连接到放大器或任何其他接地设备和保温2-3厘米的地面被剥离的另一端。 </li><li>浴电极和接地线连接到录音室的内壁无毒胶水。记录电极定位集中室和彼此相反（图1）的两个短边。 </li><li>接地电极定位在一个长边垂直记录电极（图1）的录音室。 </li><li>商会是充满了去离子水。最好的结果是获得高抗水（〜18MΩ）。 </li></ol>第3部分：浴电极记录<ol><li>记录电极的输出被放大外放大器（AM系统; 1700型）（1000倍）。从记录电极的信号进行滤波，使用低频率的组合（&lt;100赫兹）和高截止频率平衡（&gt; 5千赫）。信号，然后连接到一个开关盒和数据采集板（美国国家仪器公司）。数字化的数据记录，存储，并使用数据采集软件（Photron Motion工具）进行分析。 </li><li>另外，记录电极的信号放大可数字化之前的数字化数据记录使用其他市售的数据采集软件（例如，MDS分析技术; Axoscope）使用其他模拟数字转换器（例如，MDS分析技术; Digidata 1440）。 </li></ol>第4部分：刺激探头<ol><li>刺激探头是一个配备一双细金属丝电极的玻璃吸管（长14厘米）。电极提示暴露（0.2毫米），动物是由探头接触时产生的电子信号。这使得来衡量的刺激的确切时间。 </li><li>刺激探头的输出被放大外放大器（AM系统; 1700型）（1000倍）。信号进行过滤，使用低频率的组合（&lt;100赫兹）和高截止频率平衡（&gt; 5千赫）。信号，然后连接到一个开关盒和数据采集板（美国国家仪器公司）。数字化的数据记录，存储，并使用数据采集软件（Photron Motion工具）进行分析。 </li></ol>第5部分：视频录制<ol><li>一个高速视频摄像机（Fastcam × 1280 PCI，Photron）垂直于录音室，提供了一个侧面看。录音室里面的亮度等级，必须进行调整，以提供最好的结果，例如，使用鹅颈照明或其他可聚焦光源。 </li><li>高速录像相结合，并使用一个接线盒及数据采集板（美国国家仪器公司）的电子录音同步。从洗澡电极放大信号连接到接线盒和数据采集板，使用BNC电缆。外部的手开关触发启动的同步视频和数据采集。 </li></ol>第6部分：实验过程<ol><li>一个单一的动物是引入室，并允许5分钟，以适应环境。逃生的尾巴，翻起引起不同强度的单水龙头头部或腹部，分别。水龙头的强度是受实验者。每个转义尾巴翻转记录帧速率在1000 F /秒，数据点的电子领域的潜力是在25 kHz高速视频记录。浴和录像开始启动触发盒上的手动开关。录音时间是由选定的帧速率（例如，1000 F /秒= 4秒的总录音时间）。邮政和预触发录像时间可以选择。 </li><li> 不E：浴电极记录应结合其他方法可用录音浴电极记录每个测试的物种，一旦被证实。小龙虾，可手术植入一对银电极周围的腹神经索。可用于刺激引出逃生尾巴翻转，并记录可以相比的植入和洗澡电极的电子痕迹。 </li><li>逃生尾翻转行为和相应的神经活动，可在各种不同的背景下记录，在食肉动物的攻击，例如视觉威胁，或在斗殴遇到两个小龙虾。大小会议厅，录音模式等进行相应的调整（见讨论 ）。 </li></ol>第7部分：数据分析<ol><li>单视频帧进行运动的工具软件（Photron）。电子痕迹是用来识别每个刺激被激活的神经回路的类型。视频数据相比，生理同步录音，以确定该动物的运动和激活的神经回路。每个激活电路产生特有的电子签名（ 见代表性的结果） 。 </li><li>探针接触和神经/肌肉的反应之间的延迟计算每个实验，通过测量探头信号的发病和同浴电极记录信号的发病之间的延迟。 </li></ol>第8部分：代表结果一个单一高速视频帧和相应的逃生尾巴翻转电场录音系列在交付少年小龙虾的头部或尾部（图2）触觉刺激的反应。 图2A：强烈的触觉刺激头部诱发尾部内侧巨头电路控制的翻转。记录的巨型神经元（星号）和大的阶段性偏转扣球使非模糊识别的尾巴翻转介导的由巨大的神经元活动。向后移动视频跟踪中确定激活的神经电路（MG）的身份。 图2B：强烈的触觉刺激后尾翻转横向巨头电路介导的尾巴。向上和向前运动的视频中看到的痕迹连同同步显示的巨穗大，阶段性的初步偏转决定激活的神经电路（LG）的身份的电子跟踪。 图2C：尾翻转非巨头电路控制。胸廓的动物被送到一个更渐进的触觉刺激。虽然在视频捕捉运动不允许明确识别激活电路，电子记录缺乏一个巨大的扣球和小得多的挠度确定激活电路（非G）组成。 图3：逃跑的所有三种类型的延迟测量的尾巴，翻起。探针接触和生理反应之间的时间测量七个动物。介导的巨尾翻转引起明显快于非巨尾翻转。 <img src="/files/ftp_upload/1297/Fig1.jpg" alt="" /> 图1：总长度为2.5-3.5厘米的动物在我们的实验中用于录音室的一个例子。洗澡电极粘室的两侧，而接地线连接到室长边洗澡电极垂直。 图2：单在1000 F /秒和相应的三个不同类型的刺激的电子录音记录的视频帧。 <img src="/files/ftp_upload/1297/Fig2A.jpg" alt="" /> A）强烈的触觉刺激被送到动物的头，引起内侧巨人（MG）介导的尾翻转。 6个视频帧显示在左侧。从触摸动物探头跟踪记录显示为灰色;接触点是由黑色箭头表示。同浴电极获得的记录跟踪显示为蓝色。插图显示了前面的大阶段性变形小巨人轴突尖峰。灰色条显示在左边的视频帧对应。探针接触后，小龙虾的尾巴的第一个显着的运动发生在帧＃3，七毫秒。 <img src="/files/ftp_upload/1297/Fig2B.jpg" alt="" /> B）强烈的触觉刺激被送到动物的尾巴，引起了横向巨头（LG电子）介导的尾部翻转。 6个视频帧显示在左侧。从触摸动物探头跟踪记录显示为灰色;接触点是由黑色箭头表示。获得记录跟踪浴电极以红色显示。插图显示了前面的大阶段性变形小巨人轴突尖峰。灰色条显示在左边的视频帧对应。小龙虾的尾巴的第一个显着的运动发生在帧＃3，八毫秒后，用探针接触。 <img src="/files/ftp_upload/1297/Fig2C.jpg" alt="" /> C）薄弱，循序渐进的触觉刺激被送到动物的头，并引起了非巨人（非G）介导的尾巴翻转。 8个视频帧显示在左侧。从触摸动物探头跟踪记录显示为灰色;接触点是由黑色箭头表示。同浴电极获得的记录跟踪显示为黑色。微量元素缺乏的巨穗潜力，大量的初始挠度和更小的幅度潜力。浅灰色条显示在左边的视频帧。小龙虾的尾巴的第一个显着的运动发生在＃6帧，115毫秒后的第一次接触探头。 <img src="/files/ftp_upload/1297/Fig3.jpg" alt="" /> 图3：七男女及类似尺寸的不同动物的响应延迟测量（平均长度± stdv：3.2厘米± 0.2厘米，从讲台上尾节测量）。 MG（蓝色条）和LG（红色条）尾翻转比明显更短的响应延迟尾非G（黑条）电路翻转介导的。手段和标准差显示。同一封信酒吧没有显着差异（Wilcoxon符号秩检验成对比较，P &lt;0.05）对方。

<ol>
	<li>Herberholz, J., Issa, F. A., Edwards, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+neural+circuit+activation+and+behavior+during+dominance+hierarchy+formation+in+freely+behaving+crayfish.">Patterns of neural circuit activation and behavior during dominance hierarchy formation in freely behaving crayfish.</a> J. Neurosci. 21, 2759-2767 (2001).</li><li>Herberholz, J., Sen, M. M., Edwards, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+behavior+and+escape+circuit+activation+in+juvenile+crayfish+during+prey-predator+interactions.">Escape behavior and escape circuit activation in juvenile crayfish during prey-predator interactions.</a> J. Exp. Biol. 207, 1855-1863 (2004).</li><li>Liden, W. H., Herberholz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+neural+responses+of+juvenile+crayfish+to+moving+shadows.J.">Behavioral and neural responses of juvenile crayfish to moving shadows.J.</a> Exp. Biol. 211, 1355-1361 (2008).</li><li>Finley, L. A., Macmillan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+field+potentials+during+different+tailflip+behaviours+in+crayfish.">An analysis of field potentials during different tailflip behaviours in crayfish.</a> Mar. Freshw. Behav. Physiol. 35, 221-234 (2002).</li><li>Eaton, R. C., Lee, R. K. K., Foreman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mauthner+cell+and+other+identified+neurons+of+the+brainstem+escape+network+of+fish.">The Mauthner cell and other identified neurons of the brainstem escape network of fish.</a> Prog. Neurobiol. 63, 467-485 (2001).</li><li>Canfield, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+voluntary+C-bends+may+be+Mauthner+neuron+initiated.">Some voluntary C-bends may be Mauthner neuron initiated.</a> J. Comp. Physiol. A. 193, 1055-1064 (2007).</li><li>W&#246;hl, S., Schuster, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+predictive+start+of+hunting+archer+fish:+a+flexible+and+precise+motor+pattern+performed+with+the+kinematics+of+an+escape+C-start.">The predictive start of hunting archer fish: a flexible and precise motor pattern performed with the kinematics of an escape C-start.</a> J. Exp. Biol. 210, 311-324 (2007).</li><li>Fricke, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+habituation+in+the+crayfish+due+to+selective+weakening+of+electrical+synapses.">Development of habituation in the crayfish due to selective weakening of electrical synapses.</a> Brain Res. 322, 139-143 (1984).</li><li>Beall, S. P., Langley, D. J., Edwards, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+escape+tailflip+in+crayfish+during+backward+walking+and+the+defense+posture.">Inhibition of escape tailflip in crayfish during backward walking and the defense posture.</a> J. Exp. Biol. 152, 577-582 (1990).</li></ol>

The authors have nothing to disclose.

非侵入性的录音，单一的神经元活动或神经回路活化很难获得，奔放的动物。这里描述的方法提供了一种手段，以确定神经激活模式，基本自然发生的行为。 在过去，我们成功地使用这种技术来衡量少年小龙虾的神经逃脱电路的活动模式，在社会地位的层次1形成，在天敌2，最近的攻击，在 3种可视化的威胁。目前，我们使用同步的浴室电极录音和录?...

recordings of neural circuit activation in freely behaving animals

奔放的动物的神经活动和相应的行为表现模式之间的关系是很难建立。传统的非侵入性的方法需要至少部分内敛的研究课题，他们只允许同时激活的神经元大量的鉴定。另一方面，小合奏的神经元或个人神经元只能使用从很大程度上减少了筹备工作取得的单细胞记录。由于自然行为表达的是克制和解剖动物有限，基本控制这种行为的神经机制是难以辨认。 在这里，我提出了一种非侵入性的生理的技术，使测量在自由地表现动物的神经回路的激活。使用一对电极丝内充水室，洗澡的电极记录神经和肌肉的领域产生的少年小龙虾在自然的或实验诱发逃避反应的潜力。小龙虾的主要逃生反应介导的三个不同类型的尾翻转移动点刺激的动物。每种类型的尾巴翻转控制自己的神经回路;最快和最强大的的两个逃生反应需要两套不同的大型命令神经元的激活。浴电极录音与行为观察相结合，可以明确识别这些神经元和相关的神经回路。因此自然发生的行为背后的神经电路的活动可以测量奔放的动物，在不同的行为中。

Watch this Scientific Journal Video about Recordings of Neural Circuit Activation in Freely Behaving Animals at JoVE.com

Recordings of Neural Circuit Activation in Freely Behaving Animals

在自由的行为的动物的神经活动模式的非侵入性测量是通过结合神经生理学的高速录像录音。

自由表现动物的神经回路活化的录音

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

recordings-of-neural-circuit-activation-in-freely-behaving-animals

Research

JoVE Journal

Biology

11.5K 次观看.  University of Maryland. 在自由的行为的动物的神经活动模式的非侵入性测量是通过结合神经生理学的高速录像录音。

视频: Recordings of Neural Circuit Activation in Freely Behaving Animals

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

全细胞记录的光在视网膜切片诱发兴奋性突触电流

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

科研

生物学

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an impending predator attack.  Although it is not known whether invertebrates posses an autonomic nervous system identical to that of vertebrates, evidence shows invertebrates have a  sympathetic-like  response to regulate the internal environment and ready the organism to act behaviorally to a given stimuli.  Furthermore, this physiological response can be feasibly measured and it acts as a biological index for the animal's internal state.  Measurements of the physiological response can be directly related to internal and external stressors through changes in the central nervous system controlled coordination of the cardio-vascular and respiratory systems.   More specifically, monitoring heart and ventilation rates provide quantifiable measures of the stress response not always behaviorally observed. Crayfish are good model organisms for heart and ventilatory rate measurements due to the feasibility of recording, as well as the rich history known of the morphology of the crayfish, dating back to Huxley in 1888, and the well-studied typical behaviors.

Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state.

在自由移动的小龙虾的心脏和换气率的措施

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

RhoC的GTP酶激活法

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

建模生物膜与电路板，并在轴突测量电气信号:学生实验室练习

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

进行阴道灌洗，结晶紫染色法和阴道细胞学评价小鼠发情周期分期鉴定

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...