马克麦考利是支持由美国国立卫生研究院辅导的培训补助金5T32HL066991 - 07 Xander的Wehrens是支持由美国国立卫生研究院/ NHLBI补助1R01HL091947 01A209和3R01HL089598 - 03S109

1。遥测手术植入的制备<ol><li>插入鼠标心电图遥测之前，重要的是要确保遥测心电图是无菌的，在良好的工作秩序。通常是在无菌条件下由制造商提供新的遥测设备。可重复使用心电图遥测提供的设备是使用至少4个小时Tergazyme 1％溶液清洗。您可以用无菌水冲洗Tergazyme清洗后的遥测。此外，使用Wavicide - 01的消毒剂一夜之间消毒心电图遥测。请务必用无菌水冲洗消毒液植入到小鼠体内，48小时前。存放在无菌容器中。 </li><li>检查双方的导线和绝缘护套的完整性的遥测信息。开启遥测发射机使用磁铁在5厘米的遥测挥手，并测试一个AM广播电台，频率530信号。应强烈和明确的信号，并应不同强度和俯仰操纵的电线。记录型号，序号遥测，和心电图检查校准值。制造商通常建议的遥测应植入前24小时，为您的具体细节遥测说明书。在这个例子中，我们将使用数据科学国际（DSI）的遥测植入。 </li><li>可实现遥测导致准备削减负（白色）铅约为3.5厘米和2.5厘米积极的（红色）导致。这是在右胸负领先植入的最佳长度，并在左腹部积极的主导作用（见图2）。接下来，删除揭露7毫米的电线的绝缘护套。涂料用无菌的强力胶，如VET -债券，金属铅，然后将铅帽金属提示。这些上限将避免由于皮肤糜烂，导致安置。大约2-3毫米的金属丝应暴露电气传感本地心律。 </li></ol> 2。手术植入的遥测<ol><li>为了在小鼠手术使用的无菌技术，您将需要无菌手套，无菌的悬垂性，和6-0普理灵缝线。您还需要消毒钳，以2对，1对钝剪刀，手术刀，针驱动程序。手术器械可能被加热到250摄氏度的玻璃珠灭菌消毒。可以肯定的消毒和清洗，以前使用前的遥测。 </li><li>首先使用3％异氟醚在0.5升/分钟100％O2感应鼠标麻醉，准备手术的鼠标。使用电热垫，如T型泵，循环温水，保持麻醉小鼠的温度。直肠探头可以监测温度，如果需要的话。当鼠标充分镇静，完全剃光鼠标电动快船的腹部和胸部。重新鼠标与异氟醚麻醉之前把鼠标一个恒定流管，平卧位，头对着，对你的尾巴。磁带加热手术台下来的老鼠的爪子。用酒精棉球擦拭腹部删除剃光头发和清洁术野。适用于三个单独大衣优碘棉签消毒腹部和胸部。验证正确的麻醉水平，通过鼠标上的指甲床施加压力。鼠标已经准备手术后，使用无菌手套，适用于无菌的悬垂性手术领域。 </li><li>腹腔遥测植入提供了运动生理学实验的优势。腹腔插入，开始用手术刀在皮肤上创建一个垂直的覆腹部正中切口长度大约2.5厘米的手术。仔细分离皮肤从底层的结缔组织，用钝剪刀。然后，创建一个在白线的垂直正中切口腹膜覆盖，大约1.5厘米长。此外，创建一个小的正上方（颅）腹膜切开，这将作为皮下插座导致腹膜洞。在手术的同时，保持手术部位的湿润，偶尔滴落到操作现场无菌生理盐水。 </li><li>插入右腹腔测距仪。优越的遥测孔插入钳和拉都导致通过铅孔，使他们从腹膜突起。连续使用6-0普理灵缝合关闭腹腔1.5厘米的垂直正中切口。 </li></ol> 3。铅注入和关腹<ol><li>心电图信息被放置在导致II配置。率先与白色/透明鞘是否定的，是摆在左上腹。首先，创建一个鼠标的右上角胸部0.5厘米的皮肤切口。接下来，使用钝的剪刀，腹部切口，以创建一个隧道。通过隧道拉铅和使用6-0普理灵缝合锚导致胸肌。确保缝合的铅暴露部分的顶部，并创建一个良好的接触铅及相关肌肉之间的。使用第二个上述近端缝合，固定导致肌肉。关闭使用6-0普理灵缝合皮肤切口。 </li><li>正极（红色鞘）被放置在左腹部下方的左膜片及以下的心。铅是挂靠在底层6-0普理灵缝合腹膜组织，并应具有良好的接触与腹膜组织。另外一个切口在这一步不是必需的，因为导致植入的部位是接近垂直正中切口，因此可以很容易地与现有的外科手术领域的访问。注意：必须取消，以避免底层肠穿孔，腹膜组织。 </li><li>按顺序关闭腹部的筋膜和皮肤，用6-0层的普理灵。 </li></ol> 4。术后护理<ol><li> [1毫克/毫升]丁丙诺啡镇痛手术后立即给小鼠0.1毫升。让鼠标上加热垫手术恢复。如果鼠标已延长手术&gt; 30分钟，或如果鼠标出现干燥，您可能会注入0.2-0.3毫升无菌生理盐水补液腹膜。对于继续手术后镇痛是标准的做法，提供与丁丙诺啡小鼠三天，每天两次，再根据需要每隔八小时。 </li><li>清洁用优碘酒精棉签，以避免手术后的刺激。记住记录的手术后的重量，随后确定手术后的健康。 </li><li>手术后8-24小时，给鼠标丁丙诺啡镇痛需要的时候，因为这将减轻疼痛和减少导致鼠标扣在手术后的网站放置失败。 </li></ol> 5。代表性的成果正确执行时，鼠标应该有关闭腹部切口皮下导致皮肤下。通常情况下，鼠标需要10-30分钟，从麻醉中恢复。它大约需要7-10天之前，老鼠已经完全从手术中恢复，手术后的重量损失和流动性正常化的恢复可见一斑。 从手术中最常见的并发症是通过皮肤的侵蚀，肢体导联在手术后第10天。坚持皮下导致皮下组织或腹膜，可避免这种常见的并发症。这种技术可避免松弛信息，创建的压力和对皮肤的摩擦，当鼠标ambulates。虽然不太常见，感染和败血症，可能会出现在无菌技术，或不完整的清洁使用遥测的错误。 心电图输出可能会被记录与数据采集软件，如数据科学国际（DSI），加上一个接收器矩阵。波形结果应包括一个明确界定的P波，表示心房去极化，而且也是一个QRS波，这标志着心室除极，在图3所示。良好的品质心电图跟踪应该有一个明确界定的P波前一个明确界定的QRS波以1:1的比例。应该有低背景信号和RR间隔应定期在野生型小鼠。质量差的心电图跟踪可能有模糊P或QRS波，或背景信号，这可能使后续的计算机分析困难。一个贫穷的心电图跟踪可能会被重新植入遥测信息，通常在一次固定。要重新植入遥测导致，在麻醉状态下放置鼠标，采用手术方法，使皮肤切口直接覆在问题导致。当铅访问，切割缝合固定带头，导致前锚再次领先移动到所需位置。最后，关闭与手术部位6-0普理灵。 <img src="/files/ftp_upload/1739/1739fig1.jpg" alt="图1" /> 图1：具有正面和负面信息的小动物遥测的一个例子。在一般情况下，红铅是正极，白铅是负极。每一个领导应该有裸露电线的绝缘护套，5-7毫米，和一个塑料皑皑的提示，从而阻止导致通过皮肤的侵蚀。 <img src="/files/ftp_upload/1739/1739fig2.jpg" alt="图2" /> 图2：卡通表现出适当的遥测植入网站上的鼠标。白色（负）导致植入老鼠的右上胸，和红色（正）导致放置在左侧腹部，遥测感官“导致了两个”配置。 &lt;P类=“j​​ove_content”&gt; <img src="/files/ftp_upload/1739/1739fig3.jpg" alt="图3" /> 图3：鼠标在正常的窦性节律的代表波形。 P波代表心房去极化，QRS波群代表心室除极。一个高品质的波形应该有一个明显的P波前以1:1的比例每个Q​​RS波群。

<ol>
	<li>Cerrone, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+ventricular+tachycardia+and+fibrillation+elicited+in+a+knock-in+mouse+model+carrier+of+a+mutation+in+the+cardiac+ryanodine+receptor.">Bidirectional ventricular tachycardia and fibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiac ryanodine receptor.</a> Circ Res. 96 (10), e77-e77 (2005).</li><li>Chelu, 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=Calmodulin+kinase+II-mediated+sarcoplasmic+reticulum+Ca2++leak+promotes+atrial+fibrillation+in+mice.">Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice.</a> J Clin Invest. 119 (7), 1940-1940 (1940).</li><li>Sood, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+leak+due+to+FKBP12.6+deficiency+in+mice+facilitates+the+inducibility+of+atrial+fibrillation.">Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation.</a> Heart Rhythm. 5 (7), 1047-1047 (2008).</li><li>Swoap, S. J., Gutilla, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+changes+during+daily+torpor+in+the+laboratory+mouse.">Cardiovascular changes during daily torpor in the laboratory mouse.</a> Am J Physiol Regul Integr Comp Physiol. 297 (3), R769-R769 (2009).</li><li>Costa-Goncalves, d. a., C, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+multidomain+protein+spinophilin+in+blood+pressure+and+cardiac+function+regulation.">Role of the multidomain protein spinophilin in blood pressure and cardiac function regulation.</a> Hypertension. 52 (4), 702-702 (2008).</li><li>Wehrens, X. H., Kirchhoff, S., Doevendans, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+electrocardiography:+an+interval+of+thirty+years.">Mouse electrocardiography: an interval of thirty years.</a> Cardiovasc Res. 45 (1), 231-231 (2000).</li></ol>

此过程的关键步骤是正确的清洗和小鼠生存手术前的准备。当遥测和鼠标手术部位被正确地清理，有一个更好的手术后的结果。此外，密切关注导致安置，减少手术后的皮肤糜烂。可能修改的技术包括替代的配置，以评估横向左心室壁功能（导致我的配置），或使用不仅为遥测心电波形分析，也超出了本文的范围温度，甚至血压分析[] 。心电遥测记录的一个替代的位置是皮下导致外部导线的位置?...

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<td>Steri 250 Bead Sterilizer</td>
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ambulatory ecg recording in mice

在小鼠体内的遥测心电图记录是必不可少的了解背后的机制性心律失常，传导障碍，心脏猝死。虽然体表心电图是利用短期测量波形的时间间隔，它是不实际的心率变异或捕获罕见的心律失常发作的长期研究。植入式心电遥测提供简单的植入手术，长期卧床小鼠电图的记录，并与多种动物的同步录音的可扩展性的优势。在这里，我们提出了动态心电图记录在小鼠植入遥测一步一步的指导。认真遵守无菌技术操作是必需的植入和记录的可能性，从几周到几个月的有利的生存结果。因此，植入式心电遥测是一个有价值的关键信息在日间的动物模型，如鼠标心脏电生理检测工具。

Watch this Scientific Journal Video about Ambulatory ECG Recording in Mice at JoVE.com

Ambulatory ECG Recording in Mice

遥测心电图已成为一个必不可少的工具在评估心律失常和心脏猝死动物模型。在这里，我们提出了一个在长期的动态心电图监测在小鼠中的应用遥测心电图录音，逐步引导。

在小鼠体内的动态心电图记录

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Research

JoVE Journal

Biology

23.5K 次观看.  Baylor College of Medicine (BCM). 遥测心电图已成为一个必不可少的工具在评估心律失常和心脏猝死动物模型。在这里，我们提出了一个在长期的动态心电图监测在小鼠中的应用遥测心电图录音，逐步引导。

视频: Ambulatory ECG Recording in Mice

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

小鼠小肠移植

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

科研

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

慢性的微型驱动器阵列在体内录音：驱动器的制造

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential amplitude of action potential signatures over the channels of a tetrode allows for the isolation of single-unit activity from multi-unit signals.  The ability to precisely control the stereotaxic location and depth of the tetrode is critical for studying coordinated neural activity across brain regions.  In combination with a micro-drive array, it is possible to achieve precise placement and stable control of many tetrodes over the course of days to weeks.  In this protocol, we demonstrate how to fabricate and condition tetrodes using basic tools and materials, install the tetrodes into a multi-drive tetrode array for chronic in-vivo recording in the rat, make ground wire connections to the micro-drive array, and attach a protective cone onto the micro-drive array in order to protect the tetrodes from physical contact with the environment.



Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

慢性的微型驱动器阵列在体内录音：四极大会

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

使用卷压力记录，一条尾巴袖套法小鼠的血压测量

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

在小鼠体内的横向主动脉缩窄

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of signals to create complex, dynamic patterns as a response to environmental cues. These patterns are self-organizing and emergent; they cannot be predicted by observing the behavior of the individual cells. Using a time-lapse microcinematography tracking assay, we identified a distinct emergent pattern in M. xanthus called chemotaxis, defined as the directed movement of a swarm up a nutrient gradient toward its source 1.
In order to efficiently characterize chemotaxis via time-lapse microcinematography, we developed a highly modifiable plate complex (Figure 1) and constructed a cluster of 8 microscopes (Figure 2), each capable of capturing time-lapse videos. The assay is rigorous enough to allow consistent replication of quantifiable data, and the resulting videos allow us to observe and track subtle changes in swarm behavior. Once captured, the videos are transferred to an analysis/storage computer with enough memory to process and store thousands of videos. The flexibility of this setup has proven useful to several members of the M. xanthus community.

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

To study Myxococcus xanthus swarm behavior, we have designed a time-lapse microcinematography protocol that can be modified for different assays. It employs standard growth conditions adapted for microscopy, and yields reproducible results by the use of inexpensive, reusable silicone gaskets. We have used this method to quantify multicellular chemotaxis.

在录制多细胞行为黏细菌生物膜，使用定时Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.