这项工作得到了肌肉萎缩症协会，国立卫生研究院（NS062320）。

1。皮下给药毒蕈碱型乙酰胆碱受体拮抗剂（mAChR） <ol><li>在无菌条件下适当的剂量，mAChR拮抗剂（参表）准备通过溶解在无菌生理盐水1.5ml的反应管中的药物。以下拮抗剂：阿托品，Methoctramine，4阻尼，AFDX - 116，AFDX - 384吨7。 </li><li>绘制成a1cc胰岛素注射器的解决方案50μL，并为每个鼠标使用单独的注射器。还准备只包含每个对照组小鼠生理盐水的注射器。置于冰上直到准备注射器注入。 </li><li>麻醉与氯胺酮 - 甲苯噻嗪鸡尾酒（120毫克/公斤氯胺酮，8毫克/公斤甲苯噻嗪）的小鼠，检查通过观察反应缺乏脚趾捏适当的麻醉深度。 </li><li>经过适当的镇静，剃光头发覆盖喙头颈部的尾部区域地区，用70％乙醇擦拭其余头发远离。 </li><li>体视显微镜下，放置鼠标和插入针并行鲎肌肉，而在注射器的针头（图1A，B）的轻轻拉起。这确保针头插入覆结缔组织和鲎肌肉本身并不直接损害。插入针，使针尖涵盖鲎肌肉的尾部方面。 </li><li>非常慢慢开始注入鲎肌肉的权利的解决方案，同时撤回的注射器，注射时间应约1分钟。如果注射不当，解决方案应形成一个“巨蛋”，是直径约1厘米，上覆皮肤，将保持至少一小时。一个统一注入的解决方案将涵盖整个权鲎肌肉的喙和尾鳍地区。 </li><li>重复步骤1至7天，每12小时。对于其他研究1，我们使用相同的协议，长达21天，每12小时注射生长因子。管理麻醉2X每天长达21天的小鼠，观察无异常反应。 </li></ol> 2。鲎肌肉解剖<ol><li>安乐死与小鼠过量戊巴比妥钠（390毫克/公斤）。保证评估缺乏心跳进行开胸手术后死亡。 </li><li>拖住鼠标，在清扫菜背侧，1针，在每个爪子和1针通过鼻子。 </li><li>只能通过皮肤的第一个切口，用小弹簧剪刀削减沿左鲎从该地区实现公正近端左肩胛骨周围地区的耳朵。 </li><li>小心取出这区域的皮肤，但要避免过于靠近右耳切割，因为这是正确的鲎肌肉附着点之一。 </li><li>找到中心位置的脂肪组织脂质地带沿中线，头部浅面，左，右的鲎肌肉满足。使用小弹簧剪刀，切1厘米的脂肪组织的左鲎肌肉近端边缘（向左耳）的权利，直至达到肩（ 图1C） 。 </li><li>一旦切口，剥离回用小镊子腹侧的肌肉暴露鲎肌肉的权利。修剪的结缔组织和筋膜，一边小心翼翼地拉动右侧鲎肌肉钳（ 图 1C） 。 </li><li>周围权鲎尾切耳基地。继续切割，走向右耳喙，保持正确的鲎打开过（图1C）。这是更好地在这一步破坏鲎本身风险包括更多的组织。如果肌肉开始，干出来的，在肌肉的吸管1X PBS。 </li><li>解剖右鲎30毫米文化Sylgard最多包含1X PBS，背侧的菜。牵制与小虫子引脚四个角的肌肉。 </li><li>使用权鲎肌肉背，腹表面的小镊子和春季剪刀，清洁结缔组织。转肌和重针，以清洁腹面。 2-3肌肉可能被放置在相同的30毫米菜。 </li></ol> 3。三联免疫鲎NMJs：第1天<ol><li>免疫开始前，准备所需的所有试剂。 1X PBS中，2％牛血清白蛋白牛血清白蛋白）/ PBS中，0.1M甘氨酸在2％BSA / PBS，0.2％，2％BSA / PBS，4％多聚甲醛（PFA）在1X PBS海卫X100将是必要的。预冷的甲醇在-20 ° C冰箱。 </li><li>鲎肌肉固定在盘背侧在此过程中。倒掉1X PBS液和补充足够的4％（PFA）覆盖的肌肉。 20分钟，放在振动筛盘速度2或3。除非另有说明，应放在盘上的振动筛以下步骤。 </li><li>弃掉4％PFA和用1X PBS 3次，10&#39;每个肌肉。 </li><li>放弃最后1X PBS清洗，并添加0.1M甘氨酸在2％的BSA / PBS液30&#39;。 </li><li>丢弃0.1M甘氨酸溶液D收藏1X PBS中罗丹明标记的α-银环蛇毒素在浓度为1:200 15&#39;。 </li><li>丢弃α-银环蛇毒素的解决方案和洗1X PBS中，3次10“每个肌肉。 </li><li>通透组织加入预冷的甲醇和地方的菜，在-20 ° C冰箱正好是5分钟，没有晃动需要。 </li><li>取出甲醇洗3次，10&#39;与1X PBS。放弃最后一次洗涤与块组织0.2％Triton X - 100在2％BSA / PBS为1小时。在室温下进行洗涤和阻塞步骤。 </li><li>过夜孵育肌肉，晃动在4 ° C在在封闭液（参表）稀释的抗体鸡尾酒。 </li></ol> 4。三联免疫鲎NMJs：第2天<ol><li>洗净1X PBS 3次，10“每一个在室温下的肌肉。 </li><li>阻止在室温下1小时（参表）的解决方案中添加二级抗体鸡尾酒。这一步以后，肌肉必须得到保护，防止Alexa的福陆公司的647荧光光漂白。 </li><li>在1X PBS 3次，10&#39;每个洗的肌肉。 </li><li>清洁在1X PBS任何剩余的结缔组织，切断鲎肌肉外侧边界，并安装在幻灯片背侧，用玻璃盖单和安装媒体。使用透明指甲油，以确保在地方盖滑。 </li><li>防止光线和存储在-20 ° C，直到准备进行分析的幻灯片。 </li></ol> 5。共焦成像鲎NMJs <ol><li>徕卡计划阿婆63X石油客观上的Leica TCS 4D共聚焦显微镜（1.4NA）获得高分辨率的共聚焦图像。 </li><li>荧光素和Alexa 647信号，同时扫描激光线为488 nm（氩，488纳米，20毫瓦）和633纳米（氦氖，633纳米，10毫瓦），分别。接着，若丹明信号，然后用激光线扫描561纳米（DPSS，561，20兆瓦）。光学部分，收集了0.3微米的间隔，z平面中的图像数量从60-80各不相同。针孔调整，以艾里1（107.97 UM）。图像格式为1024 × 1024（X，Y），1 234.32微米x 234.32μm和229.05 nm的像素大小x 229.05纳米的图像尺寸的400Hz的速度结果，变焦倍数。 </li><li>徕卡TCS - NT的采集软件，然后用于重建Z系列图像到最大强度的预测。 </li><li>多面板的图像使用Adobe Photoshop的亮度和对比度的调整。 </li><li>对于一个典型的鲎肌肉，应该能够分析约75-100 EN面对 NMJs。是由突触稳定的功能，如前，近郊和突触后元素的空间对齐（即神经雪旺氏细胞和肌肉），和突触面积测量（即突触的大小）。 </li></ol>代表性的成果： 在成年哺乳动物NMJ，单一的运动神经阐述，形成高度分化的神经终端的乔木（ 图 2，绿）的罚款 ​​树枝上的一个单一的肌肉纤维，正是apposed烟碱AChRs突触后群“（图2，红）， 。 Perisynaptically，终端雪旺氏细胞紧密包括突触前神经末梢的所有分支“（ 图 2，蓝）。这三方组织的结构和功能完整性的严重不安亚型特异性mAChR抑制剂的日常应用。在这里介绍的例子（ 图3），4潮湿，mAChR拮抗剂与货币供应量M1，M3，M4和M5 mAChR亚型高亲和力，唤起整个肌肉表面的选择性消除神经末梢（ 图 3B从众多NMJs C）。此外，终端雪旺氏细胞是异常的静态8明亮S100无过程延伸标签（ 图3B，3C“）证明。突触后，肌肉纤维是正常的，没有nAChRs的损失（图3B，3C） 。 <img src="/files/ftp_upload/3124/3124fig1.jpg" alt="图1"/> 图1。鲎肌肉的解剖位置和组织。延髓（rLAL），尾椎（cLAL），鲎神经（LALn）和相应的终板带的位置（插图）。注射过程（二）尊重鲎肌肉针的位置和方向显示。切口点解剖过程（三）所示。 <img src="/files/ftp_upload/3124/3124fig2.jpg" alt="图2"/> 图2。NMJ的三方组织。鼠标NMJ高倍率焦意见。一个单一的轴突阐述罚款终端分行（一），紧紧终端雪旺氏细胞和他们的流程（A“，星号点终端雪旺氏细胞组织）覆盖。突触后在肌纤维，烟碱AChRs集群正是apposed神经末端的分支机构nals和终端雪旺氏细胞。 <img src="/files/ftp_upload/3124/3124fig3.jpg" alt="图3"/> 图3。鲎肌肉与4潮湿，mAChR拮抗剂治疗。鲎肌肉的低和高放大倍率的焦意见处理的车辆或4潮湿。对比车辆治疗肌（A - A &#39;&#39;&#39;),众多NMJs 4湿热治疗肌肉缺乏神经末梢（B - B“”C - C &#39;&#39;&#39;).图2B中的一个盒装的面积是放大​​图2C。

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没有利益冲突的声明。

这里介绍的方法，允许调查亚型特异性信号的稳定性和维护哺乳动物NMJs mAChR先前未确认的角色。此方法也将是有用的测试神经营养因子及药理制剂的影响。例如，我们的实验室发现，几乎所有的成年小鼠的鲎神经末梢引起睫状神经营养因子（CNTF <a>）</a>发芽1。这与睫状神经营养因子治疗后肢肌肉，报告中度CA发芽前研究的结果对比。 13-33％的臀大肌，外侧腓肠肌路口3 9％。我们相信这种?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>氯胺酮</td><td> Hospira公司</td><td> NDC0409 - 2051 - 05 </td><td>剂量：120毫克</td></tr><tr><td>甲苯噻嗪</td><td>劳埃德实验室</td><td> LA33806 </td><td>剂量：8mg/kg </td></tr><tr><td>阿托品</td><td> Sigma - Aldrich公司</td><td> A0132 </td><td> （纯度&gt; 98％）; 20mg/kg剂量：0.2mg/kg - </td></tr><tr><td>阿托品</td><td>福格特全球分布</td><td> AT105 </td><td>医药级</td></tr><tr><td> Methoctramine </td><td> Sigma - Aldrich公司</td><td> M105 </td><td>剂量：100 - 400M </td></tr><tr><td> 4阻尼</td><td> Sigma - Aldrich公司</td><td> D142 </td><td>剂量：2.5mg/kg </td></tr><tr><td> AFDX - 116 </td><td> Tocris生物科学</td><td> 1105 </td><td> 250M </td></tr><tr><td> AFDX - 384 </td><td> Tocris生物科学</td><td> 1345 </td><td> 165&#39; - 500米</td></tr><tr><td>吨7 </td><td>肽国际</td><td> PMT - 4340 - S </td><td> 0.1M - 1M </td></tr><tr><td> 1X的磷酸盐缓冲生理盐水，pH值7.4 </td><td> Invitrogen公司</td><td> 10010049 </td><td></td></tr><tr><td>多聚甲醛</td><td>费舍尔</td><td> T353 - 500 </td><td> 10％的解决方案，首先解散10g/100mL用去离子蒸馏水，用1X PBS 4％，调整pH至7.4 </td></tr><tr><td>钠pentobarbitol </td><td>维克动物健康</td><td> NDC - 051311 - 050 - 01 </td><td>剂量：390mg/kg </td></tr><tr><td> Sylgard </td><td>道康宁</td><td> ＃184部分</td><td>按照指示，试剂盒，可以使用多种大小的培养皿（30毫米，为60mm，100mm）的取决于需求</td></tr><tr><td> 0.1M甘氨酸</td><td> Sigma - Aldrich公司</td><td> G - 7126 </td><td>添加0.185克2％BSA / PBS至25ml </td></tr><tr><td> 2％牛血清白蛋白（2％BSA） </td><td> Sigma - Aldrich公司</td><td> A3059 - 100G </td><td>溶解到100毫升的1X PBS2克牛血清白蛋白</td></tr><tr><td>海卫X100 0.2％，2％BSA / PBS（封闭液） </td><td> Sigma - Aldrich公司</td><td> T9284 - 100ML </td><td>溶解0.2ml/100mL 2％BSA / PBS </td></tr><tr><td> α-银环蛇毒素</td><td> Invitrogen公司</td><td> T1175 </td><td>使用浓度为1:200 </td></tr><tr><td> SMI - 312 </td><td> Sternberger单克隆抗体</td><td> SMI312 </td><td>使用浓度为1:1000 </td></tr><tr><td> SV2 </td><td>发展研究杂交瘤细胞银行</td><td> SV2 -上清</td><td>使用浓度为1:10 </td></tr><tr><td> S100 </td><td> Dako公司</td><td> Z0311 </td><td>使用浓度为1:400 </td></tr><tr><td> FITC标记的羊抗鼠IgG1 </td><td>罗氏公司</td><td> 03117731001 </td><td>使用浓度为1:200，但如果背景是高的，尝试1:400 </td></tr><tr><td> Alexa的福陆公司的647标记的羊抗兔</td><td> Invitrogen公司</td><td> A21244 </td><td>使用浓度为1:200 </td></tr><tr><td> Vectashield荧光安装媒体</td><td>媒介实验室</td><td> H - 1000 </td><td>这不是一个硬设置的媒体，您将需要安全与透明指甲油的盖玻片。 </td></tr><tr><td>小弹簧剪刀</td><td>精细科学的工具</td><td> 15002-08 </td><td></td></tr><tr><td>解剖钳</td><td>精细科学的工具</td><td> 11295-51 </td><td></td></tr></tbody></table>

subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice

经常用于在哺乳动物NMJs的形成和维护必不可少的信号体内的药理研究啮齿类动物，如腓肠肌和胫前，后肢肌肉。然而，药物渗透到这些肌肉，皮下或肌肉注射后往往是不完整或不平坦的，很多NMJs可以不受影响。虽然与设备，如微型泵全身给药可改善的时空效应，这种方法的入侵性可以导致混淆的炎症反应和/或直接的肌肉损伤。此外，完整的分析后肢肌肉NMJs是具有挑战性的，因为它需要耗费时间的连续切片和广泛的免疫。 鼠标鲎是一种细长，肌肉平板位于颈部背部表面。这是一个快速肌功能移动耳廓。它包含延髓和尾鳍部分来自颅骨中线和横向延伸到每个耳廓软骨部分。肌肉是提供项目尾端，因为它退出茎乳孔面神经的一个分支。我们和其他人发现鲎是一种非常方便的准备，提供的短期和长期在体内药物的影响NMJs和肌肉的调查优势。首先，其表面的位置，方便多个本地应用程序，在浅麻醉药物。其次，它的薄（2-3层肌纤维）允许几乎所有的肌肉内NMJs的可视化和分析。第三，易于解剖与神经完好，其支配的格局， 允许在体外9,5补充电生理分析。最后，也许是最重要的是，一个小的应用量（〜50μL）轻松地覆盖了整个的肌肉表面，提供了一个其所有NMJs的统一和长时间暴露在药物和消除需要一个系统性的方法1,8。

Watch this Scientific Journal Video about Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice at JoVE.com

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

我们描述的毒蕈碱受体信号抑制剂反复提的Auris长肌（LAL）年轻的成年小鼠的肌肉和管理程序，其神经肌肉接头在随后的免疫wholemounts（NMJs）。鲎的肌肉已经露出独特的优势<em&gt;在体内</emNMJs&gt;的药理作用。

皮下给药的毒蕈碱受体拮抗剂和上睑提的Auris在小鼠体内长肌的三重免疫染色

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

subcutaneous-administration-muscarinic-antagonists-triple

Research

JoVE Journal

Neuroscience

13.6K 次观看.  Arcadia University. 我们描述的毒蕈碱受体信号抑制剂反复提的Auris长肌（LAL）年轻的成年小鼠的肌肉和管理程序，其神经肌肉接头在随后的免疫wholemounts（NMJs）。鲎的肌肉已经露出独特的优势在体内的药理作用。

视频: Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

在大鼠的药物自我管理的评估超声发声

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

科研

神经科学

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

鱼藤酮灌胃和图像分析α- synuclein的夹杂物在肠道神经系统的一个使用口服

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

光谱共焦成像的荧光标记的尼古丁受体敲在慢性尼古丁政府小鼠

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

本体感觉和张力感受器在蟹四肢：学生实验室练习

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

实时 fMRI 神经训练管理协议

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

在健康志愿者的气体麻醉剂氙和一氧化二氮的管理中记录大脑电磁活动

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...