这项工作是支持遐CIRM博士前奖学金（EDK）NARSAD青年研究者奖（DK）和镍氢智力奖（R01MH087495）（星期六）。

0.05毫克/千克皮下注射丁丙诺啡镇痛麻醉和镇痛:30分钟前麻醉，注入老鼠。启动30-40毫克/公斤腹腔钠巴比妥麻醉。在这一点上，也进一步镇痛（2毫克/公斤皮下注射）注射阿托品，以防止呼吸衰竭（0.4毫克/千克，皮下）和昔。如果5分钟后，老鼠仍是移动或脚趾掐响应，随后剂量巴比妥钠5毫克/千克（腹腔），直到老鼠对疼痛反应迟钝。在执行的第一个切口，在切口部位注射局部麻醉利多卡因（5毫克/公斤，皮）。初次注射后六至八个小时，0.05毫克/千克皮下注射丁丙诺啡镇痛注入老鼠。注射丁丙诺啡可每6-8小时以后，如果需要，虽然这通常是没有必要的。 重要的是要注意麻醉ç其他形式干扰兴奋性病变。例如，虽然氯胺酮是在啮齿类动物麻醉常用的形式，它可以干扰NMDA诱导的病变，因为它是一种NMDA受体拮抗剂。重要的是要选择一个诱导麻醉的方法，不减少病灶大小。如果需要气体麻醉，最立体的设备，包括这里所描述的，可容纳防毒面具适配器。 注:材料在特定的试剂和设备下面的表进一步描述。 1。泵和Stereotax的的制备<ol><li>填充用无菌水10μLHamilton注射器，并安装到一个6注射器可编程的泵的球童。确保年底到注射器的活塞泵上的钳制持有。 </li><li>充液气体消毒用无菌水，用针和1毫升注射器PE20管材。汉密尔顿注射器滑动开放的油管年底，被照顾FUL，以避免产生任何气泡管。 </li><li>打击30ga，平切，1英寸的油管输液针结束立体定向设备使用桶式电极操纵臂。插入输液针钳和沟槽每桶之间。确保了一个片内含有金属针，避免压扁油管和限制流体流动管钳。 </li><li> &gt; 5μL/ min的调节泵的速度，打开泵。应该出现在输液针的尖端的水珠。检查，以确保没有泄漏的油管及其接头的长度。 </li><li>珠用无菌棉签擦去水和设置泵撤回。撤回2μL气泡。上面的泡沫应该是在油管可见。 </li><li>浸入20毫克/毫升无菌0.1M PBS溶液中NMDA受体的输液针头，并撤回4-5微升。应该是无菌水和NMDA受体的解决方案之间的可见气泡在尖。重要的是要注意，NMDA受体是一种强力的毒素，应谨慎使用这一解决方案的编制和处理。在任何时候都手套和保护眼睛的建议。 </li></ol> 2。安装在立体定位装置鼠<ol><li> 图1描绘的立体装置的基本组成部分。剃须后大鼠的头部从眼睛，耳朵，加载了老鼠的门牙咬栏，老鼠的身体搁在培养基上设置一个加热垫。应挂咬栏下面的舌头，下颚。如果可以，温度反馈加热垫将是可取的。监测直肠温度也将是理想选择。 </li><li>用拇指和食指拿着老鼠的脖子，将到右耳栏上的权利的水平耳道。酒吧应该觉得这是在一个坚实的地方休息，不喜欢它下沉到头部。持稳定的头部右侧，滑动左耳扎入老鼠的左水平耳道。应该觉得这是在一个坚实的地方休息。 ，羽片应该对称，应该出现平趴在耳朵上酒吧。向上燃烧的羽片表示耳棒放置不当。头部不应摇晃在脖子上的压力。 </li><li>拧鼻子酒吧。温柔。在鼻子的骨骼很微妙，鼻栏并不需要紧。检查的老鼠仍是出发前的麻醉。 </li></ol> 3。准备手术的大鼠<ol><li>用消毒棉签，轻轻地放到每个老鼠的眼睛少量的兽医眼科润滑软膏，以保护他们从干燥和碎片。 </li><li>同心从计划切口部位到剃光地区的边缘，用2％洗必泰溶液擦拭大鼠的头皮，其次70％的乙醇三次。也可以使用，如果需要更有效的消毒剂洗必泰擦洗。完成10％碘伏溶液擦拭。现在准备的头皮是无菌的，应该只用无菌材料感动。 </li><li>唐无菌手套，作进一步的步骤，因为你将在下准备手术部位的处理。 </li><li>将无菌的悬垂性，使开窗暴露手术部位。 </li></ol> 4。创建外科窗口<ol><li>用10号手术刀刀片，使头皮的纵向中线沿切口（约2厘米长）。切口应启动后的眼睛。与非惯用手的拇指和食指，按住紧张垂直方向的切口在头皮上。 </li><li>颅骨表面覆的手术用无菌棉提示网站的边缘拉筋膜。这将需要一些力量，因此它可以被看作清除头骨。 </li><li>使用四个弯曲，无菌止血创建一个手术的窗口。对EA钳筋膜（皮肤）通道一侧的前部和后部方面的切口，然后躺在旁边的动物夹子，从而拉动打开切口，充分暴露颅骨。 </li></ol> 5。练级的头骨和创建的先导孔<ol><li>找到前囟门，前点头骨的三个主要板块相遇， 如图2所示。将输液针，使针尖刚好接触前囟门颅骨。注意:背腹坐标图3演示了如何读取Stoelting立体设备上的坐标。请注意，在这一点上，手套不再是无菌的，而不应直接接触无菌手术部位。注:触摸立体消毒的设备后，医生不再是无菌的，不应该接触的手术部位，也没有将触摸手术部位的设备零件。 </li><li>作为depicte定位的lambda，后点头骨的三个主要板块，D 图2。稍微提高了输液针，直背到拉姆达移动它。降低针，直到它刚好接触拉姆达头骨。读背腹坐标。如果头骨是水平的，这将是在0.1毫米，前囟门读数。如果它是进一步又比，移动针（的方式），松开鼻子酒吧，无论是提高或降低咬酒吧平台，根据头端太高，然后重新测量。 </li><li>移到前囟门的输液针，并记录前，后部和内侧的横向坐标。计算必须移动针的基础上的利益根据一个脑图谱12的大脑区域的坐标。血乳酸，前后的坐标减去2.8毫米，添加或减去5.1毫米/从内侧的横向坐标，取决于您是否要针对向左或向右。将输液针，以计算出的位置。 </li><li>图形界面的东东DLE的方式，照顾，以保持它位于轨道，然后钻了孔在用无菌牙钻位，使用1德雷梅尔摩托车工具的位置在直径约2毫米。仔细钻钻时通过头骨打破，往往会拉下，硬脑膜及脑组织下有可能损坏。进行划眼洞放大。 </li><li>用无菌棉签吸收血液。 </li></ol> 6。输液毒素<ol><li>摆动针进入位置回。降低针，直到它触及大脑表面。记录背腹的坐标。这是的硬膜水平。减去适当的数值，以达到利益的大脑区域。为基底外侧杏仁核，减去6.7毫米，。 </li><li>计算的水平慢慢降低输液针。然后提高它的备份，以及上述的头骨。用微量注射器将清洗线疏通输液针的尖端，小心，不要到T哎哟线在接触与非无菌手套输液针的末端。 </li><li> &gt; 5μL/ min的泵运行，并确保你看到的液体珠出来的输液针。如果没有，疏通线，并再次尝试。 </li><li>所需的背/腹水平慢慢降低输液针。 </li><li>设置为0.1μL/ min的速度对输液泵，并开始输液。注入为2分钟。 </li><li>一旦已经开始输液气泡在油管用黑色标记，标记的两端。如果两端不动，你的针被堵塞。停止输液，提高针，线的畅通，检查与高流速的流量，重新降低针，并再次尝试。 </li><li>一旦输液完成后，停泵，并等待5分钟的NMDA弥漫针尖。 </li><li>提高针0.5毫米，并再次注入1分钟。等待2.5分钟的NMDA弥漫针尖。他这额外输液LPS确保所有的血乳酸输液覆盖。这是否是必要的其他大脑区域必须确定实验。 </li><li>提高针慢慢的大脑和立体手臂摆动的方式。 </li></ol> 7。关闭切口<ol><li>唐新的无菌手套，关闭切口。 </li><li>用无菌棉提示擦去头骨上的任何多余的血液，去除筋膜夹。 </li><li>使用伦敦钳，共同推动双方的切口。应该满足和皮肤的内边缘，卷曲成切口外缘的皮肤不应该被允许。封闭而紧迫的切口，伤口剪辑应用坚决关闭前伸。继续推进沿切口的长度皮肤和主食。这通常需要3-5伤口剪辑。 </li><li>把所有的伤口剪辑后，使用的镊子，再次掐的主食，并确保他们是安全的。更安全的ţ他剪辑的可能性就越小老鼠会拉出来之前，切口愈合。 </li><li>拭装订与10％的碘伏溶液，直到流动性恢复（通常1-2小时后，麻醉，手术时间从诱导封30-45分钟），返回到其家笼和显示器大鼠切口。在这一点上，老鼠可能会返回到殖民地室。一个星期后，与异氟醚麻醉下取出主食卸妆工具的主食。 </li><li>使用热珠的灭菌消毒之前，未来的老鼠上使用的所有仪器尖端表面。沉浸在珠文书应当不超过15秒，或他们可能会变得太热处理。文书应当充分冷却后才能使用其他动物。 </li></ol> 8。复苏<ol><li>这是可能的，老鼠将展现在病变后的天癫痫发作。不同的大脑区域损坏的其他行为，如食品消费或GRO可能会受影响oming。这是重要的动物进行监测，以确保它们保持健康的恢复过程中，每天。支持治疗，如流质食物，高热量的食物，或额外的镇痛，可能需要。麻醉死亡率发生在大鼠，通常小于5％的比例很低。因其他原因死亡（感染，出血）是极为罕见的。麻醉恢复后，遇险的任何迹象排除未来的研究可能会导致，但极为罕见。 图4描述了在手术后的最初几天的减肥，因为它应预期。体重应在1周内反弹。 </li></ol> 9。代表结果 NMDA受体输液一个星期或更长时间后，病变可以可视化使用甲酚紫染液和明场显微镜。灌注冰冷的4％多聚甲醛，平衡大脑应在30％蔗糖0.1M PBS和froz恩。然后，他们应该在30-60微米切片，在一系列的1:6到1:12，并安装在之前接受的标准甲酚紫染液明胶涂层玻片。病变相对应的"秃斑"或面积下降甲酚紫染色细胞死亡后病变。 图5A和B代表病灶的血乳酸和CEA。这张照片是平板扫描仪采用了高分辨率扫描（1200 dpi）的获得。以可视化的病变，它可以帮助染液通过一系列节的大部分，所以放错了地方的病变可位于目标区域周围的组织。 <img alt="图1" src="/files/ftp_upload/4079/4079fig1.jpg" /> 图1。立体设置，这个数字显示大鼠脑立体定位与标记的相关部分。 <img alt="图2" src="/files/ftp_upload/4079/4079fig2.jpg" /> 图2。前囟门和lambda对大鼠SKULL。此图描绘的前囟门和lambda的位置。所有的坐标测量相对前囟门。 lambda是用来确保头骨是水平。 <img alt="图3" src="/files/ftp_upload/4079/4079fig3.jpg" /> 图3。阅读立体坐标。这幅画描绘了一个标准的立体阅读。要获取数字的小数位的左侧，使用右侧的标记。标记的数字对应的十位（即1 = 10，2 = 20）。之间的标有数字的散列标记对应的单一单位（1-9）。零线表示什么整数的stereotax设置。在这里，零线之间的10个和11个标记，表示读数是10和11之间。要确定小数点右侧的数字，使用左侧的标记。在左边的哈希标记的编号为0到10，每个无标签代表一个单位的差异，从哈希那些在它旁边。无论左线右侧的哈希标记最多最好的散列标记的坐标读数表示小数点第一位。在这里，左边的对应数9线与右侧的哈希标记最多最好的哈希标记，所以小数位是9。最后的读数为10.9毫米。 <img alt="图4" src="/files/ftp_upload/4079/4079fig4.jpg" /> 图4。手术后的重量变化。该图显示了大鼠接受立体定向手术组（n = 17）的平均（±标准差）重量或没有手术组（n = 6）。 0天是手术的日子。手术后，大鼠体重下降为2-3天，手术后，再增加5-6天左右。 <img alt="图5" src="/files/ftp_upload/4079/4079fig5.jpg" /> 图5。兴奋性病变例如A）此图像显示了30微米的冠状片1甲酚紫染液与"光头当场从NMDA受体病变的基底外侧杏仁核"。白色箭头勾勒出病灶。它可以较对侧没有病变B）一个中央核杏仁核类似病变的脑片。

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没有利益冲突的声明。

这里介绍的立体定向的方法可以通过注射NMDA受体的特定脑区的兴奋性病变。立体的基本方法，可适应在站点特定的方式注入各种药物和生物制剂。它也可以适应目标的大脑区域的品种，在脑图谱12立体坐标定义。小动物建立了类似的设备，可适应其他物种如老鼠。本程序为3月龄成年大鼠进行了优化，但它们可以被用来从青少年通过各种年龄的老年人。如果与年轻的动物，不得在标准脑图?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> N-甲基-D-天冬氨酸98％ </td><td> Fisher Scientific则</td><td> AC32919-0500 </td><td></td></tr><tr><td>双实验室标准立体定向w/45度。耳酒吧</td><td> Stoelting </td><td> 51653 </td><td>替代供应商:Kopf谨慎*注:如果来自多个供应商的采购，保证立体配件的兼容性</td></tr><tr><td> 10μLSYR特（* / * / *） </td><td>汉密尔顿</td><td> 701SN </td><td></td></tr><tr><td> DREMEL摩托工具</td><td> Stoelting </td><td> 58600 </td><td>替代供应商:Kopf </td></tr><tr><td>硬质合金钻，机头惠普，大小为2 </td><td>施恩牙科</td><td> 2284578 </td><td></td></tr><tr><td> ，Stoelting 6注射器方案可编程泵</td><td> Stoelting </td><td> 53140 </td><td>替代供应商:Kopf </td></tr><tr><td> 316不锈钢皮下注射例行壁管30计0.0123 0.00625"外径x内径x .003"华尔街（输液针） </td><td>小零件</td><td> HTXX-30R-06-05 </td><td></td></tr><tr><td> intramedic PE 20管（输液管） </td><td>厂商VWR </td><td> 63019-025 </td><td></td></tr><tr><td>反射剪辑，9毫米，非无菌</td><td>肯特科技股份有限公司</td><td> INS500346 </td><td>替代供应商:精细的科学工具</td></tr><tr><td>反射9毫米剪辑剪辑APPLIER </td><td>肯特科技股份有限公司</td><td> 12031-09 </td><td>替代供应商:精细的科学工具</td></tr><tr><td>弯曲哈特曼止血</td><td>罚款科学工具</td><td> 13003-10 </td><td></td></tr><tr><td>伦敦钳</td><td>罚款Sceince工具</td><td> 11080-02 </td><td></td> &lt;/ TR&gt; <tr><td> 2％洗必泰溶液</td><td> allivet </td><td> 30159 </td><td>替代供应商:PetSolutions </td></tr><tr><td> 10％的碘伏溶液</td><td> CVS的</td><td>产品代码＃739575 </td><td></td></tr><tr><td>热门珠灭菌器</td><td>哈佛仪器</td><td> 610183 </td><td></td></tr></tbody></table> 表1。表特定的试剂和设备。

stereotaxic surgery for excitotoxic lesion of specific brain areas in the adult rat

在许多哺乳动物，包括啮齿类动物和人类行为的功能是介导主要由分立的大脑区域。一个常用的方法是雪亮的行为或其他实验结果不同的大脑区域的功能来实现功能的局部消融。在人类中，经常研究与局部脑病变的患者人群赤字，希望揭示的基本功能受损区域。在啮齿类动物中，一个实验能引起特定的大脑区域的病变。 病变可几种方法来实现。电解病变可引起局部损坏，但损坏的多种细胞类型，以及从其他大脑区域发生病变部位附近穿越纤维。诱导细胞类型特异性启动子的基因技术也可能使站点的具体目标。这些技术是复杂的，总是不踏实，取决于目标的大脑区域。 excitot相比之下，采用立体定向手术的好氧病变是不破坏当地的神经胶质细胞或穿越纤维的兴奋性神经元的损毁最可靠，最实用的方法之一。 在这里，我们提出了一个协议的excitotoxin，N-甲基-D-天冬氨酸（NMDA），到基底外侧杏仁核复合体的立体输液。用解剖的迹象，我们应用立体坐标，以确定​​我们的目标的大脑区域的位置，并在上述目标的地方，降低注射针。然后，我们的大脑注入我们excitotoxin，导致附近的神经元的兴奋性死亡。虽然我们选择的实验对象是老鼠，同样的方法可以适用于其他哺乳动物，在设备和坐标进行适当的调整。 这种方法可用于各种不同的大脑区域，包括基底外侧杏仁核1-6，其他的杏仁核6，7，8海马，耳鼻喉科orhinal 9皮 ​​层和前额叶皮层10。它也可以用于注入生物化合物，如病毒载体1，11。基本的立体定位技术也可被改编为更持久的渗透泵植入，允许更长时间暴露在利益的化合物。

Watch this Scientific Journal Video about Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat at JoVE.com

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

靶向消融特定的大脑区域使用立体坐标excitotoxin输液（S）描述。这项技术也可以适用于其他化学品的输液进入大鼠脑组织。

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

stereotaxic-surgery-for-excitotoxic-lesion-specific-brain-areas-adult

Research

JoVE Journal

Neuroscience

35.3K 次观看.  University of California Berkeley. 靶向消融特定的大脑区域使用立体坐标excitotoxin输液（S）描述。这项技术也可以适用于其他化学品的输液进入大鼠脑组织。

视频: Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

啮齿动物的立体定向手术和动物福利成果改进行为神经科学

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

科研

神经科学

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化:一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

胸脊髓截断手术和大鼠开放场运动评估

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...