这项工作是由神经通信技术中心（CNCT），P41资源中心，由国家生物医学成像和生物工程研究所（NIBIB，P41 EB002030）和国立卫生研究院（NIH）支持。

1。准备固定液 （请参阅表定影液和缓冲器 ）。 2。准备灌注缓冲器 （请参阅表定影液和缓冲器 ）。 3。准备仪器和麻醉<ol><li>使用的水洗澡，温暖灌注缓冲至37°C。充满缓冲液的烧杯放置在出口阀。一个缓冲区填充50毫升注射器和重视固定液油管。冲洗管道多次被驱逐和撤出缓冲区。 </li><li>清除缓冲区的行，直到所有气泡在油管被淘汰。这是不能有气泡中的任何行灌注成功的关键。 </li><li>取下注射器和连接的4％多聚甲醛固定液（室温）容器。在管端，以避免气泡，同时定位到容器管，挤压管，所以，从笔底下降缓冲区突出O触点的容器内的液体表面。 </li><li>关闭出油口（针的末端）。将缓冲阀（蓝色）作为固定液阀的（白）相同的位置。这将允许只从缓冲区行流。 </li><li>打开出气口，通过A1 A3填充缓冲区行重复步骤。所有的气泡被淘汰后关闭出气口，取出注射器和连接的缓冲容器，照顾油管引入气泡。 </li><li>测试系统的能力举行抽水橡胶压力计blub的压力。通常是由于空气压缩系统中的一些阻力。 </li><li>设置在交通便利的秩序的手术工具。填充缓冲区灌注针，以消除气泡的可能性。 </li><li>手术前，氯胺酮/甲苯​​噻嗪的混合物（高达80毫克/公斤体重氯胺酮和10毫克/公斤体重甲苯噻嗪）管理，通过腹腔注射（27号针头注射器和1毫升）。必要时将执行麻醉额外的管理，在每个操作的过程中保持麻醉的手术平面。 </li></ol> 4。灌注术<ol><li>一旦动物已经达到了手术的麻醉平面，把它洋溢着碎冰的浅盘上。 （用脚趾捏反应的方法来判断麻醉深度。动物必须是反应迟钝，然后再继续）。 </li><li>通过体壁和腹壁下方的肋骨外侧切口5-6厘米。仔细分离膈肌肝脏。 </li><li>在使用隔膜弯曲，钝的剪刀做一个小切口。可以帮助你的手指的位置和压力​​的能力，以减少隔膜。 </li><li>继续沿整个长度的肋骨暴露胸腔膈肌切口。 </li><li>将沿肋骨一侧弯曲，钝的剪刀，仔细取代肺部，使某铜通过左肋吨上涨至锁骨。对侧上作出类似的削减。 </li><li>起重胸骨而去，精心修剪的任何组织，将它连接到心脏。胸骨与止血钳尖端，并将其放置在头上止血。如果处理得当，胸腺升降机远离心脏与胸骨，提供了一个明确的看法的主要血管。 </li><li>做一个小切口，使用虹膜剪左心室后端。 </li></ol><img alt="图1" src="/files/ftp_upload/3564/3564step7.jpg" /> <a href="http://www.jove.com/files/ftp_upload/3564/3564step7large.jpg" target="_blank">点击这里查看大图</a> 。 <ol start="8"><li>截止到升主动脉心室通过15号橄榄钝或尖灌注针。小费应该是通过主动脉壁可见，不应该达到的主动脉弓，臂丛和颈动脉分歧。 </li><li>使用止血钳，夹住心脏，这针固定，防止泄漏。如果需要，修改后的止血，可用于打击针尖周围的主动脉（这些止血留在原地，直到解剖开始，但是从未来的清晰度插图省略）。 </li><li>最后，使动物的右心房，用虹膜剪刀创造尽可能大出口不损害降主动脉切口。在这一点上的动物是准备灌注。 </li></ol> 5。灌注<ol><li>打开和出气口连接针座照顾，不引入任何气泡。 </li><li>泵的压力表灯泡到80毫米汞柱的压力，快速及均匀。维护此整个缓冲区输液期间的压力。启动定时器。 </li><li>调整针头角。针的角度是至关重要的，以实现最大流量（注意流量的变化与角度调整）。 </li><li>开关的buffER阀（蓝色）（200毫升），一旦缓冲区接近完成。流体应清楚。肝脏的结算是一个良好的灌注指标。肝脏应该清楚这一点。您的记录，显示时间。 </li><li>在几秒钟内，应遵守固定震颤，这应该被视为真正的固定时间。您的记录，显示时间。 </li><li>压力可以逐步提高到最高130毫米汞柱2，保持稳定的流量。 </li><li>关闭出口阀，一旦接近完成固定液。您的记录显示结束时间。 </li><li>在这个阶段应该是僵硬的大鼠。 </li><li>使用多聚甲醛，必须收集和储存处置根据贵机构的规定。 </li></ol> 6。解剖4,11 <ol><li>删除的头，用一把剪刀。 </li><li>使从颈部到鼻子的包膜沿中线切口和暴露头骨。 </li><li>剪掉剩余的颈部肌肉，使颅底暴露，除去残留的肌肉，用剪刀或rongeurs。 </li><li>放置到一侧的枕骨大孔的虹膜剪刀对尖底，仔细沿着头骨的内表面滑动的剪刀。 </li><li>接下来，延伸到后头骨表面的远端边缘切割。使对侧相同截。使用rongeurs扫清小脑周围的头骨。 </li><li>小心地将沿颅骨内表面剪刀的尖背远端后角头骨远端前缘，起重在刀刃上，你切割，以防止损害大脑。对面的重复。 </li><li>远离大脑头骨背表面剥离rongeurs。修剪掉的头骨两侧使用rongeurs以及。 </li><li>用铲，断绝嗅球和神经连接tions沿大脑的腹面。 </li><li>轻轻挑逗的大脑远离头部，修剪任何硬脑膜仍然连接大脑的头骨，用虹膜剪刀。 </li><li>取出大脑，并将其放置在一小瓶至少10倍体积的大脑本身的固定液含流体。偶尔漩涡小瓶。 </li></ol> 7。后固定及储存设备<ol><li>保持大脑中固定液24小时在4°C，偶尔纷飞。 </li><li> 24小时后，用大脑磷酸盐缓冲生理盐水交换媒体的3倍和纷飞每次。 </li><li>大脑可以储存在磷酸盐缓冲生理盐水或HBHS叠氮化钠，并保存在4°C。 </li></ol> 8。代表结果灌注成功的一个初步的指标是四肢，如鼻子，耳朵，爪子和内脏器官，如胸腺，肝化（IHC世界）结算。总值的检查结果显示大脑的血液血管（白色至淡黄色的外观）无效。这也将是真正在组织切片染色和免疫组化注定。灌注的结果最终指标是在组织超微结构的条件。1,6,7,8 <table border="1"><tbody><tr><td> 准备固定液： </td></tr><tr><td> 准备8％多聚甲醛股票 </td></tr><tr><td><ol><li>加入500毫升DH 2 O40克多聚甲醛加热到60的解决方案 - 65°C，而搅拌（不要超过65°C间，这样做可以产生不利影响免疫组化过程中的成功）。 </li><li>要清除的解决方案，减少热量和用滴管加2-3毫升1.0 M氢氧化钠。 </li><li>过滤器和存储长达1个月在4°C。 </li></ol></td></tr><tr><td> 准备0.2 M磷酸钠缓冲液，pH值7.4 </td></tr><tr><td><ol start="4"><li>磷酸二氢钠股票，增加至1 L DH 2 O27.8克的NaH 2 PO 4计 * 2 </li><li>磷酸钠二元股票，添加28.4克的Na 2 HPO 4至1 L DH 2 O </li><li>加入810毫升到190毫升的二元股票下一代的股票。 </li></ol></td></tr><tr><td> 准备4％多聚甲醛固定液 </td></tr><tr><td><ol start="7"><li>添加等份8％多聚甲醛股票0.2M磷酸钠缓冲</li><li>注：此修补程序的最好准备新鲜的进步，在不超过72小时。 </li></ol></td></tr><tr><td> 准备灌注和存储缓冲器： </td></tr><tr><td> 准备磷酸盐缓冲生理盐水，pH值7.4 </td></tr><tr><td><ol start="9"><li> DH 2 O的 1升</li><li> 9 g氯化钠</li><li> 144毫克KH 2 PO &lt;子&gt; 4 </li><li> 795毫克的Na 2 HPO 4 </li><li>检查pH值</li></ol></td></tr><tr><td> 肝素钠缓冲Hanks液（HBHS与叠氮化钠的pH值7.4） </td></tr><tr><td><ol start="14"><li> 990毫升DH 2 O的 </li><li> 7.5 g氯化钠， </li><li> 0.3Ğ氯化钾</li><li> 0.06克KH 2 PO 4 </li><li> 0.13克的Na 2 HPO 4 </li><li> 2克葡萄糖/葡萄糖</li><li> 2.4 G 10毫米HEPES </li><li> 0.1克MgCl 2的 6个部分DH 2 O的 </li><li> 0.05克硫酸镁 7件卫生署2 </li><li> 0.165克氯化钙 2件2 O的 DH </li><li> 90毫克，南三 </li><li>检查pH值</li></ol></td></tr></tbody></table> 表1。制备定影液和缓冲器。 <img alt="图1" src="/files/ftp_upload/3564/3564fig1.jpg" /> 图1。 </stronG&gt;标记元件的灌注装置。 <img alt="图2" src="/files/ftp_upload/3564/3564fig2.jpg" /> 图2。制备灌流装置一开始与固定液行。气泡冲洗管，填补迅速驱逐出境，并撤回到注射器，并通过行的缓冲带缓冲。关闭阀针的末端。没有引入气泡，放入固定液瓶的的固定液线。 <img alt="图3" src="/files/ftp_upload/3564/3564fig3.jpg" /> 图3。灌流装置制备。 1。打开阀针的末端。 2。旋转缓冲区阀（蓝色）定位流。 3。冲洗管气泡迅速驱逐和注射器撤出缓冲区和缓冲区填写。 4。关闭阀针端。缓冲瓶放入油管。压力测试，以确保系统正确的密封LY所抽的的压力表灯泡和看压力表。该仪器是现在的灌注过程中的准备。 <img alt="图4" src="/files/ftp_upload/3564/3564fig4.jpg" /> 图4。灌注装置在缓冲区交货的位置。 <img alt="图5" src="/files/ftp_upload/3564/3564fig5.jpg" /> 图5。灌注外科一，一）通过体壁和腹壁外侧切口。 b）使在膜片的切口，暴露心脏隔膜的跨越。使锁骨平行削减肋骨两侧。 C）胸骨与止血钳尖端，并将其放置在头上止血。 <img alt="图6" src="/files/ftp_upload/3564/3564fig6.jpg" /> 图6。灌注外科二。一）截止到升主动脉心室穿过灌注针。 B）固定灌注针使用一组止血钳的心脏。第二套的修改后的止血，也可用于打击主动脉周围，以防止泄漏的针尖。用虹膜剪刀作一小切口到左心室后端。 <img alt="图7" src="/files/ftp_upload/3564/3564fig7.jpg" /> 图7。与缓冲区灌注泵的压力表灯泡创建线的压力。打开阀门（1）和附加针枢纽。这是关键，以确保无气泡介绍。泵压力计灯泡，到80毫米汞柱的压力，并保持这种整个缓冲区输液期间的压力。 <img alt="图8" src="/files/ftp_upload/3564/3564fig8.jpg" /> 图8。一旦缓冲固定液灌注几乎完成（200毫升）切换缓冲阀（1）允许流动的固定液。压力可以逐步上升到最大的130毫米汞柱。 <img alt="图9" src="/files/ftp_upload/3564/3564fig9.jpg" /> 图9。解剖一）使用了一双剪刀除去头。 b）使沿中线从颈部到鼻子的皮肤切口，暴露颅骨。 C）剪掉剩余的颈部肌肉，使颅底暴露。按住头大开幕（枕骨大孔）在颅骨表面，使访问。小心地插入枕骨大孔的一对虹膜剪刀的尖底，并作出削减。对侧重复同样的动作。使用rongeurs扫清小脑周围的头骨。 <img alt="图10" src="/files/ftp_upload/3564/3564fig10.jpg" /> 图10。夹层。一）仔细沿着头骨的内表面滑动的剪刀。提起小费，你切割，以防止损害大脑。重复相同的对面。使用剥离颅骨rongeurs远离大脑。 B）插图的头骨和大脑暴露出来。 c）用锅铲在最前的位置大脑的嗅球和神经连接切断。小心地铲尖沿易于删除切断连接大脑底面。取出大脑，并将其放置在固定液小瓶。

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作者没有透露。

手术成功的其他注意事项： <ol><li>一旦违反隔膜，先迅速缺氧和高碳酸血症会引发不可逆转的生理变化，可能会混淆后续分析。 </li><li>当针插入夹紧到位，剩余的压力将推动血针（闪回）的基础。事实上，较大的动物可能会大力驱逐血液。这是至关重要的血液至少达到针的基础，以避免引入气泡，将提出一个障碍，完成灌注。如果没有血针基地观察，与缓冲区中删除附加到灌注钻机出口端口的针和笔芯。一旦缓冲区已通过尖端运行，针可以重新插入到心脏。 </li><li>妥善安置主动脉内针是至关重要的，它不应该达到尽可能的主动脉弓。 </li><li>如果多个灌注正在发生，它是没有必要的ţ从一开始每次O设置。用户需要有适当量的缓冲区，并在每个瓶子的固定液（200mls动物/解决方案）。如果需要的话，瓶也可以装填。最重要的方面，要考虑的是冲洗灌注线运行，从动物的设置（输出阀）。灌注完成后，这条线将在4％多聚甲醛。从缓冲瓶油管，并用50毫升注射器装满水冲洗出来的固定液。确保在一个多聚甲醛的正确处置废物收集烧杯放在出口阀。重复了三次。笔芯用同样的方法在2.3所示的缓冲。 </li><li>这种固定方法是高度适应的能力，改变缓冲区和固定其他固定剂（如电磁），根据实验的要求。 </li><li>该系统还经常被用于提取活组织。对于这个过程只需使用提供缓冲（缓冲瓶，同时还连接到整个系统被放置在一个冰桶和固定液的瓶子是空，但密封的设置）。这保证了在最佳压力冷缓冲灌注。从这里，该系统可以适应要么直接加入染料缓冲（如果数量是不是一个问题），或使用少量之间的缓冲区的缓冲区/染料溶液的注射器输液可修复染料 - 固定液的一步。 </li><li>在血管铸型的情况下，压力监测缓冲区一步是关键，然而由于铸造溶液的粘度和巩固性质无论是50ml注射器必须直接交付或一次性的二次附件仪器（油管，阀门和控股用于容器）与当前的系统接口。我们实验室的血管铸型之前灌流装置的设计和制造。因此，我们可以没有意法半导体吃，压力测量，将在交货地点的精确测量。的数字可能要进行调整，以实现粘性流体的成功交付。 </li></ol>

<table border="1"><tbody><tr><td> 设备 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 麻醉剂： </td></tr><tr><td>氯胺酮/甲苯​​噻嗪的混合物（麻醉可能会有所不同每个实验室/机构） </td><td> ketaset </td><td> NDC 0856-2013-01 </td><td> 10 ml小瓶</td></tr><tr><td></td><td></td><td></td><td> 手术治疗： </td></tr><tr><td>针尖，27遗传×1.25“ </td><td>物资服务</td><td> 25251 </td><td></td></tr><tr><td>大钝/钝弯剪刀（〜14.5厘米） </td><td>罚款科学工具</td><td> 14519-14 </td><td></td></tr><tr><td>直虹膜剪刀</td><td>罚款科学工具</td><td> 14058-11 </td><td></td></tr><tr><td&gt;标准镊子</td><td>罚款科学工具</td><td> 11027-12 </td><td></td></tr><tr><td>一双罚款（格雷夫）镊子</td><td>罚款科学工具</td><td> 11050-10 </td><td></td></tr><tr><td> 1大止血钳 - 弯或直（〜19厘米） </td><td> surgicaltools.com </td><td> 17.21.51 </td><td></td></tr><tr><td> 2标准的止血钳 - 直锯齿（14厘米） </td><td>罚款科学工具</td><td> 13013-14 </td><td></td></tr><tr><td> 1修改止血钳（15号洞，通过尖端提交） </td><td>罚款科学工具</td><td> 13013-14 </td><td></td></tr><tr><td> 15号钝或尖橄榄针（血流针） </td><td> fisnar </td><td> 5601137 </td><td></td></tr><tr><td></td><td></td><td></td><td> 灌注 ： </td></tr><tr><td> HyPerfusion系统或相当于&lt;/ TD&gt; <td></td><td></td><td></td></tr><tr><td>磷酸盐缓冲盐水，pH值7.4 </td><td></td><td></td><td></td></tr><tr><td>在0.1 M磷酸缓冲液，pH值7.4的4％多聚甲醛</td><td></td><td></td><td></td></tr><tr><td>浅的玻璃或塑料托盘，约10“×10” </td><td></td><td></td><td></td></tr><tr><td>碎冰</td><td></td><td></td><td></td></tr><tr><td>水浴（37℃） </td><td></td><td></td><td></td></tr><tr><td>定时器</td><td></td><td></td><td></td></tr><tr><td> 50毫升注射器</td><td>论文产业</td><td> NPMJD50LZ </td><td></td></tr><tr><td></td><td></td><td></td><td> 剖析 ： </td></tr><tr><td>霸IR标准的锐利/钝直剪刀（12厘米） </td><td>罚款科学工具</td><td> 14054-13 </td><td></td></tr><tr><td>中等弯曲或直rongeurs的（14-16厘米）或颅骨去除钳子</td><td>罚款科学工具</td><td> 16020-14 </td><td></td></tr><tr><td>直虹膜剪刀（〜9厘米） </td><td>罚款科学工具</td><td> 14058-11 </td><td></td></tr><tr><td>微型抹刀（双2“平面的两端，一圆形，锥形1/8”） </td><td>罚款科学工具</td><td> 10091-12 </td><td></td></tr><tr><td></td><td></td><td></td><td> 后固定及贮存： </td></tr><tr><td> 50毫升玻璃瓶</td><td></td><td></td><td></td></tr><tr><td> 40毫升肝素钠缓冲Hanks液与叠氮化钠（HBHS）（90毫克/升） </td><td></td><td></td><td></td></tr></tbodY&gt; </table>

whole animal perfusion fixation for rodents

固定的目标是迅速且均匀地保持在生命的样态组织。而直接放在组织在固定液工程小块组织，像完整的大脑，造成浸泡固定的问题更大的标本的固定液，因为没有达到该组织的所有地区在同一速率5,7。通常，在缺氧的变化开始前组织可保留12。直接通过循环系统灌注固定液的优点是化学能迅速到达使用天然的血管网的有机体的每一个角落。以最有效地利用循环系统，必须采取符合生理压力3。重要的是要注意，生理压力是依赖于所使用的物种。灌注固定的技术取决于被固定的组织，该组织将如何处理录制后。在这个视频我们描述了一个低成本，快速，控制和统一的固定程序，用4％多聚甲醛灌注，通过血管系统：通过对大鼠的心脏，以取得最好的免疫组织化学脑可能保存。这项技术的主要优点（与地心吸力系统）是最有效地利用循环系统。

Watch this Scientific Journal Video about Whole Animal Perfusion Fixation for Rodents at JoVE.com

Whole Animal Perfusion Fixation for Rodents

在这里，我们描述了一个低成本，快速，控制和统一的固定程序，用4％多聚甲醛灌注，通过血管系统：通过对大鼠的心脏，大脑获得尽可能最好的保存。

整体动物灌注固定鼠害

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

whole-animal-perfusion-fixation-for-rodents

Research

JoVE Journal

Neuroscience

379.3K 次观看.  University of Michigan. 在这里，我们描述了一个低成本，快速，控制和统一的固定程序，用4％多聚甲醛灌注，通过血管系统：通过对大鼠的心脏，大脑获得尽可能最好的保存。

视频: Whole Animal Perfusion Fixation for Rodents

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

自动化研究动物交流的交互式视频播放

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

科研

神经科学

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

在啮齿动物的行为测听的低成本设置

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

深部脑刺激与同步功能磁共振成像在鼠害

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Acrylic Resin Molding Based Head Fixation Technique in Rodents

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires surgical attachment of metallic frames on the skull. The attached frames are then clamped to a stationary platform resulting in immobilization of the head. However, metallic frames for head fixation have been technically difficult to design and implement in general laboratory environment. In this study, we provide a novel head fixation method. Using a custom-made head fixation bar, head mounter is constructed during implantation surgery. After the application of acrylic resin for affixing implants such as electrodes and cannula on the skull, additional resins applied on top of that to build a mold matching to the port of the fixation bar. The molded head mounter serves as a guide rails, investigators conveniently fixate the animal&#8217;s head by inserting the head mounter into the port of the fixation bar. This method could be easily applicable if implantation surgery using dental acrylics is necessary and might be useful for laboratories that cannot easily fabricate CNC machined metal head-posts.

Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

在鼠害丙烯酸树脂模塑基头固定技术

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

将套管植入啮齿动物的麦格纳池中

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.

Presented is a method for air-inflation with vascular perfusion-fixation of the lungs that preserves the location of cells within airways, alveoli and interstitium for structure-function analyses. Constant airway pressure is maintained with an air-inflation chamber while fixative is perfused via the right ventricle. Lungs are processed for histologic studies.

血管灌注固定的穆林肺的空气膨胀

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, ...