Name: stereotactically-guided ablation of the rat auditory cortex, and localization of the lesion in the brain
Uploaded: 2017-10-11T12:30:00.000+00:00
Duration: 9 min 29 s
Description: Watch this Scientific Journal Video about Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain at JoVE.com

这项研究得到了西班牙政府经济和竞争力部 (MINECO) 的资助, SAF2016-78898-C2-2-R。

这项研究严格按照西班牙的条例 (敕令 53/2013-法律 32/2007) 和欧洲联盟关于动物在生物医学研究中的使用和保护的指导方针 (指令 2010/63/欧盟) 进行. 
 1。大鼠的制备 注意: 我们在雄性大鼠身上进行了实验, 以避免荷尔蒙的变化. 
<ol> <li> 麻醉使用混合盐酸氯胺酮 (30 毫克/千克) 和盐酸嗪 (5 毫克/千克) 注射注; 这一剂量, 老鼠应该被深度麻醉约1小时. </li>
	<li> 捏住鼠和 #39 的脚趾; 没有撤退反射表明动物完全无意识。如果老鼠对捏的反应, 给予补充麻醉在最初剂量的三分之一. </li>
	<li> 剃光头皮, 用聚维酮碘消毒手术部位. </li>
	<li> 将该动物放在加热垫上以保持38和 #176 的温度; C、用两个耳棒和一个咬杆将动物和 #39 的头部稳定在一个立体的框架内。注意不要用耳条刺穿鼓膜. </li>
	<li> 通过在每只眼睛上应用一滴眼部凝胶或血清生理盐水来保护眼睛. </li>
</ol> 2。AC 在鼠的颞骨的位置 <ol> <li> 使用手术刀, 沿中线切开切口以露出颅骨并缩回覆盖颅骨表面的骨膜. </li>
	<li> 使用无菌棉签轻轻移除覆盖颅骨表面的任何血液, 以显示 bregma、lambda 和耳0根据鼠脑的 Paxinos 和沃森图谱 22 . </li>
	<li> 用手术刀在颅骨的背部插入颞肌附近进行切口。用针和缝合材料拉出肌肉, 将缝合材料固定在立体定向框架内;这将暴露颞骨。如果发生出血, 用冷消毒生理盐水冲洗. </li>
	<li> 在立体定向微中放置一根无菌直针, 确保其完全安全. </li>
	<li> 慢慢地放下针, 直到它在头骨的表面上方, 这样针头的尖端被设置为耳0。将此点设置为零, 并从此点确定坐标. </li>
	<li> 根据感兴趣的大脑区域, 改变立体定向坐标。通过利用鼠脑的 Paxinos 和沃森图谱 22 确定这些坐标。一旦确定了坐标, 请移动针以匹配这些坐标. </li>
	<li> 使用以下四点的坐标对 AC 进行定位: a: a/P =-5.8 mm, M/升 = +/-6.4 mm;B: A/P =-2.7 毫米, 米/升 = +/-6.4 毫米;C: A/P =-2.7 毫米, 米/升 = +/-8.67 毫米;D: A/P =-5.8 毫米, 米/升 = +/-8.67 毫米. 降低针到右上方的时间骨, 以可视化每一个这四点。使用记号笔, 标记颞骨上的点并连接它们以绘制一个长方形;该矩形将用作打开骨骼中的窗口的指南 ( 图 1 ). </li>
</ol> 3。交流的手术暴露 <ol> <li> 使用电钻和小钻头 (0.6 毫米和 #216;) 打开窗口。在 8000 rpm 的情况下钻取矩形的周长, 直到骨骼发出为止。用冷消毒生理盐水冲洗钻孔表面, 以防止损伤皮层下的结构。当骨骼让路时, 可以检测到阻力的下降。小心不要钻脑. </li>
	<li> 当边界松动时, 用细钳拉起覆盖骨, 并将其存放在冷无菌盐水中. </li>
</ol> 4。消融 AC <ol> <li> 使用外科显微镜 (10X), 用显微外科刀轻轻切开脑膜, 并用两个尖头镊子将其取出。如果发生出血, 用冷消毒生理盐水冲洗. </li>
	<li> 用外科吸入装置 (压力-0.24 bar) 轻轻吸出 AC, 并将其与无菌20克钝尖针结合。这一点是至关重要的, 需要非常小心地执行: 只吸入六皮层层, 而不是潜在的白质. </li>
	<li> 吸气直至穿孔动脉停止流血. </li>
	<li> 当吸入完毕后, 用提取的骨覆盖受伤部位, 并应用可吸收止血纱布. </li>
	<li> 让颞肌恢复其原来的位置, 然后缝合皮肤使用伤口夹 (9 毫米)。应用抗生素软膏 (请参阅 材料表 )。持续三天每日两次涂抹软膏. 
	注意: 每个应用程序都由一个薄薄的层在伤口上应用. </li>
	<li> 在手术后1小时内将丁丙诺啡皮下注射到鼠背部 (0.05 毫克/千克), 然后每8小时在72小时内注射. </li>
	<li> 将动物放在加热垫上直到它醒过来, 然后把它归还给它的外壳以恢复. </li>
	<li> 单独饲养动物以防止笼友接触缝合区, 并提供一些浓缩物品。每天更换木屑以防止感染, 并仔细检查动物是否恢复正常, 并没有出现任何不适的迹象. </li>
</ol> 5。组织集合 警告: 当处理甲醛 (粉煤灰), 固体和水, 佩戴个人防护设备 (PPE), 并使用安全柜. 
 注意: 使用热 (55 和 #176; C) 将 4% (w/v) 粉煤灰溶解为1x 磷酸缓冲溶液 (PBS), 准备750毫升甲醛溶液。用滤纸过滤甲醛溶液。通过溶解8.5 克氯化钠、0.25 克氯化钾和0.2 克 NaHCO 3 在1000毫升水中制备林格和 #39 溶液 (溶液 pH 值 = 6.9). 
<ol> <li> 让动物活下来, 只要它是研究所需要的。当研究与交流消融大鼠完成, 终麻醉它通过腹腔注射0.1 毫升戊巴比妥钠 (60 毫克/千克)。评估麻醉深度的脚趾捏和缺乏撤退反射. </li>
	<li> 当动物被深深麻醉时, 进行心内灌注 23 125 毫升的林格和 #39; s 溶液, 其次是750毫升的甲醛溶液, 使用1.8 毫米内径的针规. </li>
	<li> 当灌注完成后, 在第一个颈椎上斩首鼠. </li>
	<li> 使用剪刀从头部取出皮肤和肌肉, 并露出颅骨。使用剪刀切开和打开孔万能和去除头骨的后面. </li>
	<li> 用斯宾塞剪刀将横向切割成眶骨, 用 rongeurs 切开颅骨的顶端, 以使大脑暴露。注意不要损伤大脑. </li>
	<li> 一旦大脑暴露, 用细尖的镊子小心地取出硬脑膜。用手指轻轻地舀下并抬高大脑。提高大脑, 切断神经 unt它是免费的。将大脑浸泡在甲醛溶液中, 并将其储存在4和 #176; C 为 24 h. </li>
</ol> 6。交流病灶的定位 <ol> <li> 在 post-fixation 后, 小心地将大脑置于一个矢状大鼠脑基质中, 露出大脑的外侧表面. </li>
	<li> 将摄像头放置在皮层表面上方21厘米的位置, 使用摄像机, 选择 #34; 超级微距拍摄和 #34; 模式, 并拍摄大脑表面的图片. </li>
	<li> 将大脑置于大脑的冠状体中, 露出脑背表面, 再拍一张照片. </li>
	<li> 使用图像编辑器程序, 打开图像并将其向下缩放 50%, 以使其更容易处理。根据 Paxinos 和 Watson 坐标 19 标识图片上的 bregma、lambda 和耳0参照, 并在图片中标记它们的位置 ( 图 2 )。绘制的轮廓的消融的侧面图片的大脑。计算外围. </li>
	<li> 导入坐标图, 其中 AC 的主 (A1) 和辅助区域 (背和腹皮层) 位于与图片一起使用的编辑器程序的文件 18 中。点击地图, 并拖动它叠加到侧面照片的消融大脑. 
	<ol> <li> 使坐标映射的 bregma 和 lambda 参照与侧面大脑的图片中所标识的 bregma 和 lambda 参照一致. </li>
		<li> 使用鼻腔裂隙作为参考, 将大脑的图片调整到地图上, 并使其重合 ( 图 2B ). </li>
	</ol> </li> <li> 计算病变相对于 AC 所占区域的百分比. </li> </ol>

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提交人声明, 这项研究是在没有任何商业或财务关系的情况下进行的, 可被理解为潜在的利益冲突。

成功的脑部手术取决于两个因素: 在手术过程中和术后保持动物存活, 并准确定位感兴趣的区域。确保大鼠在手术过程中进行深度麻醉 (测试戒断反射), 并接受足够的止痛药和 non-ototoxic 抗生素有助于生存。另外, 老鼠应该被放在加热垫上, 直到它从麻醉中苏醒, 以避免体温过低。缝合将减少感染的易感性, 适当的技术是至关重要的: 动物会选择在他们的伤口夹, 所以他们应该被植入足够紧, 以防止移除, 而不会在伤口上施加太多的张力。
要准确定位 AC (或任何其他皮质区域), 重要的是要确定 bregma、lambda 和耳0的位置, 以使用它们作为计算目标区域的极限的参考。在计算坐标时的任何错误都会导致部分消融或其他周围地区的不受欢迎的吸入。因此, 针尖应只接触骨在耳 0, 然后翻译前后和译侧坐标根据本议定书所描述的。
在这篇手稿中, 我们也描述了如何手术暴露和蚀交流。有三关键步骤: 钻探过程中, 脑膜的打开和切除, 以及经穿刺消融。钻孔应以最低的速度以最小的压力进行, 因为高的钻孔速度产生的热量会影响附近的皮层结构。然而, 保持低速和冷却的钻孔区域与冷的无菌盐水应防止任何损害。此外, 最小的压力是必不可少的, 以避免突然断裂的头骨和随后的伤害的基础皮层。打开和切除覆盖 AC 的脑膜应小心, 以避免血管破裂。如果出血发生, 早期和晚期的预后通常是不赞同的, 这是值得怀疑的, 这种动物是否符合纳入标准的可靠研究。我们建议在这种情况下安乐死。最后, 吸入 (可能是最困难的方面, 在执行一个有效的损害), 必须限制在灰色物质。有两个指标可以帮助检测到白质的存在: (1) 颜色对比的变化, 因为白质比灰色的物质亮;(2) 从穿孔动脉停止出血。
在大脑中进行任何操作后, 必须评估在皮层中所做的程序的定位、大小和扩展, 以便随后对从该动物获得的数据进行分析和验证。在这篇手稿中, 我们详细介绍了如何使用我们组18之前描述的坐标图定位在皮层中进行的消融。这张地图是利用从组织学切片的连续部分重建获得的解剖参考, 与大鼠脑的 Paxinos 和沃森图谱22相关。因此, 该地图区分的主要 (A1) 和第二皮质 (背部和腹) 的 AC。这一坐标图的主要优点是, 它可以通过叠加一张从大脑外侧表面被放置在矢状脑基质中的图片来快速定位病灶。另一个优点是, 在解剖学方面经验较少的实验室可以通过将它改编成动物模型来使用这张地图。只需要设置 bregma, λ, 和耳0参考在一个控制灌流大脑的距离, 并相应地缩放地图。使用鼻腔裂隙作为参考, 调整大脑的图片到地图。此坐标图不能确定消融深度, 因此应在脑组织学切片中确定。
立体定向的方法与手术接触的交流是基本的方法, 可以很容易地适应任何研究者谁希望针对 ac 在鼠年。这可能是一个急性实验或一个需要植入永久设备。此外, 手术消融的 AC 已过去作为一个模型, 研究慢性皮质剥夺的影响, 听力。ac 消融也可用于研究单侧 ac 消融在其他皮层区施加的影响, 或作为中风模型。因此, 本文所描述的实验设计是一种有用的方法, 可以单独或组合应用于广泛的实验设计。

大鼠是听觉神经科学中最常用的动物模型之一。其行为的稳健性使其能够每天进行数百次试验。它的敏感性和光谱敏锐为听力1,2, 并且它的中央系统的解剖和功能组织, 可比较其他哺乳动物3, 使老鼠一个适当的动物模型分析各种各样的听觉神经科学的研究课题。大鼠的听觉皮质 (AC), 特别是, 一直是一些解剖和生理学研究的主题, 试图了解它的结构, 组织, 并在声音处理3的作用。现在, AC 已成为流行的神经学家感兴趣的经验依赖可塑性, 听觉知觉, 突触基础的接受外地组织, 和皮质控制的声音处理在皮层下听觉核4,5,6,7,8,9。为了应对这些新方法所带来的挑战, 能够准确定位和手术暴露交流的程序将加速研究工作。立体定向技术使大脑中的特定区域在没有生理学测试的情况下很容易本地化。虽然动物之间的大脑大小稍有不同, 但任何脑区的位置都可以用大鼠脑部颅骨上地标的立体坐标来确定。
限制消融的 AC 是手术切除的感觉区的皮层最直接相关的听觉。与其他用于阻断 ac 活动的方法相比, 如冷却或局部利多卡因注射液10,11,12, 手术切除 ac 会导致慢性功能丧失。因此, 交流消融更适合研究皮质剥夺的长期影响, 以及随后的损伤可塑性现象。立体定向方法与手术消融的结合已成功地用于研究皮质控制剥夺的生理、行为和分子效应13,14,15 ,16,17,18,19。例如, 用双侧交流消融的大鼠模型研究了皮层消融对听觉惊吓反射和听觉脑干反应 (ABR)16的影响。最近, 我们比较了单侧与双侧消融对大鼠 AC 产生的影响在 ABR 阈值, 振幅, 和潜伏期在不同时间点后的伤害18。此外 , 还利用大鼠限制流消融模型研究了 corticofugal 通路变性在下位 collicus 中的作用 :13、14、15和内耳17 ,19。在大脑中进行这种操作后, 有必要评估皮质内病变的定位、大小和外延。虽然非常有用, 但基于神经元响应的 tonotopic 映射的主要限制20,21是在大鼠大脑中定位听觉场所需的电生理学技术。由于并非所有的实验室都有必要的设备和/或专门知识来进行此类录音, 因此我们构建了一个坐标图, 该地图基于交流电的细胞极限与大脑表面的图像18的转移。这张地图是非常有用的定位 AC 没有生理学测试。
本协议描述了一种方法的 stereotactically 引导定位, 手术暴露和消融的交流在大鼠。它还描述了如何使用我们的坐标图18来轻松地将病灶的延伸定位在被消融的大脑表面的图片上。

<table><tbody><tr><td>Stereotaxic frame</td><td>David Kopf Ins.</td><td>900</td><td></td></tr><tr><td>Surgical microscope</td><td>WILD M650 Heerbrugg</td><td></td><td></td></tr><tr><td>Heating pad</td><td>DAGA</td><td></td><td></td></tr><tr><td>Dental micromotor</td><td>W&amp;amp;H elco</td><td>5118</td><td></td></tr><tr><td>Diamond burr</td><td>B Braun</td><td>GD021R</td><td>0.6 mm</td></tr><tr><td>Surgical suction device</td><td>Atmos</td><td>Atmoforte E2</td><td></td></tr><tr><td>Ketamine</td><td>Merial</td><td></td><td>30 mg/kg</td></tr><tr><td>Xylazine</td><td>Bayer</td><td></td><td>5 mg/kg</td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>SM-11</td><td></td></tr><tr><td>Scalpel</td><td>Lawton</td><td></td><td></td></tr><tr><td>Povidone iodine</td><td>Meda</td><td>Betadine</td><td></td></tr><tr><td>sterile saline serum</td><td>B.Braun</td><td></td><td></td></tr><tr><td>20G sterile needle</td><td>Terumo Neolus</td><td></td><td></td></tr><tr><td>Cotton tips</td><td></td><td></td><td></td></tr><tr><td>Suture material</td><td>B.Braun</td><td></td><td></td></tr><tr><td>Antibiotic Ointment</td><td>Quadriderm (Betametasona, Gentamicina, Clotrimazol) - Schering-Plough</td><td></td><td></td></tr><tr><td>Forceps</td><td>dimeda</td><td>10.331.12</td><td></td></tr><tr><td>Surgical needles</td><td>World Precision Instruments</td><td>501940</td><td></td></tr><tr><td>Buprenorphine</td><td>Indivior UK</td><td>Buprex</td><td>0.05 mg/kg</td></tr><tr><td>Scissor</td><td>dimeda</td><td>08.120.15</td><td></td></tr><tr><td>Spencer scissor</td><td>dimeda</td><td>08.804.14</td><td></td></tr><tr><td>Rongeurs</td><td>Lawton</td><td></td><td></td></tr><tr><td>Microsurgical knife</td><td>MSP</td><td>7503</td><td></td></tr><tr><td>Absorbable hemostatic gauze</td><td>Surgicel</td><td></td><td></td></tr><tr><td>Saggital rat Brain Matrix</td><td>Activational systems Inc.</td><td>RBM-1000DV / RBM 4000C</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Vetoquinol</td><td></td><td>60 mg/kg</td></tr><tr><td>Camera</td><td>Olympus 5.1 MP</td><td>C-5060 wide zoom</td><td>lens F2.8-4.8</td></tr><tr><td>Wound clips</td><td>Reflex 9</td><td></td><td>9 mm</td></tr><tr><td>Canvas 12</td><td>ACD Systems</td><td></td><td></td></tr><tr><td>needle gauge</td><td></td><td></td><td>diameter 1.8 mm</td></tr><tr><td>Separatory funnel</td><td>labbox</td><td>11409</td><td>500 mL</td></tr><tr><td>GluA2 primer Forward</td><td>GeneBank</td><td>NM_017261</td><td>CGGCAGCTCAGCTAAAAACT</td></tr><tr><td>GluA2 primer Reverse</td><td>GeneBank</td><td>NM_017261</td><td>TTGTAGCTGGTGGCTGTTGA</td></tr><tr><td>GluA3 primer Forward</td><td>GeneBank</td><td>NM_032990</td><td>ATTGCTGATGGTGCAATGAC</td></tr><tr><td>GluA3 primer Reverse</td><td>GeneBank</td><td>NM_032990</td><td>TTTGCATTGTCGCAAGTCTC</td></tr></tbody></table>

stereotactically-guided ablation of the rat auditory cortex, and localization of the lesion in the brain

大鼠听觉皮层 (AC) 在听觉神经学研究者中越来越受欢迎, 他们对经验依赖性可塑性、听觉知觉过程以及皮层下听觉核声音处理的控制有兴趣。为了应对新的挑战, 准确定位和手术暴露听觉皮层的程序将加速这项研究工作。立体定向神经外科通常用于动物模型的临床前研究, 以嫁接在听觉皮层的一个预定位置的针或电极。在下面的协议中, 我们采用了一种新颖的立体定向方法。我们在老鼠的颞骨表面确定了四坐标点, 以定义一个窗口, 一旦打开, 就能准确地暴露 AC 的主 (A1) 和次 (背和腹) 皮质. 使用这种方法, 我们然后进行手术消融的 AC。在进行这种操作后, 有必要评估皮质内病变的定位、大小和外延。因此, 我们也描述了一种方法, 以方便地定位的交流烧蚀使用的坐标图构造通过转移细胞限制的 ac 到大脑的表面。在坐标图中, stereotactically 引导位置和消融的交流与损伤区域的定位相结合, 有助于验证从动物获得的信息, 并导致更好的分析和对数据的理解。

我们进行了 stereotactically 引导位置, 手术暴露, 并单方面消融三大鼠的 AC。病灶的定位证实, 消融在三大鼠中的表现, 侵犯了 ac (主、背和腹侧皮质) 的主要分支, 并包括总 ac 区域的80至100% 的范围 (图 2B)。
这里描述的执行限制性 AC 消融的协议以前在我们的实验室被使用了研究皮层控制剥夺的长期作用在皮层下的听觉原子核, 并且随后现象的可塑性。在这些研究中, 通过应用生理 (ABR)、行为 (惊吓反应、脉冲抑制等) 验证了交流消融的协议;PPI) 和分子 (DNA 微阵列, qPCR, 和西部印迹) 方法13,14,15,16,17,18,19。在这里, 为了证明我们的协议的有效性, 我们让三交流消融大鼠存活一周, 并收集了耳蜗在组织收集的步骤, 以研究变化的表达最相关的 AMPA 亚基目前在成人耳蜗, GluA2 和 GluA3, 由 qPCR。交流切除大鼠和假控制动物的转录记录的比较, 所有的手术过程, 但不是皮质消融表现出了一个向下调节的 GluA2, 并对 GluA3 在两个耳蜗 (图 3), 这与我们以前的研究19是一致的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56429/56429fig1.jpg"> 
图 1: 在三不同的手术步骤中, 大鼠颞骨的图像.(A) 将 AC 的立体定向坐标转移到颞骨。四点的坐标是: a: a/P =-5.8 毫米, 米/升 = +/-6.4 mm;B: A/P =-2.7 毫米, 米/升 = +/-6.4 毫米;C: A/P =-2.7 毫米, 米/升 = +/-8.67 毫米;D: a/P =-5.8 毫米, 米/升 = +/-8.67 毫米. (B) 坐标用于在时间骨骼的表面绘制一个矩形, 以引导窗口的打开。(C) 显示钻孔后在骨骼中打开的窗口。在脑表面可以观察到具有血管的脑膜。R: 侧, D: 背。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56429/56429fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56429/56429fig2.jpg"> 
图 2: 在大鼠大脑中定位病灶的过程.(A) 用 stereotactically 植入的针头和 Bregma (根据 Paxinos 和沃森坐标19) 对 AC 消融脑的背表面的照片。虚线标记 Bregma 和耳0在 9 cm x 9 cm 格子中的位置, 以及在大脑的背表面。(B) 被消融的大脑侧面的照片叠加到 AC 的坐标图上。病变的周长在图片中被标记为红色。在地图上, AC 区域的周长被标记为黑色。在本例中, ac 消融占 ac 占总面积的百分比为84.79%。AC: 听觉皮层, IA: 耳间, FR: 鼻腔裂。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56429/56429fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56429/56429fig3.jpg"> 
图 3: 单侧 AC 消融7天后 AMPA 受体亚基 GluA2 和 GluA3 mRNA 水平的变化 post-lesion.结果显示为平均±标准偏差的褶皱变化。GluA2 成绩单的更改以蓝色表示。GluA3 成绩单的更改以红色显示。在手术后7天, 耳蜗 (信息学和对侧至消融) 明显减少 GluA2 和 GluA3 的增加, 相对于假对照无皮质消融;这与我们以前的结果19是一致的。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56429/56429fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain at JoVE.com

Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain

Stereotactically 对大鼠听觉皮层的诱导消融及脑内病变的定位

我们描述了一种方法的 stereotactically 引导的位置, 暴露, 并消融大鼠的听觉皮层。使用坐标图对消融的定位进行评估。

The rat auditory cortex (AC) is becoming popular among auditory neuroscience investigators who are interested in experience-dependence plasticity, ...

stereotactically-guided-ablation-rat-auditory-cortex-localization

Universidad San Sebastian

Research

JoVE Journal

Behavior

11.5K 次观看.  University of Salamanca. 我们描述了一种方法的 stereotactically 引导的位置, 暴露, 并消融大鼠的听觉皮层。使用坐标图对消融的定位进行评估。

视频: Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

行为学

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

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

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 ...

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 ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

微电极引导电极植入到大鼠丘脑底核的长期深部脑刺激

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

啮齿动物联合局部脑电图和局部电位的同步记录

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

一种基于微 ct 的小动物脑损伤和定位电极表征方法

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the patient&#39;s pathophysiology. Despite the long history of DBS, its mechanism of action and appropriate protocols remain unclear, highlighting the need for research aiming to solve these enigmas. In this sense, evaluating the in vivo effects of DBS using functional imaging techniques represents a powerful strategy to determine the impact of stimulation on brain dynamics. Here, an experimental protocol for preclinical models (Wistar rats), combined with a longitudinal study [18F]-fluorodeoxyclucose positron emission tomography (FDG-PET), to assess the acute consequences of DBS on brain metabolism is described. First, animals underwent stereotactic surgery for bilateral implantation of electrodes into the prefrontal cortex. A post-surgical computerized tomography (CT) scan of each animal was acquired to verify electrode placement. After one week of recovery, a first static FDG-PET of each operated animal without stimulation (D1) was acquired, and two days later (D2), a second FDG-PET was acquired while animals were stimulated. For that, the electrodes were connected to an isolated stimulator after administering FDG to the animals. Thus, animals were stimulated during the FDG uptake period (45 min), recording the acute effects of DBS on brain metabolism. Given the exploratory nature of this study, FDG-PET images were analyzed by a voxel-wise approach based on a paired T-test between D1 and D2 studies. Overall, the combination of DBS and imaging studies allows describing the neuromodulation consequences on neural networks, ultimately helping to unravel the conundrums surrounding DBS.

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

We describe a preclinical experimental method to evaluate metabolic neuromodulation induced by acute deep brain stimulation with in vivo FDG-PET. This manuscript includes all experimental steps, from stereotaxic surgery to the application of the stimulation treatment and the acquisition, processing, and analysis of PET images.

体内 正电子发射断层扫描揭示大鼠深部脑刺激诱导的活动模式

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the ...

Implanting a Wireless Bidirectional Microelectrode System in a Rat Brain

Source: Melo-Thomas, L., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56299">A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats.</a> J. Vis. Exp. (2017)
The video demonstrates a procedure for implanting a wireless bidirectional multichannel electrode unit into a rat brain to record and stimulate multichannel neural networks.

在大鼠大脑中植入无线双向微电极系统

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

A Stereotactically Guided Ablation Technique for Auditory Cortex Removal in Rats

Source: Lamas, V., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56429">Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain.</a>&#160;J. Vis. Exp. (2017).
This video demonstrates a stereotactically-guided surgical procedure for ablating the auditory cortex in a rat. The steps include identifying anatomical landmarks in an anesthetized rat secured in a stereotactic frame, creating a window on the skull above the auditory cortex, aspirating the cortical layers, and providing post-operative care to assess the impact of the ablation on sound processing.

Source: Lamas, V., et al. Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain.&#160;J. Vis. Exp. ...

Stereotactically 对大鼠听觉皮层的诱导消融及脑内病变的定位

摘要

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此视频中的章节

同时在啮齿动物脑功能磁共振成像和电

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

深部脑刺激与同步功能磁共振成像在鼠害

微电极引导电极植入到大鼠丘脑底核的长期深部脑刺激

啮齿动物联合局部脑电图和局部电位的同步记录

一种基于微 ct 的小动物脑损伤和定位电极表征方法

体内正电子发射断层扫描揭示大鼠深部脑刺激诱导的活动模式

在大鼠大脑中植入无线双向微电极系统

A Stereotactically Guided Ablation Technique for Auditory Cortex Removal in Rats

Stereotactically 对大鼠听觉皮层的诱导消融及脑内病变的定位

摘要

探索更多视频

此视频中的章节

同时在啮齿动物脑功能磁共振成像和电

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

深部脑刺激与同步功能磁共振成像在鼠害

微电极引导电极植入到大鼠丘脑底核的长期深部脑刺激

啮齿动物联合局部脑电图和局部电位的同步记录

一种基于微 ct 的小动物脑损伤和定位电极表征方法

体内 正电子发射断层扫描揭示大鼠深部脑刺激诱导的活动模式

在大鼠大脑中植入无线双向微电极系统

A Stereotactically Guided Ablation Technique for Auditory Cortex Removal in Rats

体内正电子发射断层扫描揭示大鼠深部脑刺激诱导的活动模式