我们感谢C. Montojo，J.雷耶斯，和M.装甲的技术咨询和援助。这项工作是由卫生部授予F31 NS063550美国国家卫生院（以DAL），一个罗勒奥康纳入门学者奖和补助金从Klingenstein基金和NARSAD（对某人）的支持。 SB是一个WM凯克杰出青年学者在医学研究。

动物用法获得标准的管理和使用协议批准的机构动物护理和使用委员会。当前协议是用于在5.5-10周龄成年C57BL6 / J小鼠焦照射研究开发的，如先前所述（ 图2）2。然而，其他的年龄和小动物物种（大鼠，仓鼠，松鼠， 等等 ）也可使用，其前提是有效的麻醉协议和放射线参考图谱允许识别的感兴趣区（ROI），该区域是可用的。 1。 CT引导下放射性平台的校准执行基于胶片的校准为辐射剂量预先放射性平台上的每个准直器的大小和所用的X射线管的过滤器类型。为了目标照射的电弧束在所希望的目标部位，使用的辐射剂量规划软件49 availablE在大多数商业辐射平台计算的基础上辐射剂量要求的交货时间，感兴趣区域（ROI），根据鼠标的CT扫描的区域的深度，和机器人平台的旋转速度，在此细节被排除在外这个协议，因为他们的辐射平台之间的差异。用于可视化的腹侧基底丘脑2（ 图3）与CT成像的目的，操作该X射线管具有0.4毫米的焦斑和100 kVp的的电子束能量为1 mm的铝过滤。为HPZ的焦点照射下，工作在225 kVp的13毫安的X射线管，并用0.15mm的铜过滤的1毫米焦斑，以提供辐射的10戈瑞为0.6厘米的组织深度过治疗4.6分钟的时间。不同的投资回报率之间的目标将需要计算不同的参数。 <ol><li>如果学习高脂饮食对神经发生在正中隆起的影响如前所述2，取得四周龄雌性C57BL6 / J小鼠从杰克逊实验室鼠标，每笼屋四只老鼠。从正常饮食切换到食物高脂肪的饮食五周龄。让小鼠，使其适应新的住房和食物。进行治疗的CFIR在5.5周龄。运输科达到包含放射性平台手术室。在传输过程中最大限度地减少压力水平。准备异氟醚麻醉气体室。然后，添加一个鼠标进入室内。与此同时，对于后处理准备加热垫（低设置）。 </li><li>一旦鼠标下，不再回应脚垫压缩，使被摄物体的辐射平台，并放置在机器人舞台的固定床上。将受试者的口腔进入鼻锥麻醉杯，并进入牙齿咬后卫（ 图1D）。躺在鼠标平放在固定的床和检查，看它是否继续维持反应迟钝。如果是这样，大盘向下鼠标在床上用纱布胶带。当鼠标录音下来到床上中，确保磁头平整水平面。这可以通过拉起耳朵，如果他们被拉平眼看来确定。当鼠标处于正确的位置，关闭铅防护罩。 </li><li>使用板载软件实验者的放射性平台获取所述计算机断层扫描（这将在平台之间不同），这将提供鼠标受试者的三维解剖结构的扫描。检查以查看是否鼠标头平整水平面。如果不是，请重复步骤1.2 - 1.3直到拍摄对象的头部平整。 </li><li>通过CT图像确定的投资回报率。使用一个45°角的水平面上， 如图3E中所示计算距离从ROI到颅骨的表面上。 </li><li>利用机载软件，采取从上面如图4A所示的X-射线鼠标主体。然后，从放射性平台去掉鼠标，把加热垫，并监控到主动。 </li><li>从冠状CT图像，计算至少三只小鼠，以确定输送的剂量平均的ROI解剖深度。如从先前的研究中，其中2照射10 Gy的给药至腹侧基底丘脑一个例子，从头骨（从45°角）的ROI的深度为0.66公分（参见图3E）。知道这一点，研究人员利用安装在他们的平台上放射剂量规划软件（DPS）来计算合适的治疗旋转速度和长度，以达到所需的剂量的投资回报率。 </li><li>在确定治疗的持续时间和所述机器人阶段的旋转速度随剂量规划软件，测量计算的参数嵌入在水等效塑性模拟小鼠模型GAFCHROMIC辐射敏感膜的剂量分布。要做到这一点，嵌入了四个垂直堆叠的水相当于塑料块29的之间有三个GAFCHROMIC辐射敏感薄膜图4B所示。 </li><li>将装有机器人舞台GAFCHROMIC电影模拟小鼠模型，并与新计算出的参数运行的焦点照射光束。例如，要定位的腹侧基底丘脑，输入为0.15mm的Cu过滤器，225 kVp的参数13毫安，1毫米直径的辐射束的设定，45°机架角度，130°/秒旋转，和4.6分钟实现的ROI放射剂量为10戈瑞。 </li><li>下面照射，检查薄膜图案和辐射剂量强度。对于一个360°的角度旋转，并列出为目标的腹侧基底丘脑，黑圈在等角点上面的薄膜，小脆点在等角点膜，并且在等角点下方的膜更轻环对应于所述锥形的参数照射的光束给药将观察到（ 图4B）。 </li><li>叠加等角点GAFCHROMIC薄膜在X射线的鼠标主体从该参数是卡尔计算得到。经照射的焦点在等角点应该与所需的ROI区域重叠， 如图4C所示 。 </li></ol>另一种方法：如果难以瞄准大脑的投资回报率依旧，使用含碘造影鞘内注射，以提高在CT图像心室的可视化。为简洁起见，本程序是离开了这个协议的，但是以前描述35，50。碘造影将提供额外的心室的地标（ 图5A）。 2。确定照射光束精度进一步确认CFIR光束精度通过在组织2，35，51上的辐射束的直接可视化。要做到这一点，进行免疫组化检测γH2AX51，组蛋白和DNA双链断裂的早期标志物。鼠标受试者必须进行心脏灌流固定照射后一小时内。以下辐照ΥH2Ax的振动性，DNA修复很快随之而来，和水平显著下降35。 <ol><li>重复步骤1.1 - 1.3。 </li><li>后的目标是确定在CT上，鼠标主体下的机械手控制移动对准这个目标与辐射传递光束。输入计算参数（转速和治疗时间的长度，以达到所需的剂量）从步骤1.6成剂量规划软件，并开始治疗。治疗交付与龙门指出从垂直45°，当鼠标绕一个垂直取向的轴线。 </li><li>照射后，对小鼠进行科目穿心灌注。 1小时52内进行灌注。继在4％PFA / PBS灌注，浸泡修复大脑，并在4℃下轻轻摇动过夜</li><li>第二天早上，洗5分钟，在PBS 3倍，除去多聚甲醛固定液。然后，浸泡在1×PBS中30％蔗糖上的摇杆在4℃下</li><li>轻轻摇动，在4°C（我们请手动12-16小时），直到大脑沉到管底。这可作为冷冻保护的一步。一旦大脑下沉，取出大脑穿孔勺子，以防止多余的30％sucrose/1x PBS转移。快速嵌入在干冰冷冻介质。漩涡冷冻介质使用的枪头和塑料模具使大脑。 </li><li>一旦完全冻结，脑块传送到-20℃冰箱保存。 </li><li>上时，免疫组织化学将被执行的那天，部分冠，在40微米厚，并漂浮到24孔板中含有PBS用薄画笔。段应保持在正确的串行顺序为免疫染色，以确定目标区域的照射范围。 </li><li> Prewarm的0.01M柠檬酸钠溶液至80℃，在水浴中制备用于抗原修复步骤。同时，而柠檬酸钠预热，进行5分钟清洗3倍的0.01 M的PBS。 </li><li>一旦柠檬酸钠溶液在80℃下，浸入脑切片我n中的柠檬酸钠缓冲溶液。留在水浴中于80℃下反应1小时。 </li><li>删除部分，并允许柠檬酸钠溶液达到室温，然后在0.01 M的PBS进行3个5分钟的清洗。 </li><li>阻断大脑切片进行1小时的PBS-的Triton含5％正常山羊血清。 </li><li>孵育含5％正常山羊血清以1:700浓度小鼠抗磷酸化H2AX的初级抗体在4℃下脑切片过夜在PBS-的Triton </li><li>第二天，进行15分钟，洗涤3倍于0.01M PBS-的Triton。 </li><li>孵育脑切片包含与缀合了488纳米fluorphore在1:500的浓度为2小时的山羊抗小鼠第二抗体5％正常山羊血清的PBS-的Triton。 </li><li>进行15分钟洗涤3倍的0.01 M的PBS，海卫一。 </li><li> 10分钟进行可视化核执行4&#39;，6 - 二脒基-2 - 苯基吲哚（DAPI）染色（在PBS中1:5000）。然后，洗脑切片5分钟，用PBS洗涤。 </li><li>安装在静电通道arged显微镜载玻片在PBS中浮动部分。擦去多余的PBS中，并允许片干燥。使用封固剂盖玻片的载玻片，并允许滑动到在黑暗中在室温下干燥过夜。 </li><li>利用荧光显微镜连续冠状切片图像。 ΥH2Ax免疫染色（绿色）指示网站的照射。 DAPI（蓝色）是一种核染色（ 图5B）。 </li></ol> 3。鼠标的制备主题为焦点照射检查ΥH2Ax免疫染色的结果。一旦满意的校准和照射光束瞄准，进行该项实验。在这一点上，对待鼠标（从动物设置梁竣工交付）所需的总时间是10 Gy的治疗用1毫米束约10-15分钟。 <ol><li>四阶周龄C57BL6 / J雌性小鼠杰克逊鼠标实验室。房子四只小鼠每笼，并从正常饮食食品切换到高脂肪饮食五周龄。耳朵打孔老鼠给他们独特的识别标记。每天监测小鼠的健康。注意：金属标记物不能使用，因为它们将导致在划线上的CT伪影。 </li><li>称取小鼠辐射或假治疗的前一天。小鼠分割成两个同伙的辐射或假治疗，并确保有同伙之间的重量没有显著差异。在治疗的日子，当老鼠是六周大，再次称重所有小鼠，并记录它们的质量。轻轻地运送两个同伙到放射性平台。小心，尽量减少压力水平。 </li><li>准备异氟醚麻醉气体室。麻醉两只老鼠，一个预定的照射组，而另一个在假手术对照组。准备加热垫的术后治疗低设置中设置。 </li><li>按照步骤1.2 - 1.4鼠标接收照射。对于假鼠标，鼠标保持在麻醉室，同时治疗是怎么回事。让河畔Ë的麻醉室靠近CFIR平台等环境辐射的任何影响都考虑进去后，目标被确定在CT上，移动鼠标主题下机器人控制，以使这一目标与辐射传递光束。输入计算参数（转速和治疗时间的长度）成剂量规划软件。 </li><li>一旦照射治疗不彻底，同时返回假和照射小鼠加热垫，并监视，直到他们醒来。 </li><li>同时返回假和照射小鼠动物设施。监控每一天。称取小鼠每半星期。以确认目标丘脑增殖区的照射下，管理的BrdU腹腔注射（50毫克/千克）3天后处理和检查由下列初始的BrdU曝光colabeling免疫组化的BrdU和神经元标记一个月组之间的神经发生（ 图6） 2。 </li></ol>

JW与Xstrahl，公司的一个研究经费和咨询协议

CT引导下焦照射（CFIR）是一种新型的，完整的系统方法能够使用CT-32的指导提供辐射场为目标，在机器人控制的小动物。 CFIR的交付高度集中的光束，以小动物模型的功能提供了新的研究机会弥合实验室研究和临床翻译。本文介绍了精确的辐射交付现金流利率的方法来专门针对下丘脑神经祖人口。我们在这里展示了如何通过免疫组化通过X线片和脑组织中校准和确认辐射传递特异性。 <p ...

最近的发现表明，成年哺乳动物大脑可以经历显着的塑性度。整个成年哺乳动物大脑1专门龛产生成人出生的神经元。什么是这些成年出生的神经元的功能？而越是这样，就它们在生理和行为产生影响？研究这个话题历来集中在侧脑室和海马的颗粒下层区的脑室下区，然而，最近的研究已经在其他脑区，如哺乳动物下丘脑2表征神经。神经发生已经报道在产后和成人丘脑2-10，和这些新生儿下丘脑神经元的功能仍然是调查的有源区。 成人出生的神经元的功能特性保持在一般的神经科学领域的显著挑战。规范的选择性抑制IFIC神经祖人群仍然有限，由于缺乏可用的分子标记所特有的单神经祖人口11。因此，选择性删除成人出生的通过基因靶向这些神经前体细胞神经元仍然很困难。同样，病毒递送到目标成人出生的神经元遭受潜在的混杂变量，如引入损伤和炎症到环境12。 新的放射性工具但是不断出现，，允许一个绕过这些的困惑，并通过在小动物准确和精确的解剖定位研究这些问题。电离辐射抑制新的神经元的诞生和分化，并允许非侵入性方法，针对神经祖人口13-15。最近，我们描述的哺乳动物下丘脑正中隆起（ME）的，我们称之为下丘脑增殖区（HPZ）2生发区</suP&gt;。我们发现，当年轻的成年雌性小鼠给予高脂饮食（HFD），在神经发生高脂饮食喂养的小鼠的水平比他们的正常饮食（NC）大大高于美联储控制在这个ME区域2。来测试下丘脑ME内成年神经是否调节代谢和体重，我们试图扰乱这一过程。正中隆起是一个小的单边结构在第三脑室从中调节激素释放的基极。为了抑制增殖和随后的神经发生不改变该大脑区域的其他生理功能，我们开发了一个非侵入性的焦点照射技术选择性抑制下丘脑正中隆起2刚出生的成年神经元的诞生。 一些团体已采用辐射抑制典型地区14-28神经。然而，以往的影像学方法一般都针对大面积，或ofteÑ​​无意中还针对性其中神经发生已经报道多个脑区域，因此很难明确地关联起来，在特定的神经祖种群缺陷观察到的任何行为缺陷。为更有针对性的照射能力通过结合çomputer成像制导成像与 f OCAL光束红外辐射（CFIR）传递来实现精确的解剖定位29-36放射性平台提供的。可针对特定的神经祖群35的辐射束小至0.5mm的直径。这种方法使我们能够针对下丘脑ME和逮捕增殖并阻止神经发生的小动物。下面就这些祖人口放射治疗，生理和行为测试可以进行照亮成人出生的细胞的潜在功能。焦点目标是为我们的应用程序，因为特别重要的脑垂体是靠近下丘脑正中隆起，垂体照射可能会影响荷尔蒙功能，随后混淆的结果。 用于神经照射后的抑制的生物学基础仍不清楚。上一页辐射的研究依赖于大面积的横梁，得出的结论是神经的抑制是通过炎症反应14，37介导的。因此，它是非常不清晰焦点照射是否能抑制神经发生的，因为它不会引起大幅度的炎症反应。然而，最近的研究本课题组在海马经典神经源性地区已经证明，用10 Gy的剂量高度聚焦照射可以抑制神经发生的至少4周照射35后。 询问在正中隆起成人出生的下丘脑神经元的功能，我们使用了精密的辐射ÐEVICE能够提供CT成像结合小直径的辐射光束，以抑制ME的神经发生。使用连接到机架旋转超过360°范围内的X射线管，我们提供电弧束微照射光束与使用机器人控制的试样台，其允许动物对象的过程中放射治疗的旋转（ 图1）的。高分辨率X射线探测器来采集图像时，扫描架处于水平位置33。在这项研究中，CT图像重建为0.20毫米的各向同性体素尺寸。板载CT成像允许目标的识别，而动物是在治疗位置。使用的CT导航剂量规划软件，该软件是包含在我们的市售放射性平台，目标是局部的。通过CT成像提供本地化的投资回报率后，动物是由机器人样品台有四个度移动到适当的治疗位置自由里斯（X，Y，Z，θ）。通过龙门架和机器人阶段角度的组合，光束可 ​​以从几乎任何方向上的相对输送到动物，和立体定向圆弧状的治疗是可能的29。对于这些和所有其他的成像研究中，小鼠被放置在一个固定装置，可以让递送麻醉剂异氟烷气体的同时限制移动。固定化床是CT兼容，并且连接到所述机器人样品载物台34。 我们预计现金流利率将提供观念上的进步在很多研究领域。虽然我们使用的下丘脑正中隆起的放射性靶向作为该技术的原理证明，CFIR可以用来瞄准的原则任何微小的模式生物身体的任何区域。在神经科学，例如，我们预计这种技术可以用来评价积极增殖祖人口已建议EXIS功能吨，其他周器官，如最后区38，39，穹窿40，和垂体41。关于成年神经发生的功能性作用，并确定行为的因果作用的长期争论现在也可以更好地解决。在鸣禽，这种技术可能会解决成年神经发生在维持鸟鸣42，这已经阻碍了选择性抑制神经发生在特定的大脑区域的功能的强大和季节性行为中的作用。了解这种强大的行为模式可能揭示新的洞察成年神经发生的其他调控性二态行为的作用。可替代地，在新陈代谢字段，CFIR可以用来探索的肝细胞增殖的作用和它的代谢和能量平衡的作用的方面。可能在多个研究领域的概念提前通过引入这一技术的提高。 43，44。因此，我们推广这个现金流利率协议所要求的所有研究的平台，而不是那些具体的SARRP步骤。 CFIR超过先前放射方法的优点，以抑制神经发生的是，这种技术允许定位三维体积图像的指导和剂量的靶向，适形的剂量最小化暴露于非定标大脑区域，精度高的光束的几何形状允许保形的剂量分布与锐波束边界。我们概述了如何使用CT引导下成像剂量定向到特定的解剖区域，并在这样做时，如何以可视化的辐射用免疫组织化学染色法对γ-H2AX，DNA双链断裂35，45-48的标记物剂量直接在组织中的分布。使用这种方法的神经性龛选择性照射可能对揭示的生理和疾病的新成人出生的神经元的功能作用显著影响。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>SARRP research platform</td>
			<td>Xstrahl</td>
			<td>RS225A</td>
			<td><a href="http://www.xstrahl.com/xstrahlrs225.htm" target="_blank">http://www.xstrahl.com/xstrahlrs225.htm</a></td>
		</tr>
		<tr>
			<td>SARRP irradiation bunker</td>
			<td>Xstrahl</td>
			<td></td>
			<td>Optional, but radiation exposure should be contained with alternative lead shielding</td>
		</tr>
		<tr>
			<td>GAF chromic film</td>
			<td>IPS</td>
			<td>GAFchromic ETB2</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse phantom</td>
			<td>Gammex</td>
			<td>457</td>
			<td>Purchase 0.5 cm x 30 cm x 30 cm solid water slabs from Gammex and cut to desired size.</td>
		</tr>
		<tr>
			<td>Mouse anti-phospho-histone H2AX Ser139 antibody</td>
			<td>Millipore, Inc.</td>
			<td>05-636</td>
			<td>clone JBW301</td>
		</tr>
		<tr>
			<td>High-fat rodent diet</td>
			<td>Research Diets</td>
			<td>D12492i</td>
			<td>60% of the calories as fat, food should be irradiated</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter Healthcare Corporation</td>
			<td>10019-360-40</td>
			<td></td>
		</tr>
		<tr>
			<td>0.01 M Sodium citrate</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>1.471 g of sodium citrate dissolved in 500 ml deionized water</td>
		</tr>
		<tr>
			<td>Superfrost Plus slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>nuclear counterstain</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Vectashield or Gelvatol is preferred</td>
		</tr>
	</tbody>
</table>

functional interrogation of adult hypothalamic neurogenesis with focal radiological inhibition

成人出生的神经元的功能特性仍然是一个显著的挑战。的方法来抑制成年神经发生通过病毒侵入或交付的转基因动物具有使结果的解释从这些研究很难潜在的困惑。新的放射性工具但是新兴，，，允许一个通过无创小动物准确和精确的解剖定位研究成人出生的神经元的选择组的功能。焦点电离辐射抑制新的神经元的诞生和分化，并允许特定的神经祖地区的目标。为了照亮成人丘脑神经发生中起着生理过程的调控潜在的功能作用，我们开发了一个非侵入性的焦点照射技术来选择性地抑制成年出生的神经元下丘脑正中隆起的诞生。我们描述的方法对C omputer成像制导f OCAL 红外辐射（CFIR）交付，使精确和准确的解剖小动物为目标。 CFIR使用本地化三维体积图像引导和辐射剂量的靶向，减少辐射照射到非定标大脑区域，并允许保形的剂量分布与锐波束边界。该协议允许一个问关于成人出生的神经元的功能的问题，同时也开启区域在放射生物学，肿瘤生物学，免疫学等领域的问题。这些放射性的工具将有助于发现翻译在板凳到床边。

评估CT引导下定位和准确度该系统的机械校准对于确保从不同角度的光束都相交在一个点的关键。校准是通过一半自动的基于成像的方法，其中端至端的对准精度被测量为0.2mm 29。由于下丘脑正中隆起结构的体积小2此精度是非常关键的。为了测试这一点校准，我们测量的剂量分布具有嵌入在水等效塑性模拟小鼠模型35（ 图4B）GAFCHROMIC辐射敏?...

Watch this Scientific Journal Video about Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition at JoVE.com

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

成人出生的哺乳动物神经元的功能仍然是研究的活跃领域。电离辐射抑制新的神经元的诞生。利用计算机断层扫描引导下焦照射（CFIR），特定的神经祖人群的三维解剖定位，现在可以用来评估成年神经发生的功能作用。

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

functional-interrogation-adult-hypothalamic-neurogenesis-with-focal

Research

JoVE Journal

Neuroscience

12.1K 次观看.  California Institute of Technology. 成人出生的哺乳动物神经元的功能仍然是研究的活跃领域。电离辐射抑制新的神经元的诞生。利用计算机断层扫描引导下焦照射（CFIR），特定的神经祖人群的三维解剖定位，现在可以用来评估成年神经发生的功能作用。 

视频: Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

同时脑磁图和脑电图的功能映射

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

科研

神经科学

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

方法对NF-κB依赖的成年神经发生的调制与分析

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

斑马鱼成人端脑的一刀，受伤：一个方法调查脊椎动物脑部神经发​​生与再生

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

免疫组化和多标签与同一宿主物种抗体研究成人神经发生

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) characterization of primary motor cortex (M1) excitability and inhibition. Motor response inhibition prevents unwanted actions and is abnormal in several neuropsychiatric conditions. TMS is a non-invasive technology that can quantify M1 excitability and inhibition using single- and paired-pulse protocols and can be precisely timed to study cortical physiology with high temporal resolution. We modified the original Slater-Hammel (S-H) stop signal task to create a &#34;racecar&#34; version with TMS pulses time-locked to intra-trial events. This task is self-paced, with each trial initiating after a button push to move the racecar towards the 800 ms target. GO trials require a finger-lift to stop the racecar just before this target. Interspersed randomly are STOP trials (25%) during which the dynamically adjusted stop signal prompts subjects to prevent finger-lift. For GO trials, TMS pulses were delivered at 650 ms after trial onset; whereas, for STOP trials, the TMS pulses occurred 150 ms after the stop signal. The timings of the TMS pulses were decided based on electroencephalography (EEG) studies showing event-related changes in these time ranges during stop signal tasks. This task was studied in 3 blocks at two study sites (n=38) and we recorded behavioral performance and event-related motor-evoked potentials (MEP). Regression modelling was used to analyze MEP amplitudes using age as a covariate with multiple independent variables (sex, study site, block, TMS pulse condition [single- vs. paired-pulse], trial condition [GO, successful STOP, failed STOP]). The analysis showed that TMS pulse condition (p&#60;0.0001) and its interaction with trial condition (p=0.009) were significant. Future applications for this online S-H/TMS paradigm include the addition of simultaneous EEG acquisition to measure TMS-evoked EEG potentials. A potential limitation is that in children, the TMS pulse sound could&#160;affect behavioral task performance.

We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.

在线经颅磁刺激测量与反应抑制相关的皮质生理

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...