这项工作得到了大脑韩国21加 Yonsei 大学医学科学项目的支持。此外, 这项工作得到了韩国国家研究基金会 (NRF-2017R1D1A1B03030315) 的赠款的支持。

所有程序均经 Yonsei 大学医学院适当的研究伦理委员会批准。所有实验动物都是按照 Yonsei 大学医学院实验动物护理委员会的指导方针而牺牲的。
1. 试剂的制备
注意: 多聚甲醛 (粉煤灰)、丙烯酰胺和月桂酸钠 (SDS) 是有毒刺激物, 因此应在通风罩中处理, 并配备适当的个人防护设备 (PPE; 实验室大衣, 手套, 防护眼镜)。
<ol><li>4% 丙烯酰胺 (a) 溶液 (A4P0): 将20毫升40% 丙烯酰胺溶液添加到180毫升的0.1 米磷酸盐缓冲盐水 (PBS) 中。</li>
	<li>0.25% VA-044 溶液: 添加0.5 克 2,2´-偶 [2-(2-imidazolin 2 基) 丙烷] 二盐酸盐 (VA-044) 粉到200毫升的0.1 米磷酸盐缓冲盐水 (PBS)。虽然 VA-044 粉可以储存在室温下, 它必须储存在4摄氏度后, 在 PBS 的增溶。为取得最佳效果, 请只准备实验所需的解决方案量。</li>
	<li>协议鸡尾酒解决方案: 添加10克丙烯酰胺到225毫升4% 多聚甲醛 (粉煤灰) 溶液, 并调整体积为200毫升与4% 粉煤灰。使用前, 在50毫升圆锥管中加入100毫克的 VA-044 粉, 40 毫升的混合物。</li>
	<li>协议清除解决方案缓冲区: 添加40克月桂酸钠 (SDS) 到350毫升0.1 米 pbs 和调整体积为500毫升与0.1 米 pbs。对于 mPACT 清除解决方案, 将0.5% α thioglycerol 添加到协议清除解决方案缓冲区。</li>
	<li>本研究使用的小鼠为2周大的 BALB 雄性小鼠;这项研究中使用的老鼠是2周大的雄性 SD 大鼠。</li>
</ol>2. 麻醉和灌注手术
注意: 粉煤灰和丙烯酰胺是有毒的刺激物, 因此应在通风罩内使用适当的 PPE。
<ol><li>麻醉动物在无病原体的房间, 30 毫克/千克的 zoletil 使用1毫升注射器与26口径针。监视动物 5-10 分钟。</li>
	<li>一旦动物到达麻醉的手术平面, 使用脚趾捏反应法确定麻醉深度。确认对停止响应。 注意: 以下步骤涉及鼠标和大鼠手术遵循类似的协议在以前的研究中使用11。</li>
	<li>通过外壳和腹壁, 在肋笼下面做一个5-6 厘米的切口。</li>
	<li>使用弯曲, 钝剪刀在隔膜切口, palpating 胸区预先定位切口部位。</li>
	<li>将切口延伸至胸腔的整个长度, 以暴露胸腔。</li>
	<li>小心地置换肺部。把胸腔切开到锁骨上。使用虹膜剪刀, 对左心室后端进行小切口。将18口径钝或橄榄尖的灌注针插入升主动脉, 穿过切口心室。</li>
	<li>用止血夹紧心脏, 确保针头的安全, 防止渗漏。或者, 使用修改过的止血在针尖周围夹住主动脉。</li>
	<li>在右心房做一个大切口, 注意确保对降主动脉的最小损伤。老鼠现在已经准备好灌注了。</li>
</ol>3. 大鼠全身灌注和解剖
注意: 粉煤灰和丙烯酰胺是有毒的刺激物, 因此应在通风罩内使用适当的 PPE。
注: 以下整个灌注步骤类似于前一项研究中使用的一种协议, 即 "宇et"。(2016)10,11。
<ol><li>将心脏的右心房连接到18口径的针头上, 注意不要引入任何气泡。</li>
	<li>使用50毫升注射器, 快速和均匀地泵送50毫升冷0.1 米的 PBS 溶液含肝素 (10 单位/毫升)。</li>
	<li>将18口径的针连接到蠕动泵的管上。</li>
	<li>用200毫升的冷0.1 米 PBS 溶液冲洗, 含肝素 (10 单位/毫升), 循环速度为10毫升/分。</li>
	<li>固定与250毫升冷4% 粉煤灰溶液的循环速度为10毫升/分钟。这只老鼠在这个阶段应该是僵硬的。</li>
	<li>收集并储存剩余的粉煤灰溶液进行处理。</li>
	<li>对于协议, 灌注组织与冷冻协议鸡尾酒解决方案的4% 粉煤灰, 4% 丙烯酰胺, 0.25% VA-044 粉与18口径针, 然后取出头部和脊柱。通过从颈部到鼻子的中线切开使头骨暴露出来。这种额外的灌注步骤是不必要的 psPACT 和 mPACT 方法;固定后, 立即隔离头部和脊柱, 并暴露头骨。</li>
	<li>通过除去残余的颈部和脊柱肌肉来揭露头骨的基底。</li>
	<li>用 rongeurs 和剪刀把头骨剥开。切除大脑和脊髓。</li>
	<li>将组织在4% 粉煤灰中贮存4摄氏度;组织最多可储存1周。</li>
</ol>4. 大鼠和小鼠中枢神经系统凝胶单体的注入和聚合
注意: 丙烯酰胺、SDS、α thioglycerol 和粉煤灰是刺激物, 因此应在通风罩中处理, 并适当小便。
<ol><li>
协议 (被动清算技术)
	<ol>
<li>将固定的小鼠和大鼠的整个中枢神经系统 (大脑和脊髓) 分离和培养成50毫升管, 其中含有冷冻公约鸡尾酒溶液, 4% 粉煤灰, 4% 丙烯酰胺, 0.25% VA-044 粉, 并贮存在4摄氏度为 24 h. 确保组织完全沉浸在 s 中解决方案.</li>
		<li>用与氮气罐相连的组织凝胶杂交系统将样品嵌入氮气中10分钟: 将系统设置为37摄氏度。将组织转移到含有新鲜冷 (4 °c) 协议的50毫升管, 并与管帽连接, 然后打开真空。</li>
		<li>要聚合水凝胶, 将包含样品的管放在震动孵化器 (150 rpm, 37 °c) 3 小时, 或直到聚合完成。</li>
		<li>使用印迹纸 (请参阅材料表), 移除围绕组织的剩余聚合水凝胶。</li>
		<li>将组织转移到含有清除溶液的50毫升管 (8% SDS 在0.1 米 PBS, pH 8.0)。</li>
		<li>将样品放在摇晃的孵化器中, 设置为37摄氏度和150转, 直到组织被清除。平均来说, 鼠标 CNS 需要大约20天的时间才能实现完全的清晰度。</li>
	</ol>
</li>
	<li>
psPACT (过程分离被动清除技术)
	<ol>
<li>用固定的小鼠和老鼠在干净的长凳上, 将整个中枢神经系统 (大脑和脊髓) 与骨刀和剪刀隔离。</li>
		<li>将组织转移到50毫升管, 含4% 个粉煤灰, 并贮存在4摄氏度, 24 小时. 确保组织完全浸入固定体中。</li>
		<li>在0.1 米 pbs 中清洗固定组织1小时, 然后转到 A4P0 溶液 (0.1 米 pbs 中的4% 丙烯酰胺)。存储在37°c 为24小时。</li>
		<li>在0.1 米 PBS 中清洗组织5分钟。</li>
		<li>在0.1 米的 PBS 中浸泡 0.25% VA-044 的组织。贮存在37°c 为6-24 小时, 然后转移到新鲜 0.25% VA-044/PBS 溶液。</li>
		<li>使用组织凝胶杂交系统 (参见材料表) 将样品嵌入氮气中, 并将其与氮气罐连接: 将组织转移到含有新鲜冷 (4 °c) 0.25% VA-044/PBS 溶液的50毫升管中, 并与管帽连接。打开真空。</li>
		<li>将组织转移到清除溶液中 (8% SDS 在0.1 米 PBS, pH 8.0)。</li>
		<li>在震动孵化器中孵化样品, 设置为37摄氏度和150转, 直到组织被清除。平均来说, 老鼠中枢神经系统需要大约17天的时间才能达到完全的清晰度。</li>
	</ol>
</li>
	<li>
mPACT (改进的被动清除技术)
	<ol>
<li>用固定的小鼠和老鼠在干净的长凳上, 将整个中枢神经系统 (大脑和脊髓) 与骨刀和剪刀隔离。</li>
		<li>按照 psPACT 协议的步骤 4.2.2 4.2.8。</li>
		<li>将组织转移到清除液中。请注意, 与协议和 psPACT 协议不同, mPACT 需要一个由0.5% α thioglycerol 组成的清除解决方案, 除0.1 米 PBS 中的8% 个 SDS 外, pH 为8.0。α thioglycerol 是一种不褐变剂, 有助于清除组织更迅速和有效。确保组织完全沉浸在溶液中。</li>
		<li>将样品放在摇晃的孵化器中, 设置为37摄氏度和150转, 直到组织被清除。平均来说, 老鼠中枢神经系统需要大约2周的时间才能达到完全的清晰度。</li>
	</ol>
</li>
</ol>5. 染色的折射率匹配和清除
注: n轮辋 (基于 Nycodenz 的折射率匹配解决方案) 由30毫升基缓冲器中溶解的0.8 克/毫升 Nycodenz 粉末 (0.01% 叠氮化钠和 Tween-20, 0.1 米 PBS, pH 8.0) 组成。建议将该溶液放置在37摄氏度的振动孵化箱中, 以便适当地对粉末进行溶剂化。
<ol><li>在0.1 米 pbs 中孵育 0.1% X-100 的组织为2小时, 然后在0.1 米 pbs 中用2% 牛血清白蛋白 (BSA) 阻断 6 h。</li>
	<li>洗涤部分三次在 PBST (0.1% Tween-20 在 0.1 M PBS) 为 2 h. 着色部分以主要抗体 (在这种情况下, 抗兔 PECAM-CD31 抗体, 染色血管) 2 天。</li>
	<li>染色切片与二次抗体 (在这种情况下, 山羊抗兔 IgG Cy3 荧光共轭) 在 2% BSA 2 天。</li>
	<li>在 PBST 中清洗标记的组织三次, 2 小时, 并在15毫升n轮辋中存储 2-10 天。</li>
	<li>在成像之前, 将标记的组织移动到35或60毫米组织培养皿上的少量n轮辋上。修复与硅胶周围的菜的底部边缘。</li>
	<li>添加 1.5-2 毫升新鲜的n轮辋。</li>
</ol>6. 图像处理
<ol><li>使用共焦激光扫描 microscopewith 10x 放大器获取已清除组织的图像 (请参阅材料表)。对于 Cy3-labeled 组织, 使用波长为 550-600 nm。</li>
</ol>

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作者没有什么可透露的。

尽管在协议中使用的被动、非电泳提取方法大大提高了与以前的组织清除方法一致的一致性, 如清晰度2、3、4、7,8, 该技术仍然存在几个缺点, 其中最紧迫的是实现最大组织清晰度12所需的时间长度。在当前的研究中, 我们提出了修改的协议协议, 大大减少了?...

科学和临床调查的一个基本目标是全面了解器官结构和功能;然而, 哺乳动物器官极其复杂的性质往往是完全实现这一目标的障碍1。清晰度 (透明脂交换丙烯酰胺-杂交刚性成像兼容 Tisssue-水凝胶)2,3,4, 其中包括建立一个基于丙烯酰胺的水凝胶混合从完整的组织, 达到各种器官的光学清除, 包括大脑、肝脏和脾脏, 同时保持其结构完整性5。因此, 清晰度不仅使可视化, 而且还有机会精细解剖复杂的细胞网络和组织形态, 而不需要切片。
为了达到组织间隙, 清晰度采用电泳方法去除手上样品的脂质含量。虽然已注意到生产物理稳定的组织-水凝胶杂交种, 研究表明, 其使用电泳组织清除 (等) 方法产生可变的结果, 在组织质量方面, 包括褐变, 表位损害, 和蛋白质损失5,6。为了解决这些问题, 已开发了7、8、9等修改的协议 (被动清晰度技术), 取代了被动的、基于离子洗涤剂的 delipidation 技术等处理。然而, 尽管在结果上取得了更大的一致性, 但条约需要更多的时间来获得最大的清除。此外, 这些技术尚未应用到整个中枢神经系统的形式, 或大鼠模型, 如老鼠和豚鼠。
本研究试图通过提出新的方法、psPACT (过程分离的公约) 和 mPACT (修改后的公约) 来解决这些限制, 以促进在老鼠和老鼠模型中的整个中枢神经系统和内脏器官的快速清除10。具体地说, psPACT 在水凝胶形成过程中分别在4% 丙烯酰胺和 0.25% VA-044 的两个步骤中处理组织;mPACT 基本上涉及相同的步骤, 但补充了基于 SDS 的清除解决方案与0.5% α thioglycerol 作为关键试剂。这两种技术都利用了内源系统和脑脊液循环系统, 大大减少了产生光间隙所需的时间。作为一项原则的证明, 我们展示了使用共焦显微镜分析在清除组织中的血管模式10。

<table>
	<tbody>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Affymetrix, Inc.</td>
			<td>75819</td>
			<td>Clearing solution</td>
		</tr>
		<tr>
			<td>Nycodenz</td>
			<td>Axia-Shield</td>
			<td>1002424</td>
			<td>nRIMS solution</td>
		</tr>
		<tr>
			<td>40% Acrylamide Solution</td>
			<td>Bio Rad Laboratories, Inc.</td>
			<td>161-0140</td>
			<td>Polymerization (A4P0)</td>
		</tr>
		<tr>
			<td>2,2&#180;-Azobis[2-(2-imidazolin-2-yl)propane] Dihydrochloride</td>
			<td>Wako Pure Chemical Industries, Ltd.</td>
			<td>017-19362</td>
			<td>Polymerization (VA-044)</td>
		</tr>
		<tr>
			<td>1-Thioglycerol</td>
			<td>Sigma-Aldrich</td>
			<td>M1753-100ML</td>
			<td>Clearing solution (mPACT)</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Georgiachem</td>
			<td>9005-64-5</td>
			<td>nRIMS solution</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-50ML</td>
			<td>Immuno Staining</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Bovogen</td>
			<td>BSA100</td>
			<td>Immuno Staining</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck Millipore</td>
			<td>375095</td>
			<td>Perfusion (PBS)</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-25G</td>
			<td>nRIMS solution</td>
		</tr>
		<tr>
			<td>PECAM-CD31 antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-28188</td>
			<td>Immuno Staining</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit-IgG Cy3 fluorescent conjugate</td>
			<td>Jackson ImmunoResearch Inc.</td>
			<td>111-165-003</td>
			<td>Immuno Staining</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Tech &#38; Innovation</td>
			<td>BPP-9004</td>
			<td>Perfusion, Polymerization</td>
		</tr>
		<tr>
			<td>20X Phosphate Buffered Saline (pH 7.4)</td>
			<td>Tech &#38; Innovation</td>
			<td>BPB-9121</td>
			<td>Perfusion, Buffer</td>
		</tr>
		<tr>
			<td>10 mL stripette</td>
			<td>Coatar</td>
			<td>4488</td>
			<td>Solution transfer</td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>Clearing tube</td>
		</tr>
		<tr>
			<td>35 mm Cell culture dish</td>
			<td>SPL</td>
			<td>20035</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Confocal dish</td>
			<td>SPL</td>
			<td>211350</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Korea vaccine Co., Ltd</td>
			<td>26G 1/2</td>
			<td>Anesthetize&#160;</td>
		</tr>
		<tr>
			<td>50 mL syringe</td>
			<td>Korea vaccine Co., Ltd</td>
			<td>21G1 1/4</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A3553</td>
			<td>Polymerization (A4P0)</td>
		</tr>
		<tr>
			<td>Whatman 3MM paper</td>
			<td>Sigma-Aldrich</td>
			<td>Z270849</td>
			<td>Blotting paper for gel removal</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM780</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>ZEN lite Software</td>
			<td>Zeiss</td>
			<td>ZEN 2012</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Longerpump</td>
			<td>BT100-1F</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>EasyGel</td>
			<td>Lifecanvas Technologies</td>
			<td>EasyGel</td>
			<td>Tissue gel hybridization system</td>
		</tr>
	</tbody>
</table>

novel passive clearing methods for the rapid production of optical transparency in whole cns tissue

由于清晰度的发展, 一种电化学清除技术, 允许在透明组织内的三维表型映射, 多种新的清除方法, 包括立方 (清晰, 无障碍的脑成像鸡尾酒和计算分析), 开关 (系统范围控制的交互时间和化学动力学的化学品), 地图 (放大分析的蛋白质组) 和协议 (被动清晰度技术), 以进一步扩大现有的工具箱生物组织的显微分析。本研究旨在改进和优化原始的协议程序, 为一系列完整的啮齿动物组织, 包括整个中枢神经系统 (CNS), 肾脏, 脾脏, 和整个小鼠胚胎。这些新的技术被称为 psPACT (过程分离协议) 和 mPACT (修改后的契约), 提供了非常有效的方法来映射细胞电路, 并在完整的正常和病理组织中可视化亚型结构。在下面的协议中, 我们提供了一个详细的, 分步概述如何实现最大的组织间隙, 最小的入侵他们的结构完整性通过 psPACT 和 mPACT。

利用优化的无源交换技术生成全中枢神经系统的透明模型
通过各种无源清除技术 (图 1), 快速实现了小鼠和全中枢神经组织的光间隙。图 2A显示了一段时间内组织清理的示意图。与原始的契约方法不同, psPACT (过程分离的协议) 包括处理样品与4% 丙烯酰胺 (A4P0) 和 0.25% VA-04...

Watch this Scientific Journal Video about Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue at JoVE.com

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

在这里, 我们提出了两个新的方法, psPACT 和 mPACT, 以实现最大的光学透明度和随后显微分析的组织血管在完整的啮齿动物整个中枢神经系统。

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

novel-passive-clearing-methods-for-rapid-production-optical

Research

JoVE Journal

Neuroscience

8.7K 次观看.  Yonsei University College of Medicine. 在这里, 我们提出了两个新的方法, psPACT 和 mPACT, 以实现最大的光学透明度和随后显微分析的组织血管在完整的啮齿动物整个中枢神经系统。

视频: Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

在哺乳动物的中枢神经系统视神经横断后的实验操作方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

科研

神经科学

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

新化合物调制伽玛-丁酸受体 a 型神经传递的发现方法

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

使用清晰组织清除方法对全山视网膜进行免疫染色

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...