Name: evaluation of synapse density in hippocampal rodent brain slices
Uploaded: 2017-10-06T14:00:00.000+00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about Evaluation of Synapse Density in Hippocampal Rodent Brain Slices at JoVE.com

这项工作得到了湄公河协会、欧盟 FP7、ARUK、惠康信托和帕金森英国的支持。我们还要感谢 Ms. Buchler 对共焦图像和实验室成员在手稿和方法方面的建设性投入的贡献。

所有使用小鼠和老鼠的实验都得到批准, 并使用1986《内政部动物 (科学规程) 法》所涵盖的附表1程序进行。人工脑脊液 (ACSF) 配方可以在 表 1 中找到. 
 1. 急性海马切片准备 <ol> <li> 尽快删除鼠标的大脑, 使用刮刀和锋利的剪刀切割头骨, 并把它放在 ice-cold, 含氧 (95% O 2 , 5% CO 2 ),high-sucrose ACSF (100 毫升). </li>
	<li> 将鼠标的大脑放在培养皿上, 然后用手术刀将小脑和前额皮质的一小部分移除, 然后再 hemisecting 大脑中线. </li>
	<li> 将两个半球放在刚刚切割的一侧, 并将每个半球粘附到 vibratome 的舞台上. </li>
	<li> 剪切 200-300 和 #181; 所有感兴趣区域的 m 厚部分。在海马的情况下, 应该获得大约每半球 3-4 片. </li>
	<li> 使用塑料巴斯德吸管将切片转移到浸没在含氧的腔中 (95% O 2 , 5% CO 2 ) ACSF。保持34和 #176; C 为 30 min. 
	注: 在这一阶段, 通过将制剂添加到切片室中, 可以对含有活性药理剂 (如重组 Dkk1 蛋白) 的切片进行处理. </li>
	<li> 使用画笔从腔室中取出切片, 将其放入24井板中, 并在室温 (RT) 中4% 甲醛 (粉煤灰)/4% 蔗糖中修复20分钟至1小时. 
	注意: 粉煤灰是有毒的, 穿戴适当的保护. </li>
	<li> 在 1X PBS 中清洗切片三次 (每段10分钟). 
	注意: 只要将切片保留在4和 #176 中, 则可以在这里暂停协议 1-2 天; C 1X PBS. </li>
</ol> 2。突触标记的免疫荧光分析 注意: 使用的所有缓冲区的食谱都可以在 表 2 中找到. 
<ol> <li> 在切片井中用阻塞/permeabilizing 缓冲区 ( 表 2 ) 替换 PBS, 并在 RT 中孵育 4-6 h. </li>
	<li> 在阻断/性缓冲液中稀释1:2、000稀释 vGlut1 多克隆豚鼠抗体. 
	注: vGlut1 是 pre-synaptic 兴奋性终端可视化的标志。对于后兴奋性突触识别, 使用多克隆抗体抗 PSD-95 和稀释1:500 在同一溶液中。使用最小体积300和 #181; 我在每个井。为了确定海马体的解剖位置, 使用一种抗体, 它也可以识别神经元结构, 如 MAP2 或蛋白。找出正确的浓度的所有主要抗体的突触标记的选择 (前后) 和稀释新的阻断/permeabilizing 缓冲区. </li>
	<li> 在4和 #176 中, 在一夜之间 (或在 1-2 天) 内孵育主抗体溶液中的切片; c. 使用震动平台, 剧烈运动. </li>
	<li> 在 PBS 中清洗切片三次 (每段10分钟). </li>
	<li> 在阻塞缓冲区中稀释适当的次级抗体1:500。例如, 当使用 vGlut1 豚鼠抗体时, 应用一种二次抗体, 它可以识别豚鼠的成分 ( 例如, 驴 anti-guinea-猪), 并结合到特定的荧光。当使用 PSD-95 兔抗体时, 应用一种二次抗体, 它可以识别兔子成分 ( 例如, 驴兔), 并将其共轭到不同的特定荧光. </li>
	<li> 在 RT 中对此解决方案中的切片进行 2-3 小时的孵育. 确保切片不受光照的保护, 因为二级抗体对光敏感. </li>
	<li> 在 PBS 中清洗切片三次 (每段10分钟). </li>
	<li> 使用画笔小心地从24个井板中取出切片, 并将它们均匀地放在 pre-labelled 的玻璃幻灯片上。在每个切片的顶部添加一滴安装介质, 然后轻轻地将玻璃片放在切片的顶部。避免气泡的形成。注意使用足够的安装介质 (300-400 和 #181; L), 因为数量不足可能导致干燥切片. </li>
	<li> 在 RT 中保持至少 1-2 天的干燥状态, 并使幻灯片免受光照的保护. </li>
	<li> 将幻灯片存储在4和 #176; c 在短期内, 但对于长期存储, 请将其保持在-20 和 #176; c. </li>
</ol> 3。共焦图像采集和分析 <ol> <li> 成像使用共焦激光扫描显微镜. 
	<ol> <li> 通过10x 或20x 目标标识要成像的海马区 ( 例如, 角海马1和 3 (CA1、CA3) 或齿状回 (DG)). </li>
		<li> 更改为40x 或63x 的浸油目标, 以确定连续的突起和有组织的结构, 以确保切片解剖的完整性。使用神经元标记 (如 MAP2) 作为参考 ( 图 1A ). </li>
		<li> 随后, 切换到60x 油浸目标 (NA = 〜 1.3-1.4), 并调整每个通道的设置以获得最佳信号和对比度。设置每个激光的强度, 以避免使用高/对比选项的任何像素的饱和度. 
		注意: 这些设置将取决于所使用的显微镜, 但请参阅 图 2 中使用的激光电源设置的 材料表 , 图 3 和 图 4 。为取得最佳效果, 请使用 1024 x 1024 像素分辨率。在同一实验的不同条件下, 设置应保持不变。如果需要, 可以应用其他缩放. </li>
		<li> 评估染色均匀的深度, 并获取至少8等距 (250 nm) 平面的图像堆栈。然后从每片相同的感兴趣区域中取3相邻的代表图像堆栈. 
		注: 深度可能因切片而异, 但应始终为大约 2-5 和 #181; m 从切片的表面. </li>
		<li> 按每个条件至少3片重复采集, 每个治疗组 6-8 动物. </li>
	</ol> </li> <li> 图像分析. 
	注意: 使用自动图像分析软件 (请参见 材料表 )。请注意, 有些系统需要许可证, 而另一些则是免费的, 例如 ImageJ。使用的软件必须在3D 中考虑到堆栈图像的所有平面。有关在图像分析中使用的软件的详细信息, 请参阅 材料表 ( 图 1 )。
	<ol> <li> 使用为强度阈值协议来识别所有突触点和神经元标记, 它们是在软件中手动优化和选择的 (请参见 材料表 中的特定软件协议]. 
		注意: 该软件为每个荧光标识一个最小和最大强度, 并将每个被识别对象的强度百分比关联起来。请注意, 强度的百分比将根据所使用的荧光和对突触标记的抗体而变化. </li>
		<li> 对于突触标记, 请确保在软件中选择大小过滤器 (#62; 0.1 和 #181; m 3 和 #60; 0.8 和 #181; m 3 ), 并将其应用于映像。调整参数以确保所选对象不重叠。排除任何太大或太小而不能被视为突触点的对象 (请参见 图 2B ). 
		注意: 一旦经过优化, 相同的阈值将被应用到给定实验中的所有图像中. </li>
		<li> 使用软件中选择的优化阈值协议, 对单个突触点 (或后) 的平均数、强度和体积进行量化。将点数字规范化到图像字段的体积 (大约16928和 #181; m 3 在此处使用的系统中)。将突触点强度正常化为参考神经元标记的强度. 
		注: 突触被定义为两个前后标记之间的共存。一旦后前点 h 的阈值协议建立, 软件应确定共存的突触点作为重叠的1像素或以上的单一平面. 
		注: 对于统计分析, 确认所有样本均遵循 Lilliefords 和 #967 所确定的方差的常态和均匀性; 2-测试分别。如下所述, 应通过参数测试分析显示正态分布和方差均匀性的样本。例如, 兴奋性突触点分析应使用 one-way 的变异因子进行阻断和复制。每个单独的实验应该被认为是一个块;通常, 有三独立的实验, 每一个 1-3 的老鼠从每个条件. </li>
	</ol> </li> </ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56153/56153fig1.jpg"/> 图 1: 基于图像分析的突触点分析软件和 #160; ( ) 强度阈值协议自定义的屏幕快照。给出的例子是 PSD-95, 其中所选的点具有定义的大小和 #62; 0.1 和 #181; m 3 和 #60; 0.8 和 #181; m 3 和当前最小化的重叠对象。请注意, 显示的图像是一个共焦平面, 使突触点和准确的参数选择更容易可视化。( B ) 软件分析输出的屏幕快照 (在选择优化协议之后)。一个例子的原始突触点数字测量的基础上的体积。然后将这些值导出到电子表格软件。<a href="//ecsource.jove.com/files/ftp_upload/56153/56153fig1large.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/56153/56153fig2.jpg"/> 图 2: 突触点的切片和参数设置的质量检查。( a ) 具有代表性的感兴趣区域的共焦图像 (40x 目标): 海马切片的 CA1 层 radiatum (sr) 和地层 oriens (如此) 通过 MAP2 染色进行识别。缩放条形图 = & #160; 2 和 #181; m. ( B ) 共焦图像 (60x 目标和获取软件上的额外1.3X 放大倍数) CA1 地层 radiatum, 兴奋 markers-vGlut1 (绿色) 和 PSD-95 (红色)-从海马切片。注意明确前后和点的识别。点大于0.8 和 #181; m 3 从量化 (白色箭头) 中排除。缩放条形图 = & #160; 2 和 #181; m (C) 共焦图像 (60x 目标和在采集软件上增加1.3X 放大率) CA1 地层 radiatum, 兴奋 markers-vGlut1 (绿色)-从低温切海马部分从整个大脑固定在粉煤灰。注意存在大孔和不规则, 成群 vGlut1 染色。标尺 = & #160; 2 和 #181; m. <a href="//ecsource.jove.com/files/ftp_upload/56153/56153fig2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a>

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作者没有什么可透露的。

对突触数的精确评估是神经科学领域的关键。在这里, 我们展示了如何评价 CA1 层 radiatum 区的突触密度作为一个模型系统。关于现有方法的意义, 在我们最近的一篇关于海马 Wnt 缺乏效应的研究中得到了证明, 我们也通过开路 EM10来评估突触数。突触损失的大小类似于开路 EM 和脑切片方法在这里描述的10。因此, 这一发现证明了该技术的准确性和可靠性。
重要的是, 开路 EM 和免疫突触的限制, 如这里所介绍的, 是无法准确估计的绝对突触数的特定大脑区域。事实上, 确定任何全球性的形态学变化是很重要的, 因为总体积的变化会影响突触的数量。另一个限制是, 某些抗体是不太有效的染色完整的脑区, 部分由于极端密度突触连接。我们经历了更高质量的染色 vGlut1 和 gephyrin 使用此切片准备和后续的粉煤灰固定最多1小时, 与即时固定的整个大脑在煤灰 (图 2C)。后者导致成群突触染色, 在组织中有孔。尽管如此, vGlut1 抗体的渗透仍然是限制在两种方法 (一对夫妇几十微米)。我们没有克服这个限制, 即使增加了抗体的培养, 但提供的抗体渗透大于总深度, 是成像, 不应该对最终结果的影响。此外, 应特别小心, 以确保染色甚至在整个获得的堆栈。
在我们的研究中, 我们想区分兴奋和抑制性突触。因此, 我们分别选择了 vGlut1/PSD-95 和 vGAT/gephyrin, 因为这些蛋白在成年小鼠海马中有很高的表达, 并且特定于每个突触子类型4,5。其他兴奋和抑制突触标记可以用来识别每个突触, 如在每个突触丰富的受体, 包括 NMDA 和 AMPA 受体的兴奋和 GABAA 受体抑制突触。其他的神经递质或调也可以与我们的协议, 以确保特定的抗体是可用的, 如在纹状体的多巴胺突触的显示22。此外, 减少每个切片的厚度到120µm 可以部分克服低数量的样品获得的急性切片, 这是必要的研究小大脑结构, 如杏仁核。
我们的议定书的未来应用可以在研究不同的脑区和疾病。例如, 在纹状体 (大脑中与帕金森病相关的区域), vGlut2/PSD-95 或 vGlut1/PSD-95 的组合可以用来区分从皮层或丘脑传入来的兴奋性突触,分别为22。
最后, 我们已经证明了一种可靠的方法来检查脑组织中的突触数量, 使用前后标记来定义突触。关键步骤包括制备良好的海马切片, 在粉煤灰中固定时间长达1小时, 应用足够的安装介质, 使用可靠的抗体, 适当的图像采集设置, 以及严格的突触点检测。最终, 这种方法是相对较快的, 很容易重现的任何实验室可以获得共焦显微镜。

人脑由大约 1014突触组成。突触数在中枢神经系统的发育和成熟过程中受到严格的调控。在整个发展过程中, 突触数通过突和修剪来调制, 形成功能性神经网络。学习和记忆是由突触数和强度的调节所支持的, 这一过程称为突触增强和抑郁症1。重要的是, 成熟突触的稳定对于维持神经元网络连接至关重要。此外, 在神经退行性疾病 (AD)2的早期阶段, 也报告了突触密度的缺陷。因此, 准确识别和评价突触数的能力是评估脑生理和病理的基础。
突触的极端密度和致密性使得在完整的脑区估计其精确数目具有挑战性。中枢神经系统中的化学突触由两个紧密并列的神经元组成, 由突触分裂的3分离。pre-synaptic 端, 或突触溥, 从轴突产生, 包括累积的囊泡, 其中含有神经递质, 定义突触的特异性, 即谷氨酸和伽玛丁酸 (GABA) 的兴奋性和抑制突触, 分别。囊泡中含有特定于每种神经递质的水泡转运体: 用于 gaba 的谷氨酸和水泡型 gaba 转运蛋白 (vGAT) 的水泡谷氨酸转运蛋白 (vGlut)。后终端是由多种蛋白质组成的高度致密的结构, 包括受体、黏附分子和支架蛋白。在兴奋性突触中, 后 density-95 蛋白 (PSD-95) 是最丰富的支架蛋白4, 调制兴奋性 n-甲基-d-天门冬氨酸 (NMDA) 和α-氨基-3-羟基-5-甲基-4-isoxazolepropionic 酸 (AMPA) 受体功能, 而 gephyrin 锚定抑制受体的抑制后终端5。总的来说, 蛋白质对前后室的特异性有助于突触亚型的分化和鉴定。
对突触数的评价可采用不同的方法进行。最常见的是使用电子显微镜 (EM), 这使得分析的突触在紧凑和完整的脑区, 高放大率和分辨率。然而, 这种技术是非常昂贵的, 需要复杂的样品准备, 是非常耗时。其他技术包括使用阵列层析成像6, 这是一个主要的缺点, 因为它需要专门的和昂贵的设备来执行分析。此外, 表达特定突触标记的转基因线条在评估突触数7时很有用, 但新品系的产生可能是限制性的, 代价高昂的, 而蛋白质的过度使用会导致不良的不相干型.
由于这些限制和简单性, 许多研究人员通过检查突触蛋白水平来评估突触数。然而, 在蛋白质发生重大变化之前, 突触丧失可以观察到, 因为结构突触重塑导致或后并列的扩散, 没有突触蛋白的降解。此外, 在 AD 中, 突触变性或神经元死亡后, 触点蛋白水平降低8,9。总体水平的量化并不能使这种区别。因此, 有必要建立一个可靠的, 容易接近的, 准确的方法来评价突触数在大脑中。
在这篇文章中, 我们描述如何用免疫荧光显微镜在海马鼠脑切片中彻底识别和确定突触的数量。突触的定义是定位前和后标记, 出现在一个有点分布。通过对大脑中 Wnt 信号的研究, 证明了该技术的有效性。Wnts 是分泌蛋白重要的形成, 消除和维持的突触。在体内诱导的 Wnt 叶栅分泌拮抗剂, Dickkopf-1 (Dkk1), 导致兴奋性突触丧失, 同时保留抑制性突触在海马体10。我们说明了在表达 Dkk1 的成年小鼠海马切片中的兴奋和抑制突触的识别和计数, 并强调了这种技术对其他类型组织/培养制剂的可转让性。

<table><tbody><tr><td>Vibratome</td><td>Leica</td><td>VT1000S</td><td></td></tr><tr><td>Primary antibody</td><td>Millipore</td><td>AB5905</td><td>vGlut1 (1:2000)</td></tr><tr><td>Primary antibody</td><td>Thermo Scientific</td><td>MA1-046</td><td>PSD-95 (1:500)</td></tr><tr><td>Primary antibody</td><td>Synaptic Systems</td><td>131 004</td><td>vGAT (1:500)</td></tr><tr><td>Primary antibody</td><td>Synaptic Systems</td><td>147011</td><td>Gephyrin (1:500)</td></tr><tr><td>Primary antibody</td><td>Abcam</td><td>ab5392</td><td>MAP2 (1:10000)</td></tr><tr><td>Secondary antibody</td><td>Jackson Laboratories</td><td>706-545-148</td><td>Donkey Anti-Guinea Pig Alexa Fluor 488 (1:600)</td></tr><tr><td>Secondary antibody</td><td>Abcam</td><td>ab175470</td><td>Donkey Anti-Rabbit Alexa Fluor 568 (1:600)</td></tr><tr><td>Mounting medium</td><td>SouthernBiotech</td><td>00-4958-02</td><td>Fluoromount-G</td></tr><tr><td>Confocal Microscope</td><td>Olympus</td><td>NA</td><td>FV1000 confocal microscope using a 60x 1.35 numerical aperture (NA) oil objective. For vGlut1, use a 488 laser with a percentage power of 14%. For PSD-95 use a 559 laser with a percentage power of 20%.</td></tr><tr><td>Analysis software</td><td>PerkinElmer</td><td>NA</td><td>Volocity</td></tr><tr><td>Scalpel</td><td>Swann-Morton</td><td>203</td><td>No 11</td></tr><tr><td>Super Glue</td><td>Loctite</td><td>1446875</td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>BOC</td><td>NA</td><td>Cylinder</td></tr><tr><td>24-well plate</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Glass slides</td><td>Thermo</td><td>7107</td><td>08-1.0mm thick</td></tr><tr><td>Petri Dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>iDkk1 mice</td><td>N/A</td><td>N/A</td><td>Mice were obtained by crossing tetO Dkk1 transgenic mice with CaMKII&amp;alpha; rtTA2 transgenic mice. TetO Dkk1 transgenic mice and CaMKII&amp;alpha; rtTA2 were crossed in a heterozygous state. Both mouse lines were bred in a C57BL/6J background. Single transgenic and WT littermate were used as controls.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for solutions:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>V800372</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>255793</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Ca&lt;sub&gt;2&lt;/sub&gt;Cl</td><td>Sigma</td><td>223506</td><td></td></tr><tr><td>Mg&lt;sub&gt;2&lt;/sub&gt;Cl</td><td>Sigma</td><td>M9272</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>71505</td><td></td></tr><tr><td>Kynurenic acid</td><td>Sigma</td><td>K3375</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Pyruvic acid</td><td>Sigma</td><td>107360</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G5767</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S8501</td><td></td></tr><tr><td>Triton-X</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Donkey Serum</td><td>Millipore</td><td>S30-100ml</td><td></td></tr><tr><td>PFA</td><td>Sigma</td><td>P6148</td><td></td></tr></tbody></table>

evaluation of synapse density in hippocampal rodent brain slices

在大脑中, 突触是神经元之间的特殊接合点, 决定神经元信号的强度和传播。突触的数量在发育和神经元成熟过程中受到严格的调控。重要的是, 突触数的缺陷会导致认知功能障碍。因此, 对突触数的评价是神经生物学的一个组成部分。然而, 由于突触在完整的大脑中是小而紧凑的, 对绝对数的评估是有挑战性的。本协议描述了一种使用免疫荧光显微镜轻松识别和评价海马鼠切片突触的方法。它包括一个三步的过程, 以评估突触的高品质共聚焦显微镜图像通过分析共存的前后突触蛋白在海马切片。它还解释了如何进行分析, 并给出了有代表性的例子, 从兴奋和抑制突触。该协议为神经突触的分析提供了坚实的基础, 可用于任何研究大脑结构和功能的调查。

神经退行性病变早期发生于 AD 和帕金森氏病等神经系统疾病2,11,12。然而, 这些赤字背后的分子机制仍未得到很好的理解。Wnt 信号的缺陷与 AD13、14、15、16、17有关。Wnts 是分泌的糖蛋白和发挥重要作用的突触形成和调制的突触传输18,19,20,21。最近, 我们发现 Wnts 作为神经突触维护的关键调节器在成熟神经系统10,22。为了准确地研究 Wnt 信号对转基因小鼠海马突触的影响, 我们利用脑切片制备和共聚焦显微镜对突触数的变化进行了评价。我们确定了健康的切片, 其中结构保存完好 (图 2A)。此外, 对突触点的选择进行了仔细的定义, 排除大的染色对象可能代表聚集的蛋白质 (图 2B)。该判据适用于所有图像, 并具有相同的严格分析参数。
我们使用一个转基因模型系统, 诱导在四环素控制下的成年小鼠 (iDkk1) 分泌的 Wnt 拮抗剂 Dkk1 的表达 (见材料表)10,22。我们证明, 阻断 Wnt 信号在成年海马触发兴奋性突触损失, 特别是在 CA1 地层 radiatum 地区 (图 3)。图 3A说明了具有代表性的兴奋和后标记 (分别为 vGlut1 和 PSD-95) 的共焦图像, 它们从 iDkk1 鼠的海马切片中获得, 表达 Dkk1 两周, 以及它们各自的控制。我们使用 Volocity 软件对这些图像进行分析, 并证明了点小鼠海马 CA1 区共存 vGlut1 和 PSD-95-labeled iDkk1 的兴奋性突触数量显著减少。诱导 Dkk1 两周 (图 3B, * p 和 #60; 0.05;克鲁斯卡尔-瓦利斯试验;每型6只小鼠)。在 CA1 地层 radiatum, 兴奋性突触点数每1000µm3减少了500的控制小鼠到300在 iDkk1 小鼠。相反, 诱导 Dkk1 表达不影响抑制突触的数量 (由共存之间的后标记 vGAT 和 gephyrin 分别确定), 如图 4A-b所示 (p & #62; 0.05;克鲁斯卡尔-瓦利斯试验;每型3只小鼠)。
重要的是, 我们进行了统计功率分析, 以估计适当数量的切片和动物所需的产生这些可靠的结果。使用 R, 我们计算, 每个条件需要大约35片 (3 图像 x 3 切片 x 6 动物 = 36), 以检测一个大的影响大小 (f = 0.4) 与80% 信心 (功率 = 0.8) 在 one-way 方差分析与阻塞和复制, 如步骤3.2.6 中所述。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56153/56153fig3.jpg"> 
图 3: iDkk1 小鼠海马切片的兴奋性突触丧失.(A) CA1 层 radiatum 的具有代表性的共焦图像显示兴奋性突触, 由 vGlut1 和 PSD-95 之间的共存 (白色箭头) 识别。下面板表示突出显示的矩形的放大倍数。缩放条形图 = 2 µm. (B) 控制和 iDkk1 之间兴奋性突触的百分比是使用材料表中提到的软件 (* p & #60; 0.05;克鲁斯卡尔-瓦利斯试验;每型6只小鼠)。数据代表± SEM。此图已从引用10中进行了修改。<a href="//ecsource.jove.com/files/ftp_upload/56153/56153fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56153/56153fig4.jpg"> 
图 4: iDkk1 小鼠的海马切片中的抑制突触不变.(A) 具有代表性的 CA1 层 radiatum 的共焦图像, 由共存之间的 vGAT 和 gephyrin (白色箭头) 识别的抑制性突触。下面板表示突出显示的矩形的放大倍数。缩放条形图 = 2 µm (B) 控制和 iDkk1 小鼠之间的抑制性突触百分比, 使用 Volocity 软件进行量化 (p & #62; 0.05;克鲁斯卡尔-瓦利斯试验;每型3只小鼠)。数据代表± SEM。此图已从引用10中进行了修改。<a href="//ecsource.jove.com/files/ftp_upload/56153/56153fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>高蔗糖 ACSF 成分</td>
			<td>浓度</td>
			<td>注意</td>
		</tr>
		<tr>
			<td>nacl</td>
			<td>75毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>25毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>氯化钾</td>
			<td>2.5 毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4, 2H2O</td>
			<td>1.25 毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>Kynurenic 酸</td>
			<td>1.25 毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>丙酮酸</td>
			<td>2毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>edta</td>
			<td>0.1 毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1毫米</td>
			<td>溶液加氧后添加</td>
		</tr>
		<tr>
			<td>氯化镁2</td>
			<td>4毫米</td>
			<td>溶液加氧后添加</td>
		</tr>
		<tr>
			<td>d-葡萄糖</td>
			<td>25毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>蔗糖</td>
			<td>100毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>沉水室 ACSF 组件</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>nacl</td>
			<td>125毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>25毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>氯化钾</td>
			<td>2.5 毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4, 2H2O</td>
			<td>1.25 毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1毫米</td>
			<td>溶液加氧后添加</td>
		</tr>
		<tr>
			<td>氯化镁2</td>
</tr></tbody></table>			<td>1毫米</td>
			<td>溶液加氧后添加</td>
		
		<tr>
			<td>d-葡萄糖</td>
			<td>25毫米</td>
			<td></td>
		</tr>
	

表 1: ACSF 组成。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>阻塞/permeabilizing 缓冲区</td>
			<td>浓度</td>
			<td>注意</td>
		</tr>
		<tr>
			<td>驴血清</td>
			<td>10%</td>
			<td></td>
		</tr>
		<tr>
			<td>海神-X</td>
			<td>0.50%</td>
			<td></td>
		</tr>
		<tr>
			<td>pbs</td>
			<td>1x</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td></td>
			<td>pH 值为7。4</td>
		</tr>
		<tr>
			<td>nacl</td>
			<td>1.37 M</td>
			<td></td>
		</tr>
		<tr>
			<td>氯化钾</td>
			<td>27毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>100毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>2PO4</td>
			<td>18毫米</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA/4%sucrose</td>
			<td></td>
			<td>pH 值为7。4</td>
		</tr>
		<tr>
			<td>pbs</td>
			<td>1x</td>
			<td>从10x 稀释</td>
		</tr>
		<tr>
			<td>粉煤灰</td>
			<td>1.33 M</td>
			<td></td>
		</tr>
		<tr>
			<td>蔗糖</td>
			<td>117毫米</td>
			<td></td>
		</tr>
	</tbody>
</table>
表 2: 用于荧光染色的缓冲器。

Watch this Scientific Journal Video about Evaluation of Synapse Density in Hippocampal Rodent Brain Slices at JoVE.com

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

本文概述了一种用免疫荧光法准确识别和分析海马切片突触的协议。

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

evaluation-of-synapse-density-in-hippocampal-rodent-brain-slices

Research

JoVE Journal

Neuroscience

16.8K 次观看.  University College London. 本文概述了一种用免疫荧光法准确识别和分析海马切片突触的协议。

视频: Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

科研

神经科学

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

生物化学

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

从成人和老化小鼠的河马切片中尽量减少低氧

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

急性小鼠脑切片研究自发海马网络活动

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

鼠脑水平海马片

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

<a href='https://app.jove.com/v/56153/evaluation-of-synapse-density-in-hippocampal-rodent-brain-slices'>Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).</a>This video demonstrates a method for visualizing synaptic markers in hippocampal brain slices using confocal immunofluorescence analysis. The slices are immunostained with primary antibodies targeting presynaptic and postsynaptic markers, followed by fluorophore-conjugated secondary antibodies. The labeled markers are then visualized using confocal microscopy to evaluate the synaptic architecture through their colocalization.

Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).This video demonstrates a method for ...