这项工作是由神经生物有限公司资助的。我们想感谢 Ferrati 博士和塞尔希奥·比埃拉·德梅洛博士圣乔瓦尼罗 (神经生物学) 对手稿的评论和建议。

所有动物研究都是根据批准的议定书进行的。
注意: 在本节中, 提供了在实验过程中执行的主要阶段的顺序和建议的时间间隔 (图 1)。此外, 对该议定书的一步一步的描述由一个说明性的小组补充, 显示了在潜伏期后从脑切除到组织均匀化的关键行动 (图 2)。前面介绍了用于构建该设备的材料和说明的详细信息以及对世行分析的后续阶段的细节22。
1. 外科工具、Vibratome 设置和治疗准备
注意: 在开始实验过程之前, 请执行以下步骤:
<ol><li>准备两种不同的人工脑脊液 (aCSFs), 一个用于脑切片, 另一个用于孵化步骤, 如前所述的29。简单地说, 两个 aCSFs 的工作浓度 (毫摩尔) 为 "切片" aCSF: 120 氯化钠, 5 氯化钾, 20 NaHCO 3, 2.4 CaCl 2, 2 MgSO 4, 1.2. 2 PO 4 和10葡萄糖;6.7 HEPES 盐和 3.3 HEPES 酸;pH 值: 7.1。而为 "录音" aCSF: 124 氯化钠, 3.7 氯化钾, 26 NaHCO3, 2 CaCl2, 1.3 MgSO4, 1.3 2 PO 4 和10葡萄糖;pH 值: 7.1。</li>
	<li>混合解决方案, 然后放在冰和氧切片 aCSF, 以便让它冷, 而删除大脑和后续切片。保持室温 (RT), 并向录音 aCSF 提供氧气。</li>
	<li>将器械置于解剖罩下, 用于斩首和脑切除,即、断头台、手术解剖包 (剪刀、镊子、刀片) 和刮刀。</li>
	<li>通过选择切片厚度 (300 µm) 来设置 vibratome 参数, 频率为 7, 速度为6。将 vibratome 腔插入适当的空间, 并将其与冰环绕。</li>
	<li>打开卡波金 (95% O2, 5% CO2) 阀, 氧在脑切片过程中的 "切片" (aCSF)。选择最小流量, 大约2毫升/分钟。</li>
	<li>用两个15毫升的管子来准备测试的条件。添加一个管10毫升的 "记录" aCSF 单独 (对照组) 和其他, 10 毫升的 "录音" aCSF 丰富的 T30 集中2µM (治疗组)。涡流两管, 并保持他们在冰上, 直到他们使用在安装准备阶段 (3 节)。</li>
</ol>2. 麻醉、脑提取和切片
注: 本研究采用9种野生 P14 雄性大鼠。脑提取和后续切片构成了该议定书的一个关键部分, 因为它们应迅速执行 (10 分钟以内), 以防止或至少减慢组织退化过程。
<ol><li>在棉层添加1.5 毫升 100% w/瓦的异氟醚到感应腔内, 将动物放在里面, 然后关闭盖子。等待, 直到动物被深深麻醉, 确认没有踏板反射, 然后斩首动物使用断头台。</li>
	<li>把动物的头放在装有冰冷切片 aCSF 的罐子里, 在脑移除步骤中 (图 2A -c)。</li>
	<li>用手术刀片切开头骨上的皮肤, 然后用剪刀在中线后切开头骨, 从后部开始前方直到到达 OBs 上方的骨骼 (图 2A)。</li>
	<li>用一对镊子将头骨两侧侧拉, 以便能够进入大脑 (图 2B)。插入一个铲腹部到大脑, 轻轻地舀出来, 并保持它在冰冷的 "切片" aCSF 水合约5分钟 (图 2C)。</li>
	<li>使用刀片 (图 2D) 将大脑放在滤纸上, 并切断小脑。将大脑垂直粘附在 vibratome 盘上, 并将其插入切片腔内, 用冰冷 "切片" aCSF 填充, 并提供氧气 (图 2E)。</li>
	<li>调整切割间隔, 然后进行脑切片 (图 2F)。收集包含感兴趣区域的部分, 如前面描述的22所述的内侧隔膜 (MS)、DBB 的对角线带和 innominata (SI)。从上到下, 三切片包含 BF (图 2G) 的延髓 (R)、中间 (I) 和尾部 (C) 部分。</li>
	<li>沿中线除以小剪刀, 获得两个镜面 hemislices (图 2H), 分别用作同一解剖平面上的控制和处理条件。</li>
	<li>将 hemisections 在 vibratome 室中保持 5-10 分钟, 然后用玻璃吸管将其转移到含有记录 aCSF 的鼓泡锅中。保持在 RT 约30分钟恢复。</li>
	<li>同时填写三个玻璃瓶, 3 毫升记录 aCSF (以前在步骤1.6 中编写) 为对照组和三瓶与3毫升的 aCSF 丰富的 T30 (以前在步骤1.6 中编写) 为治疗组。</li>
</ol>3. 安装准备
<ol><li>将 hemisections 转移到相应的玻璃小瓶 (图 2I) 中, 以便为控制组 (绿色框架、图 2J) 连续三个 hemislices (包括延髓、中间和尾部高炉区域) 及其匹配处理组的对应项 (红色框架,图 2J)。</li>
	<li>用两个不同的刷子转移脑组织, 一个用于控制 hemislices, 另一种用于治疗的对应物, 以防止条件之间的任何污染。</li>
	<li>用相应的盖子封住每个玻璃瓶, 呈现氧气针 (21G) (图 2J)。将设备的主管连接到卡波金源 (图 2K)。在整个潜伏期提供氧气到 hemislices 的最低流速为2毫升/分钟, 以避免脑组织的任何移动和可能的机械损伤在实验期间。开始5小时的孵化。在5分钟内执行步骤 3.1-3.3。 注: 经常检查 (每 15-20 分钟) 是否将氧气供应给所有的瓶子, 其流量不会从底部移动组织。</li>
</ol>4. 均匀化样品
<ol><li>停止孵化后的氧气流, 并从瓶子里取出所有盖子。使用适当的画笔将 hemislices 转移到含有250µL 溶解缓冲器的1.5 毫升管中, 在冰上保持管 (图 2L)。为了使裂解缓冲, 混合 PBS 1x 与磷酸酶和蛋白酶抑制剂鸡尾酒都稀释 1:100, 并融汇组织与不同的杵, 以防止污染之间的样本。在 10-15 分钟内完成这些操作。</li>
	<li>离心机的脑裂解在 1000 x g 5 分钟4摄氏度。将上清液转移到新管中, 将样品保持在-80 摄氏度。</li>
</ol>5. 蛋白质浓度和印迹分析的阅读
<ol><li>确定每个样品的蛋白质浓度, 并随后为西方印迹分析准备整除数, 如前所述的 22.</li>
	<li>进行统计学分析, 评价治疗对蛋白质表达的影响。</li>
</ol>

<ol>
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作者宣布了相互竞争的金融利益。苏珊. 绿地是一家私营公司的创始人和总裁, 拥有公司的股份。艾玛努艾拉 Brai 和安东内拉 Cogoni 是神经生物有限公司的员工。

该协议的主要方面是基于已建立良好的 "体外" 大脑技术, 允许同步测试两个镜面 hemislices, 从同一解剖平面获得, 监测其在应用特定条件 (控制或治疗);因此, 这提供了一个实验范式, 尽可能严密控制。如在神经退行性事件22中所见, 以时间、剂量和地点特定方式评估与神经元损伤有关的不同 neurochemicals 的可能性是一个基本特征。此方法将体内生理环境的优点与体外准备工作的优势联系起来, 例如在获取感兴趣区域和创建理想的胞外环境方面的准确性。该协议代表了用于前体电生理记录25,26,27和光学成像28,的普通脑切片程序的适应性.29、30或体外脊髓切片, 通过启用结构和突触组织23,24 , 模拟更接近的在体内情况.,31,32。
此外, 这一程序可以帮助减少行为研究中使用的动物数量, 因为它部分复制了体内类似条件, 可用于确认体外数据, 如细胞培养和免疫组化分析, 然后预测体内实验。
拟议的方法也提出了一些限制, 如截面厚度和完整性, 如果不适当, 可以改变最终结果。潜伏期也是这一技术的一个基本组成部分, 因为它可能会影响对照组和治疗一。此外, 在该议定书的所有步骤中都可以遇到一系列程序或技术问题, 例如: (1) 在大脑中提取或切片机械损伤, 通过精确和温和的组织处理可以避免这种情况;(2) 漂浮的 hemislices 在孵化期间由于过量的氧气流量, 它可以机械地损害组织, 如果它触及氧气针淹没在 aCSF, 从而影响其完整性。这可以通过调整到最低速率的冒泡来防止;(3) 缺少或不恒定的氧气流, 可与空罐、油管系统中的损坏或断开、氧气针的插头或未正确浸入 aCSF 中的连接。由于恒定的氧合及其在整个过程中的流动是本协议最关键的方面之一, 所以经常监视所有这些参数, 以保持被调查组之间的均匀状态是很重要的。
协议中的其他关键步骤是由执行每个操作所需的时间窗口表示的, 随后将尽可能保留组织的完整性和功能。此外, 为了评估这些参数, 可以执行免疫组化分析来检测与神经元生存能力和电生理记录相关的特定标记, 如前面所描述的23,29。
除了此处描述的结果之外, 还可以为特定的调查设计即席方法. 例如, 它可以应用于: (1) 监测和比较不同的化合物在几个脑区的活动, 孵化他们与选择的解决方案, 如 aCSF 或 Neurobasal 培养基;(2) 对缺氧进行研究, 因为氧流量可以有选择地控制在每个横切;(3) 从不同疾病的转基因动物模型研究脑组织的性质;(4) 同时探索在同一解剖水平上,体内单边脑注射的效果, 并与控制半球进行比较;(5) 探索其他器官或组织的性质, 因此有可能促进和减少药物筛选试验的时间长短。

AD 是一种慢性病理学特征的逐步神经退行性损伤影响不同的大脑区域, 如嗅皮质 (EC), BF, 海马 (HC), 和嗅觉灯泡 (OB)1,2,3, 4,5。AD 开发的后期阶段导致了逐渐的认知下降, 使这种疾病成为最常见的痴呆症, 大约占所有病例的 70%6。尽管人们广泛尝试了解导致 AD 的最初阶段, 但目前还没有一个明确的实验性指示来阐明它们。此外, 最流行的理论-"淀粉样假说"-越来越受到质疑, 因为它没有提供一个完整的形象解释广告病理, 也没有一个药物的目标已证明有效的7,8 ,9。
另一种被越来越关注的理论表明, 变性期间发生的初始机制与主要易受 AD3、10、11的神经元簇有关.,12,13,14. 这种异构的蜂窝中枢包含在 BF、中脑和脑干中, 项目到多个区域, 如 EC、HC 和 OB15,16。尽管它的神经元形态学和神经递质合成的多样性, 这一核心细胞在表达疼痛的共同特征, 也可以有一个非酶功能17,18。这种非经典的角色作为一个新的信号分子介导钙 (Ca2 +) 流入神经元, 可以接受营养或有毒事件有关 Ca2 +剂量, 可用性和神经元年龄17,18,19。
在变性期间, 观察到的细胞损失可能因此与这个非酶函数17,18,20, 这是归因于30mer 肽 (T30) 从疼痛 C 总站裂了20. 根据以前的结果, 进行细胞培养和光学成像18,21的准备工作, 我们通过一种新的方法, 通过一种基于前体大鼠脑切片的高炉结构, 表明 T30 诱导类似于 AD 的配置文件22。具体地说, 这种新的方法提供了比细胞培养更具生理的情况, 因为它保持了完整组织的许多特征, 从解剖到电路保存, 尽管时间窗口小时。我们应用这个协议来探索在变性的早期阶段发生的事件, 监测 T30 应用时的急性反应。
尽管大量的文献, 使用脑切片来调查隐含在神经损伤或神经发生的分子通路23,24, 此协议首次提供更直接和敏感的读出比较脊髓切片的常用用法。然而, 正如脊髓脑部分的情况一样, 这种急性切片程序也可以用于几个目的, 如评估神经保护或毒性分子, 发现在特定过程中发生的主要分子变化,中枢神经系统相关病理学的免疫组化分析和药理学测定。

<table><tbody><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich, Germany</td><td>S7653</td><td>Reagent for aCSF preparation</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich, Germany</td><td>P9333</td><td>Reagent for aCSF preparation</td></tr><tr><td>Sodium bicarbonate (NaHCO3)</td><td>Sigma-Aldrich, Germany</td><td>S5761</td><td>Reagent for aCSF preparation</td></tr><tr><td>Magnesium sulphate heptahydrate (MgSO4 (7H2O))</td><td>Sigma-Aldrich, Germany</td><td>63138</td><td>Reagent for aCSF preparation</td></tr><tr><td>Potassium phosphate monobasic (KH2PO4)</td><td>Sigma-Aldrich, Germany</td><td>P5655</td><td>Reagent for aCSF preparation</td></tr><tr><td>Hepes salt</td><td>Sigma-Aldrich, Germany</td><td>H7006</td><td>Reagent for aCSF preparation</td></tr><tr><td>Hepes acid</td><td>Sigma-Aldrich, Germany</td><td>H3375</td><td>Reagent for aCSF preparation</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich, Germany</td><td>G7528</td><td>Reagent for aCSF preparation</td></tr><tr><td>Calcium chloride dehydrate</td><td>Sigma-Aldrich, Germany</td><td>223506</td><td>Reagent for aCSF preparation</td></tr><tr><td>T30 peptide</td><td>Genosphere Biotechnologies, France</td><td></td><td>AChE-derived peptide tested</td></tr><tr><td>Surgical dissecting kit</td><td>World Precision Instruments, USA</td><td>Item #: MOUSEKIT</td><td>Brain removal step</td></tr><tr><td>Surgical blades</td><td>Swann-Morton, UK</td><td>BS 2982</td><td>Brain removal step</td></tr><tr><td>Filter paper</td><td>Fisher Scientific, USA</td><td>11566873</td><td>Brain preparation for slicing</td></tr><tr><td>Glue</td><td></td><td></td><td>Brain preparation for slicing</td></tr><tr><td>Vibratome</td><td>Leica, Germany</td><td>VT1000 S</td><td>Slicing</td></tr><tr><td>Brushes</td><td></td><td></td><td>Tissue handling</td></tr><tr><td>Oxygen canister</td><td></td><td></td><td>Sectioning and incubation phase</td></tr><tr><td>1x Phosphate buffer saline (PBS)</td><td>Fisher Scientific, USA</td><td>BP2438-4</td><td>Homogenization step</td></tr><tr><td>Phosphatase inhibitors</td><td>Fisher Scientific, USA</td><td>1284-1650</td><td>Homogenization step</td></tr><tr><td>Protease inhibitors</td><td>Roche complete PIC, USA</td><td>4693116001</td><td>Homogenization step</td></tr><tr><td>Pestles</td><td>Starlab, UK</td><td>I1415-5390</td><td>Homogenization step</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>Pierce 660 nm Protein Assay</td><td>Thermo Scientific, USA</td><td>22660</td><td>Protein concentration</td></tr></tbody></table>

an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices

尽管许多研究试图开发可靠的动物模型, 反映了变性的主要过程, 但很少有人被广泛接受。在这里, 我们提出了一个新的程序, 适应于已知的前体脑切片技术, 它提供了一个更接近的在体内类似的情况下, 比的试管准备, 以调查早期事件触发细胞变性, 作为观察到阿尔茨海默病 (AD)。这个变异包括简单和容易地可再生的步骤, 使保存选定的脑区域的解剖细胞构筑和它的地方功能在一个生理环境中。不同的解剖区域可以从同一大脑中获得, 提供了在一个站点、剂量和时间依赖性的方式下进行多项实验的机会。可能影响与此方法有关的结果的潜在限制与组织的保存有关,即,在切片和孵化步骤和剖面厚度期间保持其解剖完整性, 这能影响生化和免疫组化分析。这种方法可以用于不同的目的, 如探索分子机制参与生理或病理条件, 药物筛选, 或剂量反应化验。最后, 该议定书还可以减少在行为研究中使用的动物数量。在这里报告的应用最近首次被描述和测试的体外大鼠脑切片包含基底前脑 (BF), 这是一个主要影响的脑区的 AD. 具体地说, 它已经证明, 从乙酰胆碱酯酶 (疼痛) 的 C 终点产生的毒性肽的管理可能会提示一个 AD 样的轮廓, 触发, 沿 antero-后轴的 BF, 差异表达AD 中的蛋白质改变, 如 alpha7 烟碱受体 (α7 nAChR), 磷酸化头 (p 头) 和淀粉样蛋白β (Aβ)。

这里提出的协议表明, 在含 BF 的部分 (图 3A) 中, 对有毒肽 T30 的管理, 以与站点相关的方式调节α7-nAChR、p 头和 Aβ的表达式。烟碱受体显示, 延髓处理的 hemislice 显著增加, 与它的控制对应 (切片 1, p = 0.0310) (图 3B), 而中间切片不显示任何变化的两个条件 (切片 2, p = 0.1195) (图 3B)。在后段中, T30-exposed 部分在其控制端有显著的减少 (切片3、 p = 0.0476) (图 3B)。在 T30 应用时, p 头水平在前面区域明显地增加对控制匹配边 (切片 1, p = 0.0158) (图 3C)。另一方面, 其他两个包含 BF 的部分不显示条件之间的任何显著差异 (切片 2, p = 0.1014; 切片 3, p = 0.6405) (图 3C)。T30 暴露后, Aβ在延髓和中间 hemisections 显著增强, 与未处理的对侧部分相比 (切片 1, p = 0.0136; 切片 2, p = 0.0109) (图 3D)。在尾部切片中, 两种处理 (切片3、 p = 0.1231) 之间没有任何变化 (图 3D)。对 p-头的主要抗体识别含有磷酸化 Ser202 残留的表位。对 Aβ的主要抗体检测到多种亚型, 从 Aβ37到 Aβ42。数据被分析为以前描述的22使用两个尾配对t测试和表示为 SEM. N = (hemislices 每组, 鼠) 是 (27, 9)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57507/57507fig1.jpg"> 图 1: 在实验过程中执行的主要步骤的序列和指示时间.完成第一个步骤和第三步的时间间隔可能因操作员的经验级别而异。<a href="/files/ftp_upload/57507/57507fig1large.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/57507/57507fig2.jpg"> 图 2: 说明协议进程的顺序步骤.(A-D)从头骨中提取大脑, 从大脑的其余部分分离小脑。(E) 将大脑附着在 vibratome 盘上, 并转移到解剖室, 被冰包围, 并在切片过程中不断加氧, 并充满冷 aCSF。(F) 冠状脑切片。(G) 切片的集合, 其中包含延髓 (R)、中间 (I) 和基底前脑的尾鳍 (C) 结构。(H) 两个匹配 hemislices 中的节的分隔。(I) 每个横切都放置在相应的小瓶中。(J) 瓶子放置在支架内, 在仪器箱内单独关闭。(K) 在系统连接到氧气源 (黑圆圈) 的潜伏期。(L) 处理后样品的均匀化。此数字从22中修改。<a href="/files/ftp_upload/57507/57507fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57507/57507fig3.jpg"> 图 3: T30 介导的α7-nAChR、p 头和 Aβ沿 BF rostro 尾轴的特定于站点的表达式.(A) 脑日冕切片的表示, 包括基底前脑核 (白色虚线), 分为两个互补部分并暴露在不同的条件下。(B) 在 T30 暴露后, 烟碱受体显示前细分 (T30 1) 的显著增加, 后一个 (T30 3) 的显著减少与相应的控制侧 (ctrl 1 和 ctrl 3) 相对应。中间切片不显示两个条件之间的任何更改 (ctrl 2, T30 2)。(C) 在处理的延髓部分 (T30 1) 上, p 头表达式明显高于其控制匹配对应项 (ctrl 1), 而其他两个切片则不显示两组中的任何差异 (切片2和切片 3)。(D) Aβ在前部和中间处理的细分 (T30 1 和 T30 2) 中显示显著增加, 与相应的未治疗侧 (ctrl 1 和 ctrl 2) 相对应。在尾部部分, 两组之间没有变化。误差线表示 SEM. * p < 0.05.n = (每组 hemislices, 大鼠) 为 (27, 9)。刻度条为1毫米。此数字已从22中修改。<a href="/files/ftp_upload/57507/57507fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices at JoVE.com

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

我们提出了一种方法, 它可以进一步深入了解基础变性的早期事件, 并基于建立的前体脑技术, 结合体内和试管内实验的优点。此外, 它是一个独特的机会, 直接比较治疗和未经治疗的小组在同一解剖平面。

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

an-alternative-approach-to-study-primary-events-neurodegeneration

Nanyang Technological University

Research

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Neuroscience

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

6.9K 次观看.  Culham Science Centre. 我们提出了一种方法, 它可以进一步深入了解基础变性的早期事件, 并基于建立的前体脑技术, 结合体内和试管内实验的优点。此外, 它是一个独特的机会, 直接比较治疗和未经治疗的小组在同一解剖平面。

视频: An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

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 ...

神经科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

医学

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

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 ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

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 ...

Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model

Source: Carvalho, M., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61330">A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures.</a> J. Vis. Exp. (2021)
This video demonstrates the technique of assessing neuronal death in rhinal cortex-hippocampus organotypic slices. Neuronal death occurs via evolving epileptic seizures &#8212; resembling in vivo epilepsy &#8212; and is detected through dual-staining of the dead cells using propidium iodide and immunofluorescence.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

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