动物使用遵循批准的育种和动物使用协议, 符合动物保育和使用的准则, 科罗拉多州立大学。
注: 下面的协议描述了长期孵化和间歇性海马切片成像的制备和培养方法。一个单一的海马切片是连接到一个专门准备光刻片使用等离子凝块, 然后片被密封在平坦的一侧的 drilled-out 辊管, 这是保持在滚筒孵化器。血浆凝块在病毒感染前以溶溶解, 用于荧光蛋白表达和高分辨率成像。一种荧光神经元生命染料被用来在切片中成像神经元。
1. 滚筒管架的制备
<ol>
	<li>使用显示在图 1A中的模板打印到缩放栏上显示的大小。用钉子, 打孔小孔 (足够大的罚款点标记) 在模板为中心的洞。</li>
	<li>将模板设置在 15 cm 组织培养皿的底部 (公称直径为14厘米), 并标记孔的位置。在第二道菜上重复这个。</li>
	<li>钻头设计用于塑料, 钻六1.5 厘米直径孔在每个菜的六角阵列 (4.8 厘米中心) 与孔中心2.5 厘米从盘子的边缘。钻三孔 (直径3毫米) 12 mm 从等距的边缘放置在两个较大的孔之间, 如图 1A所示。</li>
	<li>与每道菜的底部面对面, 放置一个2.5 英寸长的机器螺丝 (3/16 英寸直径) 与一个平垫圈通过一个小孔, 其次是第二个平垫圈, 一块聚乙烯管材 (间隔, 4.7 厘米), 另一个平垫圈, 第二个组织培养皿, 另一个平垫圈, 一个锁紧垫圈, 和一个螺母。</li>
	<li>重复步骤1.4 对其他两个机器螺丝, 并收紧只是松散, 直到所有的机器螺丝到位。然后安全地拧紧螺母。</li>
	<li>工作扣眼 (5/16 英寸厚, 5/8 英寸孔直径) 到底部盘孔获得最后的滚子管架 (图 1B; 显示为两个管到位)。在每个机架上放置一个带有唯一编号的标签。</li>
</ol>
2. 滚子管和片的制备
<ol>
	<li>滚子管孔钻夹具的制作
	<ol>
		<li>在一个 2 x 4 x 5.5 英寸木块的中心一侧钻一个 1.5 cm 孔8厘米深, 这样, 当插入 (图 2a) 时, 滚子管的扁平侧将与该块几乎平行。</li>
		<li>使用圆木文件扩大孔, 使孔扩大和锥度, 允许管插入 (滚柱管直径稍大, 靠近瓶盖) (图 2B)。</li>
		<li>钻1.5 厘米直径的垂直孔, 从块的一侧5.5 厘米, 并以侧孔为中心 (图 2C)。</li>
		<li>当侧面孔足够锥形时, 插入一个在所需位置标记的滚子管, 以使切片的孔居中, 并定位管, 以便在1.5 厘米的垂直孔中放置有标记的点。</li>
		<li>取下管子, 测量从现场到管子末端的距离。从夹具中的孔的中心处标记此距离, 并插入一根钉子, 以提供停止正确定位钻杆的位置 (图 2C中的箭头)。</li>
		<li>用钢锯将木块表面的钉子切掉, 防止受伤。</li>
		<li>在跳汰机底部添加弹簧夹, 如果有带有允许锚定的槽的钻床 (图 2D黑色箭头)。否则, 使用 C 形夹具将夹具牢固地固定在钻床上。</li>
	</ol>
	</li>
	<li>使用上述夹具举行和定位一个平坦的 11 cm 塑料文化管与平坦的一面 (图 2a), 钻一个6毫米直径孔与中心 1.0 cm 从底部和中心之间的管两侧。 注: 应使用为塑料设计的钻头。</li>
	<li>使用旋转的去毛刺工具, 平滑孔的边缘 (图 2E), 并在孔的内侧边缘 (图 2e, 嵌入) 上4凹槽, 以便在转动过程中使孔排出。</li>
	<li>用 12 mm 孔冲床, 从无毒双面胶片中切割12毫米直径的圆盘。使用标准的一个孔纸冲床 (6 毫米直径), 使每个磁盘的中心孔。</li>
	<li>用70% 乙醇冲洗钻孔管, 在生物安全柜中将空气烘干, 并在生物安全柜中将管子和穿孔的胶盘消毒40分钟, 在紫外线灯下 (30 W, 平均距离为70厘米)。</li>
	<li>在20分钟后重新定位管子和圆盘, 使所有暴露的表面都被消毒。在无菌条件下, 剥离从粘附盘的白背, 并将硅橡胶粘贴到管外, 对准孔 (图 2F)。 注意: 为了避免紫外线照射, 在打开 uv 灯前要戴上眼睛保护和关闭机箱。</li>
	<li>清洁12毫米直径光刻 (100 中心编号1毫米正方形) 德国玻璃片。用镊子轻轻握住片, 用绝对乙醇蘸水, 然后再用绝对乙醇, 最后将片在火焰中浸泡, 以燃烧乙醇。让片冷却。</li>
	<li>用钳夹住片, 在丙酮中浸入 2% 3-aminopropyltriethoxysilane 十年代. 用超纯水冲洗片, 使空气干燥。</li>
	<li>将片放在生物安全柜内的无菌滤纸上, 然后打开紫外线灯。将片的每一侧曝光20分钟。 注意: 为了避免紫外线照射, 在打开 uv 灯前要戴上眼睛保护和关闭机箱。</li>
</ol>
3. 海马切片的制备
<ol>
	<li>在开始解剖前, 为组织斩波器准备一半双刃刀片。用手指小心地将刀片对折, 然后将其折断一半。</li>
	<li>用棉签冲洗刀片的半部分, 用棉签清洗, 然后在绝对乙醇和空气干燥中漂洗。</li></ol>在组织斩波机上安装半刃前, 用70% 乙醇漂洗消毒。
	<li>遵循机构动物保育和使用委员会批准的协议;在异氟醚麻醉后, 安乐4-7 天的老老鼠或大鼠被斩首, 用剪刀或断头台除去头部。 注意: 脑切片培养协议独立于鼠或大鼠品系或基因型。许多转基因小鼠品系具有不同的遗传背景。</li>
	<li>用70% 乙醇冲洗头部, 并将其放在60毫米的培养皿中。直到最后安装的大脑切片到辊筒上, 所有以下步骤都在层流罩中进行, 以保持不育。</li>
	<li>使用 #21 手术刀片, 使一个矢状切口通过皮肤和头骨。用一个 #5 的钳, 剥去皮肤和头骨, 露出大脑。</li>
	<li>用闭合的镊子, 轻轻地梳理整个大脑, 通过捏钳通过在小脑后面的脑干中释放它。将大脑放在一个不育的60毫米碟中, 其中含有4° c Gey 的平衡盐溶液/0.5% 葡萄糖 (决定/葡萄糖)。</li>
	<li>使用解剖显微镜来可视化大脑 (图 3a), 将大脑的背侧向上, 用外科刀片 (图 3B) 将脑部和小脑的前三分之一切断。
	<ol>
		<li>用镊子, 保持修剪脑后侧向上和腹侧对培养皿一侧的稳定。轻轻地把脑膜周围的矢状中线, 并删除的中脑组织使用罚款倾斜 #5 钳 (图 3C, 虚线圆圈)。</li>
		<li>沿大脑一侧进行两次切口将其展开 (图 3C, 虚线)。一旦大脑被放置在背侧向下并张开, 海马的裂缝应该是可见的 (图 3D, 箭头)。</li>
		<li>将分散开放的大脑转移到一块 polychlorotrifluoroethylene 的塑料薄膜上, 并将其放置在组织斩波器的舞台上进行切片。用决定/葡萄糖湿刀片, 将海马切成300µm 厚的切片。</li>
		<li>与转移吸管, 冲洗切片的大脑从塑料薄膜成一个新鲜的60毫米碟含有决定/葡萄糖 (图 3E)。用细尖的镊子将剩余的脑膜和其他 non-hippocampal 组织 (图 3F) 从切片中轻轻捏开和逗走 (图 3G, H)。</li>
	</ol>
	</li>

4. 电镀片
<ol>
	<li>一旦切片获得, 放置2µL 鸡血浆的中心光刻一侧的准备片。将等离子体稍微扩散, 以达到直径3-4 毫米的斑点。 注: 光刻侧是片的顶面, 当通过解剖显微镜观察到这些数字是正确定向的。</li>
	<li>将1脑切片与不育的窄尖端刮刀 (图 4a) 转移到等离子点 (图 4B)。用闭合钳将切片从决定/葡萄糖上抬起。</li>
	<li>触摸刮刀到片上的等离子点, 用闭合钳将切片推到片上。</li>
	<li>混合2.5 µL 血浆与2.5 µL 凝血酶在一个单独的管。快速地将这种混合物的2.5 µL 放在切片的周围, 然后吸管上下轻轻地混合在一起 (图 4B)。 注: 血浆在10-15 秒内会凝结, 因此必须迅速完成。如果切片黏附性是一个问题, 混合5µL 血浆与5µL 凝血酶和使用4-5 µL 在切片上, 去除一些在混合以后, 以便切片说谎平在片。</li>
	<li>从先前贴在滚筒管上的硅橡胶粘合剂的外露面上取下透明的塑料覆盖物, 并将片与脑切片放在孔内的粘合剂上 (图 4C)。</li>
	<li>为了确保附着力, 应用软, 甚至压力的片与拇指通过按片下来均匀, 并持有约1分钟, 而转移到生物安全柜。</li>
	<li>在生物安全柜中, 将0.8 毫升完全 Neurobasal 的培养基 (材料表) 添加到每个管中 (图 4D)。</li>
	<li>流动一个 5% CO2/95% 空气混合物通过一个无菌棉塞的巴斯德吸管持有安全钳。用气体混合物冲洗滚筒管, 并迅速将管子从吸管周围抽出。</li>
	<li>用切片号和机架编号对管进行标签。将管子插入滚筒架中, 确保其几何平衡。如果有一个奇怪的管数, 添加管平衡。</li>
	<li>将机架放置在一个35° c 辊筒箱中, 滚轮在大约 10-13 RPH (图 4E) 上转动滚轮架。为了使培养基保持在管子的底部, 在板上抬高它的前部, 使孵化器向后倾斜大约5。</li>
	<li>在电子表格上输入切片和管号, 用于记录切片处理和观察日期的所有信息。</li>
	<li>在大约6天在文化, 增加1µL (0.002 U) 的活跃溶对每管。</li>
	<li>血块完全溶解后 (通常在几个小时内), 取出培养基, 用新鲜培养基代替, 不溶。如有必要, 切片可在一夜之间用溶孵育, 第二天则改变培养基。</li>
	<li>切片通常孵育至少7-10 天, 然后再用于实验。吸气培养基并在3或4天更换, 再在7天和此后每7天。</li>
</ol>
5. 基因表达的病毒载体的制备
注: 转基因在切片培养神经元中的表达是通过利用基因工程啮齿动物的大脑或通过感染重组复制缺陷病毒引入转基因的方法实现的。腺 (AV), 腺相关病毒 (AAV), 和重组慢载体都已被用于我们的海马切片培养不同的荧光蛋白合体在脑切片的表达。
<ol>
	<li>根据其他地方13,14中描述的方法, 准备复制缺陷 AV 来表示感兴趣的 RNA。使用病毒表达蛋白抗体的串联稀释法对感染的 U/mL 病毒进行效价, 如所描述的14。为了观察 cofilin 聚集体和 cofilin 肌动蛋白棒的形成, 利用 cofilin-R21Q-mRFP cDNA (质粒 #51279)16。 注: 突1启动子是一个很好的选择神经元特定表达式15, 而巨细胞病毒启动子是非常有用的驱动高表达水平在许多细胞类型的13。</li>
	<li>准备 AAV 由 co-transfection 的转移质粒含有感兴趣的基因和一个代表/cap 质粒, 有或没有辅助质粒, 进入 HEK293 包装细胞, 提供病毒 E1 基因, 如先前所描述的17,18。 注: 重组 AAV 也可用于定向插入宿主细胞基因组19。对于转移质粒, 我们使用人类 cofilin 1 与 C 终端 mRFP1 荧光蛋白标记 (质粒 #50856) 克隆成一个突启动子, 包含 AAV 质粒下游从钙传感器 GCaMP5G20。在转移质粒制备过程中, P2A self-cleaving 肽序列的 DNA 编码片段由 GCaMP5G 和 cofilin-RFP 之间的 PCR 插入, 以提供单 AAV 转录21的两种蛋白表达。</li>
	<li>制备重组慢载体 co-transfection 的转移质粒含有基因或 cDNA 的兴趣和集成信号, 连同第三代慢包装混合物, 把病毒性的插科打诨, 政客, 转速, 和 vsv g 基因到三分离质粒22,23。</li>
	<li>对于转移质粒, 使用单步克隆系统24将突启动子和 cofilin-R21Q-mRFP cDNA (从质粒 #51279) 组合成 pLKO. 1-GFP (质粒 #30323), 突启动子和 cofilin-R21Q-mRFP 取代 hPGK促进剂和 GFP cDNA 分别。</li>
	<li>用磷酸钙染将最终质粒转化为 HEK293T 细胞, 如前所述23。</li>
	<li>收集培养基从四10厘米的菜肴, 集中到500µL 使用 150 k 截止离心浓缩器, 并储存在-80 ° c 的最后慢在小等分后, 快速冷冻液氮。只为感染细胞解冻一次分。</li>
	<li>根据经验确定每种病毒类型的体积, 以达到所需的表达程度, 通过设置多个不同的切片区域性来跟踪病毒感染后的转基因表达。 注意: 通常每切片使用1-10 µL 病毒。</li>
</ol>
6. 切片处理
<ol>
	<li>感染切片与病毒
	<ol>
		<li>工作在生物安全内阁批准为病毒工作在生物安全水平适当为传染媒介, 混合病毒的分 (通常1-10 µL) 与0.8 毫升完全媒介。</li>
		<li>用不育的巴斯德吸管将培养基从切片中抽出, 放入含有漂白剂的收集陷阱中。在第一个阱和真空源之间总是使用二次阱。</li>
		<li>将此介质替换为上面准备的含有病毒的分, 将培养管返回到机架, 并放置在孵化器中。</li>
		<li>在生物安全柜中孵育病毒切片2-5 天后, 用无菌转移吸管将含有病毒的培养基移除, 并将其放入含有经批准的抗病毒药物的瓶子中, 以杀死其毒株。</li>
	</ol>
	</li>
	<li>生命染料对神经元的染色
	<ol>
		<li>准备和储存荧光神经元生命染料的等分25快速冷冻4µL 的等分在液氮浓度为100µM 和存储这些在-20 ° c。不要多次冷冻/解冻染料。</li>
		<li>通过荧光显微镜将神经元标记为可视化, 将神经元生命染料的一个分解冻, 在完全 Neurobasal 培养基中稀释为4毫升 (最终染料浓度为 100 nM)。</li>
		<li>从切片中取出培养基 (如果培养基中含有病毒, 则用转移吸管), 用含有 100 nM 神经元生命染料的0.8 毫升培养基取代。将切片返回到孵化器中的滚筒装置。</li>
		<li>在孵化2小时的切片后, 在生物安全柜中抽吸含有染料的培养基, 用0.8 毫升的新鲜完整培养基代替。 注: 用生命染料切片对神经元进行标记需要几个小时的潜伏期。第一个图像通常采取24小时后染料处理。虽然神经元是明确标记, 有背景荧光下降超过2-3 天, 以提供更好的神经元成像。72小时后, 重要染料的强度下降。</li>
		<li>随着时间的推移切片形态学的变化, 每7天重新切片。</li>
	</ol>
	</li>
</ol>
7. 切片成像
<ol>
	<li>在倒置显微镜上查看切片。对于最明亮的荧光成像, 在24小时前成像, 交换培养基与完整的 Neurobasal 一个媒介没有酚红色 pH 值指示器。 注: 在这里报告的实验中, 切片在倒置旋转圆盘共焦荧光显微镜上查看, 配有线性编码的 x、y 阶段和压电 z 控制和灵敏的高分辨率数码相机。</li>
	<li>将该管与切片培养图像从滚子管装置到 custom-made 管持有者 (图 5A), 它放置在舞台适配器 (图 5B) 中, 以使片垂直于目标和保持切片在相同的方向在重复成像会议上长时间间隔。</li>
	<li>将舞台适配器上的滑块紧压在管上, 以保持管的位置 (图 5B)。</li></ol>使用加热条和内置在 custom-made 级适配器中的调节控制器 (图 5C), 在成像过程中保持35° c 的切片温度。 注: 管架、舞台适配器和加热器的详细信息可访问: https://vpr.colostate.edu/min/custom-machining/roller-tube-holder/。
	<li>使用低功耗 (e. g, 4X) 目标和明亮字段透视, 将焦点放在切片下的光刻网格模式 (图 6a) 下。对于初始成像会话, 快速扫描切片以查找并记录需要更高放大率成像的各个区域的编号 (例如、角海马 (CA) 1、CA3、齿状回 (DG)、等)。</li>
	<li>将舞台移至包含感兴趣区域的第一个网格正方形。切换到20X 空气目标, 并找到一个基准标记 (e. g, 一个蚀刻数的尖端) (图 6B)。</li>
	<li>切换到更高的功率目标 (40X 或 60X), 本地化基准标记, 然后记录舞台的 x 和 y 位置。移动舞台以查找附近的感兴趣的字段, 并从基准标记 (图 6B, 箭头) 中记录它们的 x 和 y 偏移量。如果需要, 在其他网格区域重复。 注意: 这些抵消的位置从基准标记允许一致的精确定位的片位置时, 切片是成像, 即使原始 x, y 设置的基准标记改变时, 管或舞台适配器被删除和更换。</li>
	<li>使用显微镜物镜控制或压电舞台控制, 在每个选定的字段中捕获一个图像 Z 栈 (如果可用)。 注意: 图像平面通常以0.5 µm 到2µm 的间隔获得, 具体取决于图像特征的大小和所需分辨率。构建高质量的3D 映像要求图像特征延伸到多个采集面, 因此, 为了可视化较小的特征, 飞机之间需要更小的间隔。</li>
	<li>保持每个切片的总成像时间尽可能短。 注: 这里报告的大多数成像会话低于18分钟/片。然而, 我们已经拍摄了一些切片十或更多次, 甚至只要40分钟, 在一个单一的会话, 没有明显的损害, 长期生存的切片。</li>

<ol>
	<li>Brewer, G. J., Torricelli, J. R., Evage, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35 (5), 567-576 (1993).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+a+review.">Organotypic brain slice cultures: a review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-182 (1991).</li><li>G&#228;hwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+monolayer+cultures+of+nervous+tissue.">Organotypic monolayer cultures of nervous tissue.</a> J Neurosci Methods. 4 (4), 329-342 (1981).</li><li>Gogolla, N., Galimberti, I., DePaola, V., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+imaging+of+neuronal+circuits+in+organotypic+hippocampal+slice+cultures.">Long-term imaging of neuronal circuits in organotypic hippocampal slice cultures.</a> Nat Protoc. 1 (3), 1223-1226 (2006).</li><li>Roo, M. D., Ribic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+structural+plasticity+of+dendritic+spines+in+organotypic+slice+culture.">Analyzing structural plasticity of dendritic spines in organotypic slice culture.</a> Methods Mol Biol. 1538, 277-289 (2017).</li><li>Lee, K. F. H., Soares, C., Thivierge, J. -. P., B&#233;&#299;que, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+synaptic+inputs+drive+dendritic+calcium+amplification+and+cooperative+plasticity+during+clustered+synapse+development.">Correlated synaptic inputs drive dendritic calcium amplification and cooperative plasticity during clustered synapse development.</a> Neuron. 89 (4), 784-799 (2016).</li><li>Davis, R. C., Maloney, M. T., Minamide, L. S., Flynn, K. C., Stonebraker, M. A., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cofilin-actin+rods+in+stressed+hippocampal+slices+and+the+role+of+cdc42+in+amyloid-beta-induced+rods.">Mapping cofilin-actin rods in stressed hippocampal slices and the role of cdc42 in amyloid-beta-induced rods.</a> J Alzheimers Dis. 18 (1), 35-50 (2009).</li><li>Clark, R. E., Squire, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarity+in+form+and+function+of+the+hippocampus+in+rodents,+monkeys,+and+humans.">Similarity in form and function of the hippocampus in rodents, monkeys, and humans.</a> Proc Natl Acad Sci U S A. 110, 10365-10370 (2013).</li><li>Minamide, L. S., Striegl, A. M., Boyle, J. A., Meberg, P. J., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+stimuli+induce+persistent+ADF/cofilin-actin+rods+that+disrupt+distal+neurite+function.">Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function.</a> Nature Cell Biol. 2 (9), 628-636 (2000).</li><li>Rahman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofilin+rods+and+aggregates+concur+with+tau+pathology+and+the+development+of+Alzheimer's+disease.">Cofilin rods and aggregates concur with tau pathology and the development of Alzheimer's disease.</a> J Alzheimers Dis. 42 (4), 1443-1460 (2014).</li><li>Bamburg, J. R., Bernstein, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+dynamics+and+cofilin-actin+rods+in+Alzheimer+disease.">Actin dynamics and cofilin-actin rods in Alzheimer disease.</a> Cytoskeleton(Hoboken). 73 (9), 477-497 (2016).</li><li>He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W., Vogelstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+system+for+generating+recombinant+adenoviruses.">A simplified system for generating recombinant adenoviruses.</a> Proc Natl Acad Sci U S A. 95 (5), 2509-2514 (1998).</li><li>Minamide, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+use+of+replication-deficient+adenovirus+for+transgene+expression+in+neurons.">Production and use of replication-deficient adenovirus for transgene expression in neurons.</a> Methods Cell Biol. 71, 387-416 (2003).</li><li>K&#252;gler, S., Kilic, E., B&#228;hr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+synapsin+1+gene+promoter+confers+highly+neuron-specific+long-term+transgene+expression+from+an+adenoviral+vector+in+the+adult+rat+brain+depending+on+the+transduced+area.">Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area.</a> Gene Ther. 10 (4), 337-347 (2003).</li><li>Mi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+reporter+for+real-time+imaging+of+cofilin-actin+rods+in+living+neurons.">A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.</a> PLOS ONE. 8 (12), 83609 (2013).</li><li>Wang, L., Blouin, V., Brument, N., Bello-Roufal, M., Francois, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+recombinant+adeno-associated+vectors.">Production and purification of recombinant adeno-associated vectors.</a> Methods Mol Biol. 807, 361-404 (2011).</li><li>Matsushita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+vectors+can+be+efficiently+produced+without+helper+virus.">Adeno-associated virus vectors can be efficiently produced without helper virus.</a> Gene Therapy. 5 (7), 938-945 (1998).</li><li>Ward, P., Walsh, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+integration+of+rAAV+vector+into+the+AAVS1+region.">Targeted integration of rAAV vector into the AAVS1 region.</a> Virology. 433 (2), 356-366 (2012).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> J Neurosci. 32 (40), 13819-13840 (2012).</li><li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PLOS ONE. 6 (4), 18556 (2011).</li><li>Benskey, M. J., Manfredsson, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production+and+purification.">Lentivirus production and purification.</a> Methods Mol Biol. 1382, 107-114 (2016).</li><li>Huang, L., Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoprocessing+of+human+immunodeficiency+virus+type+1+protease+miniprecursor+fusions+in+mammalian+cells.">Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells.</a> AIDS Res Ther. 7, 27 (2010).</li><li>Bordat, A., Houvenaghel, M. C., German-Retana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibson+assembly:+an+easy+way+to+clone+polyviral+full-length+infectious+cDNA+clones+expressing+an+ectopic+VPg.">Gibson assembly: an easy way to clone polyviral full-length infectious cDNA clones expressing an ectopic VPg.</a> Virol J. 12, 89 (2015).</li><li>Er, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuO:+a+fluorescent+chemical+probe+for+live+neuron+labeling.">NeuO: a fluorescent chemical probe for live neuron labeling.</a> Angew Chem Int Ed Engl. 54 (8), 2242-2246 (2015).</li><li>Bernstein, B. W., Chen, H., Boyle, J. A., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+actin-ADF/cofilin+rods+transiently+retards+decline+of+mitochondrial+potential+and+ATP+in+stressed+neurons.">Formation of actin-ADF/cofilin rods transiently retards decline of mitochondrial potential and ATP in stressed neurons.</a> Am J Physiol Cell Physiol. 291 (5), 828-839 (2006).</li><li>Cichon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofilin+aggregation+blocks+intracellular+trafficking+and+induces+synaptic+loss+in+hippocampal+neurons.">Cofilin aggregation blocks intracellular trafficking and induces synaptic loss in hippocampal neurons.</a> J Biol Chem. 287 (6), 3929-3939 (2012).</li><li>Stine, W. B., Dahlgren, K. N., Krafft, G. A., LaDu, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+conditions+for+amyloid-beta+peptide+oligomerization+and+fibrillogenesis.">In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis.</a> J Biol Chem. 278 (13), 11612-11622 (2003).</li><li>Maloney, M. T., Minamide, L. S., Kinley, A. W., Boyle, J. A., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-secretase-cleaved+amyloid+precursor+protein+accumulates+at+actin+inclusions+induced+in+neurons+by+stress+or+amyloid+beta:+a+feedforward+mechanisms+for+Alzheimer's+disease.">Beta-secretase-cleaved amyloid precursor protein accumulates at actin inclusions induced in neurons by stress or amyloid beta: a feedforward mechanisms for Alzheimer's disease.</a> J Neurosci. 25 (49), 11313-11321 (2005).</li><li>Davis, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+dimers/trimers+potently+induce+cofilin-actin+rods+that+are+inhibited+by+maintaining+cofilin+phosphorylation.">Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin phosphorylation.</a> Mol Neurodegener. 6, 10 (2011).</li><li>Walsh, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-&#946;+and+proinflammatory+cytokines+utilize+a+prion+protein-dependent+pathway+to+activate+NADPH+oxidase+and+induce+cofilin-actin+rods+in+hippocampal+neurons.">Amyloid-&#946; and proinflammatory cytokines utilize a prion protein-dependent pathway to activate NADPH oxidase and induce cofilin-actin rods in hippocampal neurons.</a> PLOS ONE. 9 (4), 95995 (2014).</li><li>Woo, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RanBP9+at+the+intersection+between+cofilin+and+A&#946;+pathologies:+rescue+of+neurodegenerative+changes+by+RanBP9+reduction.">RanBP9 at the intersection between cofilin and A&#946; pathologies: rescue of neurodegenerative changes by RanBP9 reduction.</a> Cell Death Disease. , 6 (2015).</li><li>Shaw, A. E., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+regulation+of+cofilin+activity+in+the+CNS:+a+novel+therapeutic+approach+for+treatment+of+multiple+neurological+disorders.">Peptide regulation of cofilin activity in the CNS: a novel therapeutic approach for treatment of multiple neurological disorders.</a> Pharmacol Ther. 175, 17-27 (2017).</li></ol>

这里描述的滚子管法允许长期培养和高分辨率活体影像切片脑组织。切片技术所应用的一个主要问题是切片的安装和维护。片涂层, 支持切片粘附, 促进切片细化, 通过提高突起的生长和细胞迁移的切片;因此, 我们避免使用这些基板。通过 3-aminopropyltriethoxysilane 的治疗, 将氨基基团插入玻璃上, 提高了切片的粘附性, 但在片上太少或太多的鸡血浆也会导致粘附问题导致切片损失。适当的粘附所需的血?...

从啮齿动物脑区分离神经元的原代培养是研究人员用来观察病理性牵连刺激反应的重要工具。然而, 这种研究的缺点是只在2D 和没有他们的神经胶质支持系统的情况下观察神经元。此外, 除非在非常高密度的条件下生长 (640 神经元/mm2或大约16% 的表面积), 在这种情况下, 它不可能跟随树枝状或轴突的随机突起, 而从其细胞体, 海马4周以上的神经元存活能力明显下降1, 限制了游离培养对年龄相关病症的扩展研究。从啮齿动物大脑中制备的切片是一种有吸引力的选择, 它通过维持一个有组织的细胞结构和存活数周或数月来克服这些限制。在切片培养中维持啮齿动物大脑许多不同区域的条件已被描述为2。
两种主要的方法被广泛应用于脑切片的长期培养: 在气界面的膜上培养3或在密封管上培养片, 允许在滚筒孵化器中旋转以提供曝气的4。在膜上培养的切片可以直接成像高分辨率荧光显微镜使用直立显微镜和水浸泡目标5。另外, 在膜上培养的切片也被转移到玻璃底盘, 用倒置显微镜6实现树突棘的良好分辨率。然而, 两种在膜上生长的成像切片方法都是开放系统, 需要进行中等程度的改变, 并且经常使用抗真菌和/或抗生素来防止或减少污染5,6。膜上的切片在 air-medium 界面保持优良的形态学和生存, 但返回到精确的位置在重复成像在高放大率是非常困难的, 除非实验只遵循小小组的细胞表示荧光标记。虽然在膜上生长的切片已使用与病毒介导的表达转基因5,6, 生物安全协议可能需要使用封闭的文化系统, 用于某些病毒载体表达荧光标记的蛋白质和细胞生理学记者。此外, 沉浸目标要求在培养5中遵循的样本之间进行净化。膜界面培养的一个主要应用是结合高分辨率成像与电生理学在单时间点7。
在塑料管内片的滚子管法不允许任何电生理学或高分辨率成像, 不排除片。因此, 这种方法最常被应用到长期研究中, post-fixation 观测已经被做为8。这里描述的是一种利用辊筒培养技术的方法, 但对片上的 drilled-out 管, 只要保持文化, 就可以反复成像。封闭的系统不需要介质变化的成像和利用光刻片提供的基准标记, 允许成像在高放大率, 几天或几周后, 精确的领域以前成像。
我们应用这种方法来检查啮齿动物海马的变化, 这是一个主要的大脑区域, 涉及记忆和学习。啮齿类动物的海马体通常被研究为在认知障碍发展过程中观察到的病理或年龄相关变化的模型9, 如 AD 中发生的那些。我们的方法特别适合于研究随着环境变化而在单个切片内发展的病理变化, 如 a 肽的增加, 这是 AD8的特征。一个病理与人类和啮齿动物 AD 脑是存在的 cofilin 肌动蛋白聚合体和棒, 后者含有束的花丝, 其中 cofilin 和肌动蛋白是在1:1 摩尔比10,11,12. 在 a 治疗后的大鼠海马固定切片上观察到了棒状体, 以及在表达 cofilin-GFP 受缺氧8的活鼠脑切片内, 它们可能会导致 AD 中出现的突触功能障碍。和中风。在这里, 我们用这种新的培养方法观察不同病毒引入的外源嵌合荧光蛋白切片中的时间过程和分布。然后利用 cofilin 记者构造的神经元特异性表达, 跟踪 cofilin 棒和海马切片的聚集病理, 以应对可溶性 a 寡聚体 (ao) 的治疗。

<table><tbody><tr><td>Bottoms from 15 cm culture dishes</td><td>VWR Scientific</td><td>25384-326</td><td></td></tr><tr><td>Phillips Head Machine Screws (#10-32)</td><td>Ace Hardware</td><td></td><td>2.5&quot; long and 3/16&quot; in diameter</td></tr><tr><td>Flat Washers #10</td><td>ACE Hardware</td><td></td><td></td></tr><tr><td>Machine Screw Nuts (#10-32)</td><td>ACE Hardware</td><td></td><td></td></tr><tr><td>Rubber Grommets&amp;nbsp;</td><td>ACE Hardware</td><td></td><td>5/16&quot;, thick; 5/8&quot;, hole diameter; 1.125&quot;, OD</td></tr><tr><td>Polyethylene tubing (5/16&quot;; OD, 3/16&quot;; ID)</td><td>ACE Hardware</td><td></td><td>Cut to 1.8&quot; length</td></tr><tr><td>Lock Washer #10</td><td>ACE Hardware</td><td></td><td></td></tr><tr><td>Drill Press, 5 speed&amp;nbsp;</td><td>Ace Hardware</td><td>ProTech Model 1201</td><td></td></tr><tr><td>Nunclon Delta Flat-Sided Tubes</td><td>VWR</td><td>62407-076</td><td></td></tr><tr><td>Drill bits, 3 mm, 6 mm and 15 mm&amp;nbsp;</td><td>Ace Hardware</td><td>Diablo freud brand</td><td>Drill bits for cutting plastic.</td></tr><tr><td>Drill bits for wood, 1.5 cm and 1 mm</td><td>Ace Hardware</td><td></td><td></td></tr><tr><td>Wood file, 1/4&quot; round</td><td>Ace Harware</td><td></td><td></td></tr><tr><td>Spring clips, 16 mm snap holder</td><td>Ace Hardware</td><td></td><td></td></tr><tr><td>Swivel Head Deburring Tool, 5&quot;</td><td>Ace Hardware</td><td>26307</td><td></td></tr><tr><td>Adhesive Silicone Sheet (Secure Seal)</td><td>Grace Bio-Labs</td><td>666581</td><td>0.5 mm Thickness</td></tr><tr><td>6 mm hole punch</td><td>Office Max</td><td></td><td></td></tr><tr><td>12 mm hole punch</td><td>thepunchbunch.com</td><td></td><td></td></tr><tr><td>70% Ethanol</td><td></td><td></td><td></td></tr><tr><td>Phototeched Coverslips, 12 mm diameter</td><td>Bellco Glass, Inc.</td><td>1916-91012</td><td></td></tr><tr><td>Bunsen Burner</td><td></td><td></td><td></td></tr><tr><td>Absolute Ethanol</td><td></td><td></td><td></td></tr><tr><td>Nanopure Water</td><td></td><td></td><td></td></tr><tr><td>3-aminopropyltriethoxylane</td><td>Sigma-Aldrich</td><td>A3648</td><td></td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124</td><td></td></tr><tr><td>#5 Dumont Forceps</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>McIlwain Tissue Chopper</td><td>Ted Pella, Inc.</td><td>10180</td><td></td></tr><tr><td>Double Edge Razor Blades</td><td>Ted Pella, Inc.</td><td>121-6</td><td></td></tr><tr><td>Whatman Filter Paper</td><td>VWR</td><td>28450-182</td><td>Cut into 5.8 cm diameter circles</td></tr><tr><td>Poly-chloro-trifluoro-ethylene (Aclar)</td><td>Ted Pella, Inc.</td><td>10501-10</td><td>Cut into 5.8 cm diameter circles</td></tr><tr><td>#21 Surgical Blade</td><td>VWR Scientific</td><td>25860-144</td><td></td></tr><tr><td>#5 Dumont Forceps</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Spatula, stainless with tapered end</td><td>VWR</td><td>82027-518</td><td></td></tr><tr><td>Gey&#039;s Balanced Salt Solution</td><td>Sigma-Aldrich</td><td>G9779&amp;nbsp;</td><td></td></tr><tr><td>&amp;nbsp;Glucose</td><td>ThermoFisher Scientific</td><td>15023-021</td><td>25% (w/v) Solution, 0.2 mm filter sterilized</td></tr><tr><td>Chicken Plasma</td><td>Cocalico Biologicals</td><td>30-0300-5L</td><td>Rehydrate in sterile water, centrifuge at 2500 x g 30 min at 4 &amp;deg;C, quick freeze aliquots in liquid nitrogen and store at&amp;nbsp; -80 &amp;deg;C.</td></tr><tr><td>Thrombin, Topical (Bovine)</td><td>Pfizer</td><td>Thrombin-JMI</td><td>Quick freeze aliquots in liquid nitrogen at 1,000 international units/mL in diluent provided and store at -80&amp;deg;C. Use at 250 units/mL.</td></tr><tr><td>Cell Roller System</td><td>Bellco Biotech</td><td>SciERA</td><td></td></tr><tr><td>Roller Incubator</td><td>Forma</td><td>Model 3956</td><td></td></tr><tr><td>N21-MAX</td><td>ThermoFisher Scientific</td><td>AR008</td><td></td></tr><tr><td>Pen/Strep (100X)</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>200 mM Glutamine</td><td>ThermoFisher Scientific</td><td>25030081</td><td></td></tr><tr><td>Glucose</td><td>ThermoFisher Scientific</td><td>15023-021</td><td>25% (w/v) Solution, 0.2 mm filter sterilized</td></tr><tr><td>Neurobasal A</td><td>ThermoFisher Scientific</td><td>10888-022&amp;nbsp;</td><td>Complete Medium: 48 mL Neurobasal A, 1 mL N21-MAX, 0.625 mL 200 mM Glutamine, 0.180 mL 25% Glucose, 0.250 mL 100x pen/strep.</td></tr><tr><td>Third generation lentivirus packaging</td><td>Life Technologies</td><td>K4975-00</td><td></td></tr><tr><td>159 K cutoff centrifugal filters (Centricon)</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>Lentiviral cloning system (InFusion)</td><td>Clonetech</td><td></td><td></td></tr><tr><td>Plasmids 30323, 50856, 51279</td><td>Addgene</td><td></td><td></td></tr><tr><td>Neuronal cell viability dye (NeuO)</td><td>Stemcell technologies</td><td>1801</td><td>Thaw once and quick freeze in 4 &amp;micro;L aliquots. Store at -20 &amp;deg;C</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX83</td><td></td></tr><tr><td>Microscope objectives</td><td>Olympus</td><td>air: 4X, 20; oil: 40X, 60X,</td><td></td></tr><tr><td>Spinning disc confocal system</td><td>Yokagawa</td><td>CSU22</td><td></td></tr><tr><td>Microscope EMCCD camera</td><td>Photometrics</td><td>Cascade II</td><td></td></tr><tr><td>Linear encoded (x,y), piezo z flat top stage</td><td>ASI</td><td></td><td></td></tr><tr><td>Microscope lasers and integration</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr><tr><td>HEK293T cells</td><td>American Type Culture Collection</td><td>CRL-3216</td><td></td></tr><tr><td>Human Plasmin</td><td>Sigma Aldrich</td><td>P1867</td><td>0.002 U/mL in 0.1% bovine serum albumin (0.2 mm filter sterilized), quick freeze in liquid nitrogen and store at -80 &amp;deg;C.</td></tr></tbody></table>

modified roller tube method for precisely localized and repetitive intermittent imaging during long-term culture of brain slices in an enclosed system

培养的啮齿动物脑切片有助于研究神经元和胶质细胞在维持许多正常的体内相互作用的环境中的分子行为。从各种转基因小鼠线获得的切片或使用病毒载体表达荧光标记蛋白质或记者在野生型脑切片允许高分辨率成像荧光显微镜。虽然有几种方法已经开发成像脑切片, 结合切片培养的能力, 以执行重复高分辨率成像的特定细胞在活片长期的时间已经提出了问题。当病毒载体被用于外源蛋白的表达时尤其如此, 因为这是最好的在一个封闭的系统, 以保护用户和防止交叉污染。简单的修改对辊筒管脑切片培养方法, 允许重复高分辨率成像的切片在一个封闭的系统多周。在光刻片上培养切片允许使用基准标记快速准确地将舞台重新定位到不同处理前后的同一域中。例示为使用此方法结合特定的神经元染色和表达观察海马切片结构的变化, 荧光蛋白的病毒介导的神经元表达, 以及 cofilin 病理的发展,这是先前观察到的阿尔茨海默氏病 (AD) 的海马反应的切片治疗与低聚体淀粉样β (a) 肽。

为了确定如何准确地使用基准标记来重新同一字段中的同一单元格, 我们检查了在光刻片 (图 6A) 上生长的切片。神经元被染色与一个重要染料 (100 nM 为2小时; 不染色神经细胞细胞), 它从神经元随着时间的推移而消失, 而不损害细胞25。我们在一个格子正方形中确定了一个基准标记 (图 6a, B</stro...

Watch this Scientific Journal Video about Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System at JoVE

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

本文介绍了一种改进的滚子管法培养和间歇高分辨率成像啮齿动物脑切片多周, 精确重新定位光刻片。神经元的生存能力和切片形态学保持良好。提供了这种完全封闭的系统使用病毒的细胞类型的特定表达的应用。

封闭系统中脑切片长期培养中精确定位和重复间歇成像的改进辊筒法

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

modified-roller-tube-method-for-precisely-localized-repetitive

Research

JoVE Journal

Neuroscience

10.6K 次观看.  Colorado State University. 本文介绍了一种改进的滚子管法培养和间歇高分辨率成像啮齿动物脑切片多周, 精确重新定位光刻片。神经元的生存能力和切片形态学保持良好。提供了这种完全封闭的系统使用病毒的细胞类型的特定表达的应用。

视频: Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

注册生物成像，纳米材料可用于诊断和治疗监测

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

科研

生物工程

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

神经科学

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

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

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

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

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

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

修改一个Colliculo  - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

微胶质过程对急性脑片 atp 或血清素的双光子成像

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

多发性硬化患者综合解剖计划

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

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 ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

封闭系统中脑切片长期培养中精确定位和重复间歇成像的改进辊筒法

摘要

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胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

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胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像