我们感谢 f. Benfenati (无依儿童意大利 di Tecnologia) 的支持和卡梅拉维塔利帮助进行示范。这项工作由 Compagnia 和圣保罗 (9734 号赠款) 资助。

所有的实验都是按照欧洲共同体理事会制定的指导方针进行的 (2014年3月4日指令 2010/63/欧盟), 并得到意大利卫生部的批准。
1. RNA 干扰 microRNAs 的设计及其在异源表达系统中的有效性评价
注意: 此协议需要了解以下已建立良好的方法: 分子克隆、DNA 测序、细胞系维持、磷酸钙转染、定量实时 pcr (qRT pcr)、细胞株裂解物细胞的制备和西方的印迹。
<ol><li>确定感兴趣的基因是否受替代剪接。对于基因的一般击倒, 选择专属本构外显子;对于一个特定的交替拼接异构体的击倒, 选择相关的交替拼接外显子。</li>
	<li>使用专用软件 (例如https://rnaidesigner.thermofisher.com/rnaiexpress/) 设计人工的大鹏湾, 以实现 RNA 对兴趣序列的干扰。 注意: 使用基本的本地对齐搜索工具 (爆破) 来检查同一物种内可能存在的目标是很重要的。</li>
	<li>选择顶部三排列的目标基因的大鹏湾。</li>
	<li>从合适的公司订购所选的大鹏湾的上下股。对于负控制, 请使用选定的大鹏湾或和平号的一个被炒版本, 预测不针对实验中使用的物种的任何已知基因 (e. g. 对于脊椎动物基因, 和平号在 pcDNA6.2-GW/EmGFP-no) 中存在。</li>
	<li>将选定的大鹏湾的顶部和底部进行退火, 并将其克隆成质粒, 用于表达大鹏湾 (例如pcDNA6.2/EmGFP), 使用标准的克隆策略。 注意: 目的是创建一个和平号表达式卡带, 由 5 ' 和平号的侧翼区域、选定的和平号序列和一个 3 ' 和平号侧翼区域组成, 可在 RNA 聚合酶 ii 型启动器控制下的一个记者基因的 3 ' UTR 中表达。</li>
	<li>使用 DNA 测序验证选定的大鹏湾的正确插入。</li>
	<li>用 DMEM 培养基在湿润细胞培养孵化器 (37 °c, 5% CO2) 中生长 HEK293 细胞, 辅以10% 胎牛血清、1毫米 NaPyruvate、10 mm HEPES (pH 7.39) 和1x 青霉素/链霉素 (笔/链球菌)。</li>
	<li>当 HEK293 细胞为70% 的汇合时, 用磷酸钙方法6与每一个和平号表达载体和一个向量, 用1:1 的 DNA 比值表达目标基因的染。包括以下控制: (i) untransfected 细胞, (ii) 转染的载体与载体 DNA (例如pBluescript ii (+)) 或 (iii) 和平号控制相结合的向量。 注: (i) 所表达的靶向基因必须来自与大鹏湾设计的同一物种, 除非大鹏靶的序列在两种核苷酸水平上绝对守恒。(二) 可使用 HEK293 以外的细胞线。(三) 可采用替代的转染方法, 如电穿孔或脂质体的方法。</li>
	<li>
48小时后转染, 溶解细胞, 运行蛋白凝胶, 执行西方印迹和分析蛋白质含量与抗体识别靶蛋白。目的为≥50% 的击倒效率。 注: 击倒效率可以或者, 或者另外, 被评估 (i) 通过 ELISA 分析, (ii) 通过测量目标蛋白质的活动如果适用 (例如离子通道的当前密度) 或 (iii) 通过 qRT PCR 在 mRNA 水平如果适当抗体不可用 (协议步骤 3)。<ol>
<li>将文化菜肴放在冰上, 用冰冷的磷酸盐缓冲盐水 (PBS) 清洗细胞。</li>
		<li>吸入 PBS, 然后加入冰冷马国贤缓冲 (50 毫米三盐酸 pH 值 7.4, 150 毫米氯化钠, 2 毫米 EDTA, 1% NP40, 0.1% 月桂酸钠 (SDS), 含蛋白酶和磷酸酶抑制剂; 1 毫升为一盘的直径100毫米, 0.5 毫升为一盘直径60毫米)。</li>
		<li>用细胞刮刀刮掉盘子上的粘附细胞, 轻轻地将细胞悬浮液转移到预冷的离心管中。</li>
		<li>离心管在 1.5万 x g 15 分钟在4°c, 转移上清在一个新的预冷离心管和丢弃的颗粒。</li>
		<li>使用可鉴定蛋白检测试剂盒或其他合适的方法 (例如布拉德福德化验) 确定蛋白质浓度。 注: 样品可以冻结在-20 摄氏度或-80 摄氏度以后使用, 或立即处理。</li>
		<li>将适当数量的裂解物转移到离心管, 使所有样品具有相同的蛋白质浓度, 并添加足够的冰冷裂解缓冲, 以弥补所有的裂解物到同一体积。 注:30 至50µg 总蛋白应足以满足大多数蛋白质的需要, 但应根据感兴趣的蛋白质的丰度确定适当的数量。</li>
		<li>添加适量的 2x Laemmli 缓冲液 (4% SDS, 10% 2-巯基乙醇, 20% 甘油, 0.004% 溴酚蓝, 0.125 米三盐酸, pH 6.8), 并煮沸的样品在95摄氏度为5分钟。</li>
		<li>将样品装在丙烯酰胺凝胶上, 再加上分子量标记。用100伏的凝胶1-2 小时。 注: 凝胶比例取决于感兴趣蛋白质的大小。</li>
		<li>组装转移三明治, 并将蛋白质从凝胶转移到 100 V 的膜上2小时。膜可以是硝化棉或 PVDF。用甲醇活化 PVDF 1 分钟, 在准备堆栈前用转移缓冲器冲洗。</li>
		<li>用阻断缓冲器 (PBST 5% 牛奶 (PBS + 0.1% Tween-20) 在室温下阻断1小时的膜, 然后在4°c 隔夜孵化膜, 并在阻塞缓冲器中稀释原抗体。</li>
		<li>在 PBST 三次洗涤膜10分钟, 并在室温下用酶共轭二级抗体在阻塞缓冲液中孵育1小时。</li>
		<li>在 PBS 四次清洗膜10分钟, 然后将化学发光基板应用于膜, 并使用 CCD 相机成像仪捕获化学发光信号。</li>
		<li>使用图像分析软件对击倒效率进行量化。</li>
	</ol>
</li>
	<li>选择两个最有效的大鹏靶向非重叠序列, 进一步功能化验。 注意: 对于任何和平号, 非目标效果永远无法完全排除。然而, 如果两个独立的大鹏湾有类似的效果, 这是极不可能的, 这是因为一个相同的非目标击倒。</li>
	<li>如果选定的大鹏湾的击倒效率不令人满意 (< 50%), 则为其他大鹏湾的屏幕。或者, 在串联中表示最有效的和平号,即用同一表达式卡带中的多个副本表示它们, 这可能会导致更高的击倒效率7。</li>
</ol>2. 重组腺相关载体的构建 Optogenetic 探针和 microRNAs 的联合表达
注意: 此协议需要了解以下已建立良好的方法: 分子克隆、DNA 测序和 rAAV 生产。
<ol><li>将每种最有效的和平号表达式磁带复制到 3 ' UTR 中, 用于生产重组 rAAVs 和兴奋 optogenetic 探针的表达, 如在 pAAV-Syn-ChETA-TdT-X (图 1A) 中, 其中神经元特定的突触素启动器驱动器的表达式超快 channelrhodopsin ChETA, 红色荧光记者 TdTomato 和和平号 (s) 插入 NheI 和 EcoRI站点5 之间。 注: (i) 不要超过 rAAVs 的包装限制, 它的长度大约是 4.7 Kb, 从 ITR (倒置终端重复) 到 ITR (图 1A, 橙色)。(二) 需要在停止密码子和 WPRE 之间插入和平号表达盒 (土拨鼠肝炎病毒转录后水平管理单元);图 1A)。(三) 列入荧光蛋白, 如 TdTomato, 非常有用, 因为它允许随时监测感染的局限性和强度 (议定书步骤 4)。</li>
	<li>使用 DNA 测序验证和平号磁带的正确插入。</li>
	<li>根据以前发布的协议8rAAV1/2 生成和滴度, 每个选定的和平号都有一个 rAAV1/2。一个典型的实验为被击倒的一个基因将包括两个独立大鹏反对同一基因和一个和平号控制。针对≥109病毒基因组 (vg)/µL 的病毒滴度。 注意: rAAVs 需要在生物安全等级 1 (BSL-1) 设施中进行处理。请与机构生物安全委员会查询详细信息。 注: 如果实验室没有病毒设施或病毒滴度不令人满意, rAAV 生产可以外包。例如, 访问 https://www.med.upenn.edu/gtp/vectorcore/或 https://vcf.charite.de/en/metas/。</li>
</ol>3. 从原代神经元培养中提取 RNA qRT PCR 评价内源基因的击倒效率
注: (一) 本议定书要求了解以下已确立的方法: 初级神经元培养和 qRT PCR 的制备和维持。(二) 重复击倒效率的量化 (步骤 3.1–3.14) 至少3次 (生物复制)。(iii) 在对蛋白质含量的分析被排除时, 例如当击倒另一种拼接的亚型, 而特定抗体不可用的时候, qRT PCR 在 mRNA 水平上的分解效率是合适的, 如在敲掉5。
<ol><li>
从感兴趣的大脑区域准备主要的神经元培养。按照以前发布的9中给出的皮层区域性的协议进行以下修改:
	<ol>
<li>6井板中的板神经元, 密度为每井50万个神经元。使用2.5 毫升/井的附件介质和3.3 毫升/良好的维修介质。</li>
		<li>如果观察到星形胶质细胞过度生长, 添加0.5 毫升/井的维护培养基补充7.5 µM 胞嘧啶β d-arabinofuranoside (为最终浓度1µM) 在3–4天体外(DIV)。</li>
	</ol>
</li>
	<li>
在 5-6 DIV 中,每和平号三口井 (技术复制) 感染. 使用最低的感染剂量, 这将感染神经元的≥99%.
	<ol>
<li>从每个井中取出2毫升的培养基, 并在50毫升的猎鹰管中收集。</li>
		<li>将病毒直接添加到神经元, 轻轻混合, 将盘子放回孵化器中37摄氏度。</li>
		<li>将收集到的培养基存放在孵化器中。松开猎鹰管帽, 使气体平衡。</li>
		<li>24小时后, 在每个井 (1.9 毫升/井) 中加入被去除的介质。</li>
	</ol>
</li>
	<li>
At 17-18 DIV, 溶解用于 RNA 提取的神经元. 注意: 裂解试剂和氯仿具有剧毒。在化学罩下工作, 穿戴防护用具;根据您的国家和机构指南处理废物。 注意: 对于步骤 3.3–3.13, 请在无 RNAse 条件下工作。戴上手套, 使用无 RNAse 玻璃器皿和一次性 plasticware。有关如何处理 RNA 的一般预防措施, 请参考 RNeasy 微手册的附录 A。<ol>
<li>孵化玻璃巴斯德吸管一夜之间在烤箱180摄氏度。</li>
		<li>使用与真空泵连接的玻璃巴斯德吸管, 将盘子倾斜, 从每个井中完全吸出介质。</li>
		<li>立即添加700µL 的裂解试剂给每个井。</li>
		<li>用手仔细地摇晃盘子, 均匀地展开溶液。</li>
		<li>将溶液向上和向下第4-5 倍, 直至得到均匀悬浮。</li>
		<li>把溶液从每个井转移到一个单独的1.5 毫升离心管。 注: 细胞裂解物可以储存在-80 摄氏度或立即处理。</li>
	</ol>
</li>
	<li>如果需要, 解冻细胞的裂解物在 RT, 并立即进行步骤3.5。</li>
	<li>将140µL 氯仿添加到每个细胞裂解物样品中。 注: 氯仿易挥发, 不易吸管, 应尽快关闭离心管。</li>
	<li>大力摇动离心管十五年代或直到样品完全乳化。</li>
	<li>保持样品在 RT 最小或直到液相开始分离。</li>
	<li>离心样品在 1.2万 x g 15 分钟在4°c。</li>
	<li>将含有 RNA 的上水相 (~ 320 µL) 转移到新的离心管上, 并丢弃剩余的液体。 注: 不要接触下粉红色有机相或白色环之间的两个阶段与尖端的吸管。</li>
	<li>在水相中加入1.5 卷100% 乙醇 (480 µL), 并将溶液慢慢向上和向下搅拌3次。</li>
	<li>使用可用于从小样本中纯化 rna 的商用试剂盒纯化 rna。</li>
	<li>用分光光度法定量测定 RNA 浓度和样品纯度。 注: (一) 预期≥3.5 µg/井产量;260/280 和260/230 的纯度比蛋白质和有机化合物应该是≥1.9。(ii) RNA 样品可以储存在-80 摄氏度或立即处理。</li>
	<li>Retrotranscribe 250, 500 或 1000 ng 的 RNA 与一个商业上可用的套件。</li>
	<li>用 qRT PCR 方法定量分析感兴趣的内源基因的击倒效率。有关详细协议, 请参见引用10。针对≥60% 的击倒效率 (图 2)。将数据规范化到≥2的家政基因 (e. g。GAPDH、ACTB、TUBB3、PPIA)使用多个内部控制基因方法11。 注: 如果在大鼠工作, 使用以下 PCR 引物的管家基因: GAPDH: 5 ' GGTGCTGAGTATGTCGTGGA 3 ' 和 GAPDH-转速: 5 ' GATGATGACCCTTTTGGC 3 ';ACTB: 5 ' CATCACTATCGGCAATGAGC 3 ' 和 ACTB-转速: 5 ' TCATGGATGCCACAGGATT 3 ';TUBB3-fwd: 5 ' GCCTTTGGACACCTATTCAG 3 ' 和 TUBB3-rev: 5 ' TCACATTCTTTCCTCACGAC 3 ';PPIA: 5 ' CACTGGGGAGAAAGGATTTG 3 ' 和 PPIA-转速 5 ' CCATTATGGCGTGTGAAGTC 3 '。</li>
</ol>4. 通过对光遗传学的靶向刺激, 评估突触前蛋白在完整神经元回路中的作用
注意: 下面的协议需要以前的经验, 在急性脑切片的电生理记录和使用电生理设置。
<ol><li>Stereotactically 在上一个发布12中的详细协议后, 向大脑注入 rAAV1/2。使用用于鼠标13或鼠脑14的立体定向图谱确定感兴趣的大脑区域的立体定向坐标。
	<ol>
<li>使用微拉拔 micropipettes 与长柄Ø7–9µm;用剪刀夹在小腿上的注射 micropipettes。使用一个薄的标记和图表纸来放置校准标记在注射 micropipettes 每2毫米。</li>
		<li>麻醉的动物与异氟醚, 并修复它在立体定向设备。</li>
		<li>在整个手术中保持动物的温暖, 加热垫设置在37摄氏度。</li>
		<li>用眼润滑剂保护眼睛。</li>
		<li>用电动剃须刀刮头上的皮毛。</li>
		<li>用棉花芽将聚维酮碘涂在剃头上。</li>
		<li>在解剖显微镜下, 进行中线切口。</li>
		<li>用棉花芽清洁头骨表面, 使 bregma 和 lambda 可见。</li>
		<li>把注射微放到它的支架上。将支架连接至立体定向臂。</li>
		<li>确定注射相对于 bregma 和/或 lambda 的位置的 x 和 y 坐标。</li>
		<li>用钻头将头骨薄在靶区。 注意: 使用柔和的圆周运动, 避免钻过头骨, 因为这会损害大脑的表面。</li>
		<li>如果有出血, 用毛巾纸吸收过量的血液。 注意: 过量的血液会使你很难正确地确定 z 坐标。</li>
		<li>通过毛细管作用将病毒加载≤2µL 注射微。</li>
		<li>将注射微到注射部位的 x 和 y 坐标。</li>
		<li>从硬脑膜中计算 z 坐标, 将吸管慢慢地移入大脑。</li>
		<li>当达到 z 坐标时, 等待3分钟, 以允许组织调整。</li>
		<li>使用1毫升注射器通过软管连接到注射微的背面, 应用低正压。通过解剖显微镜直观地监测注射微中病毒的射出速度, 并用标定标记作为参考点。 注: 病毒需要注射在缓慢的速度 (< 100 nL/分钟), 以避免组织损伤。</li>
		<li>注射完毕后, 等待5分钟, 取出注射微0.2 毫米, 再等5分钟, 以避免病毒回流。</li>
		<li>从大脑中缓慢而完全地提取注射微, 并将其处理成装满漂白剂的容器。</li>
		<li>用生理溶液将头骨湿润, 用3-4 针缝合皮肤。</li>
		<li>将庆大霉素软膏应用于伤口。</li>
		<li>单把动物放在一个干净的笼子里, 里面有食物小球和一盏热灯, 直到它完全恢复。</li>
	</ol></li>
	<li>≥15天后注射, 斩首动物下深异氟醚麻醉。</li>
	<li>用毒气 (95% O2) 准备 Vibratome 感兴趣的脑区的急性脑切片, 5% CO2) 冰冷 aCSF 溶液, 含 (毫米): 123 氯化钠, 1.25 氯化钾, 1.25 的 2 PO 4, 1.5 氯化镁 2, 1 CaCl 2, 25NaHCO3、2 NaPyruvate 和18葡萄糖 (渗透调整为 300 mOsm)。例如, 如果目的是分析从 CA3 到 CA1 锥体神经元的突触传递, 则准备海马形成的矢状切片 (350 µm 厚)。 注: 从这个步骤开始工作在低光条件下, 以避免激活 optogenetic 探头的环境光。</li>
	<li>让切片恢复30分钟, 在37°c 在同一 aCSF 中的一个房间内设计, 以保持脑切片。将切片在室温下保持在同一脑切片室中, 直到录音。 注意: 使用这些条件, 切片可以保持健康, 6–8 h。</li>
	<li>将一个切片转移到一个浸没的记录室, 并将其与用于恢复的相同 aCSF 的2毫升/分钟 superfuse, 辅以1.5 毫米 CaCl2 (总 Ca2 +: 2.5 毫米) 和没有 NaPyruvate。 注意: 切片是在低浓度的 Ca2 + (1 毫米) 中制备和维护的, 以最小化毒性。在2.5 毫米 Ca2 +中执行录制以支持囊泡释放。</li>
	<li>简要检查表示的荧光记者的信号 (e. g。TdTomato;图 3B)确定感染的定位和强度。</li>
	<li>用含有 (毫米) 的胞内溶液填充贴片电极: 110, 葡萄糖酸钾, 22 氯化钾, 5 NaCl, 0.5 EGTA, 3 氯化镁2, 4 毫克 ATP, 0.5 Na3-GTP, 20 K2-肌酸磷酸, 10 HEPES-KOH (pH 7.28, 290 mΩ)。 注: 使用带 5–6 mΩ的吸管电阻的贴片电极。</li>
	<li>在红外线照射下, 从受感染神经元接收突触输入的神经元中, 达到严密密封的全细胞构型。例如, 如果 CA3 锥体神经元被感染, 则在 CA1 区域的近端至内侧通道上贴上锥体神经元 (图 3C)。 注意: 系列电阻可以左无偿, 但应该是恒定和低 (≤20 MΩ)。</li>
	<li>用药理学来隔离被调查的突触电流。例如, 如果目的是研究兴奋性突触传递, 阻断抑制突触传递与10µM 荷包牡丹碱。</li>
	<li>唤起突触电流, 例如, 兴奋性突触电流 (EPSCs), 使用 473 nm 蓝色激光耦合到光纤 (Ø≤250µm) 定位在胞体的受感染的神经元 (e.g。CA3 锥体神经元)。
	<ol>
<li>将刺激长度调整到最低限度, 以减少每光脉冲产生不止一个动作电位的可能性。如果使用 ChETA, 请将其设置为2毫秒15。</li>
		<li>调整激光的刺激强度, 以产生小而清晰检测到的突触电流 (≤50 pA 峰值振幅为 EPSCs 记录在-70 mV 之间的持有电位在 CA3 和 CA1 锥体神经元之间;图 3D)。如果使用 ChETA 和250µm 直径的光纤, 则应适当地在纤维出口1–3兆瓦的刺激强度。 注: 如果激光强度不能调节, 使用中性密度过滤器。</li>
		<li>将473纳米激光照射到受感染神经元的胞体上, 而不是它们的轴突。例如, 在 CA3 胞体上发光, 远离谢弗络, 以避免轴突的直接退极化。</li>
		<li>应用河豚毒素 (TTX; 0.5 µM), 一个阻滞剂的钠通道, 到样品确认的渠道是行动潜力驱动。</li>
		<li>在不同的录音中, 应用选择性阻滞剂对被调查的突触电流进行确认, 以确定刺激是选择性的。 注: 例如, 将 NBQX (10 µM) 应用于 AMPA 型谷氨酸受体介导的 EPSCs。</li>
		<li>比较在同一急性脑切片中的光学和电诱发突触电流对重复刺激 (≤20 Hz ≥2刺激) 的反应。在使用控制和平号的时候, 在电和光刺激之间应该观察到类似的突触促进或抑郁, 这就暗示了类似的细胞机制是由两种激励作用激活的。 注意: 上述步骤是必要的, 以确保光刺激不会导致直接去极化突触前 boutons, 从而绕过一些机制的突触传递。</li>
	</ol></li>
	<li>比较突触传递的反应, 以回应单一和重复刺激 (≥2在≤20 Hz) 之间的控制和平号和大鹏湾的突触前蛋白质的调查。</li>
</ol>

<ol>
	<li>Lam, J. K., Chow, M. Y., Zhang, Y., Leung, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+Versus+miRNA+as+Therapeutics+for+Gene+Silencing.">siRNA Versus miRNA as Therapeutics for Gene Silencing.</a> Mol Ther-Nucl Acids. 4, e252 (2015).</li><li>Kohl, M. M., 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=Hemisphere-specific+optogenetic+stimulation+reveals+left-right+asymmetry+of+hippocampal+plasticity.">Hemisphere-specific optogenetic stimulation reveals left-right asymmetry of hippocampal plasticity.</a> Nat Neurosci. 14 (11), 1413-1415 (2011).</li><li>Maejima, 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=Postnatal+loss+of+P/Q-type+channels+confined+to+rhombic-lip-derived+neurons+alters+synaptic+transmission+at+the+parallel+fiber+to+purkinje+cell+synapse+and+replicates+genomic+Cacna1a+mutation+phenotype+of+ataxia+and+seizures+in+mice.">Postnatal loss of P/Q-type channels confined to rhombic-lip-derived neurons alters synaptic transmission at the parallel fiber to purkinje cell synapse and replicates genomic Cacna1a mutation phenotype of ataxia and seizures in mice.</a> J Neurosci. 33 (12), 5162-5174 (2013).</li><li>Mark, M. D., 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=Delayed+postnatal+loss+of+P/Q-type+calcium+channels+recapitulates+the+absence+epilepsy,+dyskinesia,+and+ataxia+phenotypes+of+genomic+Cacna1a+mutations.">Delayed postnatal loss of P/Q-type calcium channels recapitulates the absence epilepsy, dyskinesia, and ataxia phenotypes of genomic Cacna1a mutations.</a> J Neurosci. 31 (11), 4311-4326 (2011).</li><li>Thalhammer, 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=Alternative+Splicing+of+P/Q-Type+Ca2++Channels+Shapes+Presynaptic+Plasticity.">Alternative Splicing of P/Q-Type Ca2+ Channels Shapes Presynaptic Plasticity.</a> Cell Rep. 20 (2), 333-343 (2017).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-phosphate-mediated+Transfection+of+Eukaryotic+Cells+with+Plasmid+DNAs.">Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs.</a> CSH Protoc. 2006 (1), (2006).</li><li>Chung, K. 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=Polycistronic+RNA+polymerase+II+expression+vectors+for+RNA+interference+based+on+BIC/miR-155.">Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155.</a> Nucleic Acids Res. 34 (7), e53 (2006).</li><li>McClure, C., Cole, K. L., Wulff, P., Klugmann, M., Murray, A. J. <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+titering+of+recombinant+adeno-associated+viral+vectors.">Production and titering of recombinant adeno-associated viral vectors.</a> J Vis Exp. (57), e3348 (2011).</li><li>Cingolani, L. 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=Activity-dependent+regulation+of+synaptic+AMPA+receptor+composition+and+abundance+by+beta3+integrins.">Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins.</a> Neuron. 58 (5), 749-762 (2008).</li><li>Huang, H., Xu, Y., Cheng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+alternative+splicing+during+epithelial-mesenchymal+transition.">Detection of alternative splicing during epithelial-mesenchymal transition.</a> J Vis Exp. (92), e51845 (2014).</li><li>Vandesompele, 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=Accurate+normalization+of+real-time+quantitative+RT-PCR+data+by+geometric+averaging+of+multiple+internal+control+genes.">Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.</a> Genome Biol. 3 (7), (2002).</li><li>Cetin, A., Komai, S., Eliava, M., Seeburg, P. H., Osten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nat Protoc. 1 (6), 3166-3173 (2006).</li><li>Paxinos, G., Franklin, K. 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> The Mouse Brain in Stereotaxic Coordinates. , (2012).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (2013).</li><li>Gunaydin, L. 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=Ultrafast+optogenetic+control.">Ultrafast optogenetic control.</a> Nat Neurosci. 13 (3), 387-392 (2010).</li><li>Bourinet, E., 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=Splicing+of+alpha+1A+subunit+gene+generates+phenotypic+variants+of+P-+and+Q-type+calcium+channels.">Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.</a> Nat Neurosci. 2 (5), 407-415 (1999).</li><li>Chaudhuri, D., 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=Alternative+splicing+as+a+molecular+switch+for+Ca2+/calmodulin-dependent+facilitation+of+P/Q-type+Ca2++channels.">Alternative splicing as a molecular switch for Ca2+/calmodulin-dependent facilitation of P/Q-type Ca2+ channels.</a> J Neurosci. 24 (28), 6334-6342 (2004).</li><li>Soong, T. W., 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=Systematic+identification+of+splice+variants+in+human+P/Q-type+channel+alpha1(2.1)+subunits:+implications+for+current+density+and+Ca2+-dependent+inactivation.">Systematic identification of splice variants in human P/Q-type channel alpha1(2.1) subunits: implications for current density and Ca2+-dependent inactivation.</a> J Neurosci. 22 (23), 10142-10152 (2002).</li><li>Berndt, 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=High-efficiency+channelrhodopsins+for+fast+neuronal+stimulation+at+low+light+levels.">High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels.</a> P Natl Acad Sci USA. 108 (18), 7595-7600 (2011).</li><li>Klapoetke, N. 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nat Methods. 11 (3), 338-346 (2014).</li><li>Lin, J. Y., Lin, M. Z., Steinbach, P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+engineered+channelrhodopsin+variants+with+improved+properties+and+kinetics.">Characterization of engineered channelrhodopsin variants with improved properties and kinetics.</a> Biophys J. 96 (5), 1803-1814 (2009).</li><li>Zhang, Y. P., Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+induction+of+synaptic+plasticity+using+a+light-sensitive+channel.">Optical induction of synaptic plasticity using a light-sensitive channel.</a> Nat Methods. 4 (2), 139-141 (2007).</li><li>Fowler, D. K., Williams, C., Gerritsen, A. T., Washbourne, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+knockdown+from+artificial+microRNAs+in+an+enhanced+miR-155+backbone:+a+designer's+guide+to+potent+multi-target+RNAi.">Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.</a> Nucleic Acids Res. 44 (5), e48 (2016).</li></ol>

作者没有什么可透露的。

optogenetic 探针和和平号对突触前基因的共同表达提供了一种强有力的方法来描述基因击倒对完整神经元回路中突触前功能的影响。对于这种实验方法, 重要的是要识别和特征的大鹏, 是高效和选择性的, 以降低基因的兴趣在本机系统。如果可能的话, 两个或两个以上的独立的大鹏对同一 mRNA 的利益应该被用来控制最终的非目标效果。在系统中重新引入抗和平基因的抢救实验, 可作为特异性的控制。
使用相同的构造来表达一个 optogenetic 探针和一个和平号 , 只允许刺激光学上只突触前神经元的纵。这是不可能的电刺激, 因为他们不区分感染和非感染的神经元, 从而产生混合和稀释结果。因为光刺激可以诱发多个动作电位15, 所以在扩展的调色板15、19、20、21中选择最快的 optogenetic 探头是很重要的。此外, 必须确保光刺激不会导致突触前 boutons 的直接退极化, 以避免绕过突触传输的一些步骤你希望调查22。
我们在这里描述的实验方法, 使得有可能在有限的时间内 (第4-6 月) 平行地评估多个突触前基因的生理相关性。然而, 重要的是要记住, knockdowns 很少达到100%。此外, rAAVs 需要至少两个星期的时间, 以允许最大的击倒和充分表达的 optogenetic 探针, 这可能是一个时间限制, 在调查早期发展过程。虽然有更多的时间消耗, 条件击倒小鼠仅限于突触前神经元, 一般导致完全删除的兴趣基因, 因此提供了有效的补充方法。
和平号技术的一个具体优势是, 它能够从同一启动子中表达多个大鹏湾。该属性主要用于通过在同一目标基因上插入相同的和平号或不同的大鹏湾的多个拷贝来提高击倒效率。然而, 它也可以用来击倒多个基因通过表达大鹏对不同的目标基因7,23。这个属性可能被用来沉默多个突触前蛋白质解剖突触前信号通路。
在这里, 我们结合光遗传学与人工大鹏湾的特点的影响, 基因击倒对突触前功能的急性海马切片。同样的方法也可用于操作和探测神经元电路在体内。此外, 人工大鹏湾与 chemogenetic 方法相结合, 将使人们能够在较长的时间尺度上对神经元电路进行审问。

本协议描述了一种新的方法来表征基因击倒对突触前功能的影响, 在完整的神经元电路。在完整的神经元回路中研究突触前功能是很有挑战性的, 因为许多突触前 boutons 太小, 远离躯体, 允许联合分子和电生理干预。虽然 RNA 干扰提供了强大和灵活的手段, 以击倒突触蛋白1, 这种方法已被谨慎使用, 以调查突触前功能, 因为它是很难发现的影响, 使用传统的击倒电刺激, 不区分操纵和幼稚突触前boutons2。在这里, 我们描述如何结合人工 microRNA (和平号) 介导的 RNA 干扰与最近开发的 optogenetic 技术, 以实现选择性刺激操作突触前 boutons 在急性脑切片。
虽然条件击倒小鼠结合电生理学也可以用来研究突触前蛋白的功能3, 4, 我们的战略不需要发展和维持基因修改过的鼠标线, 也可以很容易地使用, 以摧毁特定的亚型基因。相对于较常用的短发夹 rna (shRNAs), 人工大鹏, 我们在这里使用, 提供了关键的优势, 以击倒在神经元。与 shRNAs 不同, 它们可以在聚合酶 II 启动子1的控制下表达。因此, 一个单一的启动器可以用来驱动和平号和 optogenetic 探针的表达, 连同一个荧光记者。这样, 构造的大小可以保留在重组腺相关病毒的包装范围内 (rAAV,图 1A)。此外, 使用单一的结构和单一的启动器减少实验的变异性, 因为它允许表达的和平号, optogenetic 探针和荧光记者的固定比率。
我们最近应用这一技术来研究突触前钙通道的交替拼接亚型在海马5 中的作用。这种策略一般适用于研究其他 presynaptically 表达的蛋白质在任何感兴趣的脑回路中的生理相关性。

<table>
	<tbody>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma</td>
			<td>Acrylamide/Bis-acrylamide, 30% solution</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Animal Temperature Controller with heat pad</td>
			<td>WPI</td>
			<td>ATC2000</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>BLOCK-iT Pol II miR RNAi Expression Vector kit</td>
			<td>ThermoFisher Scientific</td>
			<td>K4936-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain slices Prechamber</td>
			<td>Harvard Apparatus</td>
			<td>BSC-PC</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera-based imager</td>
			<td>Bio-Rad</td>
			<td>ChemiDoc&#8482; MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture reagents</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescent substrate</td>
			<td>GE Helthcare</td>
			<td>ECL Western Blotting Reagents, RPN2106</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabinofuranoside</td>
			<td>Sigma</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Foredom</td>
			<td>K.1030</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin ointment</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>54459</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection micropipettes</td>
			<td>Narishige</td>
			<td>GD1</td>
			<td></td>
		</tr>
		<tr>
			<td>Inorganic salts &#38; detergents</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K2-creatine phosphate</td>
			<td>Calbiochem</td>
			<td>237911</td>
			<td></td>
		</tr>
		<tr>
			<td>KGluconate</td>
			<td>Fluka</td>
			<td>60245</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Laserglow Technologies</td>
			<td>LRS-0473-GFM-00100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane</td>
			<td>Amersham</td>
			<td>Protran&#8482; 0.2 &#181;m NC</td>
			<td></td>
		</tr>
		<tr>
			<td>MgATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Narishige</td>
			<td>IM-H1</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3GTP</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>NaPyruvate</td>
			<td>Sigma</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular lubricant</td>
			<td>Local pharmacy</td>
			<td>Lacrigel</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate inhibitors</td>
			<td>Sigma</td>
			<td>P0044, P5726</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Sigma</td>
			<td>cOmplete&#8482;, EDTA-free Protease Inhibitor Cocktail</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein gel electrophoresis and blotting devices</td>
			<td>Bio-Rad</td>
			<td>Mini-Protean III Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Providone iodine</td>
			<td>Local pharmacy</td>
			<td>Betadine</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiazol Lysis Reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>QuantiTect Reverse 
			Transcription Kit</td>
			<td>Qiagen</td>
			<td>205311</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic apparatus</td>
			<td>WPI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>1069</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
	</tbody>
</table>

combining optogenetics with artificial micrornas to characterize the effects of gene knockdown on presynaptic function within intact neuronal circuits

本协议的目的是描述基因击倒对完整神经元回路中突触前功能的影响。我们描述了如何将人工 microRNA (和平号) 介导的 RNA 干扰与光遗传学相结合的工作流程, 以实现对急性脑切片操作的突触前 boutons 的选择性刺激。实验方法包括使用单一的病毒构造和单个神经元特定的启动子来驱动 optogenetic 探针和人工和平号对突触前基因的表达。当 stereotactically 注射在感兴趣的大脑区域时, 表达的构造使它成为可能的刺激以光完全地被研究的基因的表达减少的神经元。这种策略不需要开发和维护基因修饰的鼠标线, 原则上可以应用于其他有机体和任何选择的神经元基因。我们最近应用它来研究如何在突触前 P/Q 型电压门控钙通道 (VGCCs) 的替代拼接异构体的击倒如何调节 CA3 对急性海马切片 CA1 兴奋性突触的短期突触可塑性。同样的方法也可以用来操纵和探测神经回路在体内。

上述程序提供了一个可靠的方法来评估突触传递是如何影响到突触蛋白在突触前神经元。突触前Cav 2.1 (P/Q 型) VGCCs 对 CA1 兴奋性突触的短期突触可塑性有何影响, 并以此为例, 给出了可供选择的不同剪接异构体的结果。
Cav2.1 (P/Q 型) 通道是中枢神经系统中最快速突触的主要突触前 VGCCs。另一种拼接的互斥外显子37a 和37b 的孔隙形成α1亚基的 Cav2.1 (α1A) 产生两个主要变体, cav2.1 [普及] 和 cav2.1 [EFb]16,17 ,18。要确定 cav2.1 [EFa] 和 cav2.1 [EFb] 差异调节大鼠海马锥体神经元的突触传递和可塑性, 我们首先开发了异构体特定的大鹏湾以选择性地击倒 cav2.1 [普及教育] 或 Cav2.1 [EFb]5。尽管在外显子37a 和37b 之间的短尺寸 (97 bp) 和高相似性 (核苷酸水平的61.86% 个身份), 我们可以设计三和平号序列对大鼠 Cav2.1 [EFa] (和平号 EFa1: TCCTTATAGTGAATGCGGCCG; 和平号 EFa2: ATGTCCTTATAGTGAATGCGG;和平号 EFa3: TTGCAAGCAACCCTATGAGGA) 和两对老鼠 Cav2.1 [EFb] (和平号 EFb1: ATACATGTCCGGGTAAGGCAT; 和平号 EFb2: ATCTGATACATGTCCGGGTAA), 预测的高击倒效率。作为负控制 (和平号控制), 我们使用了 pcDNA6.2-GW/EmGFP-和平号质粒, 包含一个序列, 不针对任何已知的脊椎动物基因。根据 HEK 293 单元格对异源通道的第一个屏幕, 我们选择了和平号 EFa1、和平号 EFa3 和和平号 EFb2 并将其表达式磁带复制到矢量 UTR-合成 pAAV-ChETA-X 的 3 ' TdT 中, 它是为生产 rAAVs 而设计的, 在突触素启动子驱动 channelrhodopsin ChETA、红色荧光蛋白 TdTomato 和插入的和平号(图 1) 的表达式。我们还复制了和平号 EFb2 的表达盒, 以提高该和平号的击倒效率。
接下来, 我们为上述四种构造 rAAV1/2, 并利用异构体特异性 qRT PCR 技术, 对原代大鼠神经元培养的击倒效率和选择性进行量化。和平号 EFa1 和和平号 EFa3 将本机 Cav2.1 [EFa] 的 mRNA 减少了 ~ 70%, 而不是 Cav2.1 [EFb], 虽然和平号 EFb 将本机 Cav2.1 [EFb] 的 mRNA 降低了 60%, 但不是 Cav2.1 [EFa] (图 2) 的。
然后, 我们 stereotactically 注射四 rAAV1/2 到 P18 鼠海马的 CA3 区 (图 3A-c), 其坐标为 (A-P/米/d-V 从 Bregma) −2.6/@ 2.9/−2.9。注射后十五至二十四天, 我们准备了 rAAV1/2-injected 大鼠的急性海马切片, 并使用 TdTomato 荧光来确认 rAAVs 的表达和定位 (图 3B)。要调查 cav2.1 [EFa] 还是 cav2.1 [EFb] 的突触前击倒是否影响了 CA3 对 CA1 突触的短时突动可塑性, 我们刺激有选择性地感染了 473 nm 激光光脉冲 (2 毫秒长的 CA3 神经元);图 3C), 并通过修补 CA1 区域的近端至内侧通道中的锥体神经元 (图 3C) 来记录由此产生的 EPSCs。我们发现, cav2.1 拼接亚型的击倒影响了对配对脉冲刺激的响应, 相反方向: cav2.1 [EFa] (和平号 EFa1 或和平号 EFa3) 的击倒使配对脉冲简化 (PPF), 而对 ca 的击倒v2.1 [EFb] (和平号 EFb2) 取消了它 (图 3D, E)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57223/57223fig1.jpg"> 图 1: 针对 Cav2.1 [EFa] 和 cav2.1 [EFb] 的超快 optogenetic 探头 ChETA 和异构体特定的大鹏湾组合表达式的构造方案。(A) rAAV 构造 pAAV-ChETA-TdT-X 的映射, 其中包含一个突触素启动子 (Syn), 超快 channelrhodopsin ChETA 融合到 TdTomato, 在近 3 ' UTR, 一个Cav 2.1 拼接异构体特定的和平号。限制酶显示的是单一刀具。(B)顶部, 表达式卡带的方案。突触素启动子驱动 ChETA TdTomato 和异构体特定的和平号的表达式。底部, 21 核苷酸目标序列的反向补充, 构成和平号卡带的一部分。对于和平号 EFb2, 两个相同的和平号磁带被串联在一起。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57223/57223fig1large.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/57223/57223fig2.jpg"> 图2。异构体专用大鹏湾的击倒效率和选择性的评价v2.1 [EFa] 和 cav2.1 [EFb].异构体特异性 qRT PCR 分析 17-18 div 原代感染 6 div 的 RNA, rAAVs 表达大鹏湾的目标是 cav2.1 [普及教育] (和平号 EFa1 和和平号 EFa3) 或 cav2.1 [EFb] (和平号 EFb2)。数据被规范化为负控制 (和平号控制)。和平号 EFa1 和和平号 EFa3 显著和有选择地减少了 Cav2.1 [普及教育] (n = 8 和7种文化的 mRNA, 分别), 而和平号 EFb 显著并有选择地减少 Cav2.1 [EFb] (n = 4 种文化的 mRNA; ** p < 0.001; 单向分析方差测试后, Tukey-克莱默后测试)。数据显示为均值电子扫描电镜。这个数字已经从 Thalhammer, A.等等中进行了修改。5.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57223/57223fig2large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57223/57223fig3.jpg"> 图3。通过有针对性地刺激光遗传学的击倒神经元, 评估 ca v 2.1 [EFa] 和 ca v 2.1 [EFb] 在本机海马中的作用。用于体内感染的 rAAV 构造的表达式卡带的(A)方案。(B)海马部分显示 TdTomato 荧光仅限于 CA3 区域及其投射。(C)实验配置: 将激光束定向到 CA3 胞体, 并从 CA1 锥体神经元进行膜片钳记录。(D) 2 毫秒蓝 (473 nm) 激光光脉冲照射在20赫兹唤起 EPSCs, 其 PPF 由大鹏湾目标 cav2.1 [EFa] 增加, 并由和平号为 cav2.1 [EFb] 而取消。(E)实验的配对脉冲比摘要 (D), 显示了和平号 EFa1 和和平号 EFa3 的 PPF 增加, 以及和平号 EFb2 相对于和平号控制的减少 (n = 9-11 录音; * p = 0.02; ** p = 0.01; ** p < 0.0004; 协方差分析)。数据显示为均值电子扫描电镜。这个数字已经从 Thalhammer, A.等等中进行了修改。5.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57223/57223fig3large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>

Watch this Scientific Journal Video about Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits at JoVE

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

该协议提供了一个工作流, 如何将人工 microRNA 介导的 RNA 干扰与光遗传学相结合, 以刺激特定突触前 boutons 在完整的神经元回路中的选择性基因表达减少。

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

combining-optogenetics-with-artificial-micrornas-to-characterize

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.1K 次观看.  Istituto Italiano di Tecnologia. 该协议提供了一个工作流, 如何将人工 microRNA 介导的 RNA 干扰与光遗传学相结合, 以刺激特定突触前 boutons 在完整的神经元回路中的选择性基因表达减少。

视频: Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

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 ...

科研

神经科学

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

测绘激光扫描Photostimulation抑制神经回路

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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

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

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

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

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

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

使用电生理评估功能，在啮齿动物的视网膜和远程肢体缺血预适应的保护作用

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

光遗传学玻璃光纤化学传感在醒鼠中的神经型鉴定

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...